Determination of the encapsulation efficiency of lipid nanoparticles

Abstract

Disclosed herein is a method for assessing the percentage of total mRNA present encapsulated in lipid nanoparticles using a hypochromic shift in the absorbance band of a phenothiazinium dye.

Description

[Background technology]

[0001] Related Applications This application claims the benefit under 35 U.S.C. §119(e) of U.S. Provisional Application No. 63 / 290,027, filed December 15, 2021, and U.S. Provisional Application No. 63 / 329,808, filed April 11, 2022, the entire contents of each of which are incorporated herein by reference.

[0002] Lipid nanoparticles (LNPs), liposomes, and lipoplexes have been shown to be effective in delivering various payloads, such as small molecules, proteins, or nucleic acids, to cells. LNPs have shown particular promise in a variety of pharmaceutical applications, such as the delivery of mRNA vaccines. Analytical procedures for characterizing LNP compositions are challenging due to their unique properties and characteristics. Summary of the Invention

[0003] The present disclosure is based at least in part on the discovery that phenothiazinium dye (e.g., methylene blue) assay can be used to determine the percent encapsulation efficiency of lipid nanoparticles (LNPs).Accordingly, the present disclosure provides in some embodiments a method for measuring the free nucleic acid and / or encapsulation efficiency (%EE) of a sample that comprises nucleic acid and an encapsulating agent, comprising contacting the sample that comprises nucleic acid and an encapsulating agent with a phenothiazinium dye (e.g., methylene blue), measuring the absorbance of the solution that comprises nucleic acid, encapsulating agent, and a phenothiazinium dye (e.g., methylene blue), and determining the amount of free nucleic acid and / or the %EE based on the absorbance value.

[0004] In a further aspect, the present disclosure provides a method for determining the amount of free nucleic acid and / or encapsulation efficiency (% EE) of a sample comprising nucleic acid and a mounting agent, comprising measuring a hypocolor shift in the absorbance of the sample resulting from an interaction between the nucleic acid and a phenothiazinium dye (e.g., methylene blue).

[0005] In some embodiments, the nucleic acid is DNA or RNA. In some embodiments, the RNA is mRNA, siRNA, shRNA, snRNA, snoRNA, or lncRNA. In some embodiments, the RNA is mRNA.

[0006] In some embodiments, the encapsulating agent comprises a lipid nanoparticle (LNP), lipoplex, or liposome. In some embodiments, the encapsulating agent comprises an ionizable amino lipid. In some embodiments, the encapsulating agent further comprises a PEG lipid. In some embodiments, the encapsulating agent further comprises a structured lipid. In some embodiments, the encapsulating agent further comprises a phospholipid. In some embodiments, the encapsulating agent comprises an ionizable amino lipid, a PEG lipid, a structured lipid, and a phospholipid. In some embodiments, the encapsulating agent comprises a ratio of 20-60% ionizable amino lipid, 5-30% phospholipid, 10-55% structured lipid, and 0.5-15% PEG-modified lipid. In some embodiments, the encapsulating agent comprises a ratio of 20-60% ionizable amino lipid, 5-25% phospholipid, 25-55% structured lipid, and 0.5-15% PEG-modified lipid.

[0007] In some embodiments, the encapsulating agent comprises a lipid nanoparticle (LNP). In some embodiments, the encapsulating agent comprises a liposome. In some embodiments, the encapsulating agent comprises a lipoplex.

[0008] In some embodiments, the sample is formulated in an aqueous solution. In some embodiments, the aqueous solution has a pH of 5-8 or about 5-8, including a pH of about 5, 5.5, 6, 6.5, 7, 7.5, or 8. In some embodiments, the aqueous solution comprises a phosphate buffer, a tris buffer, an acetate buffer, a histidine buffer, or a citrate buffer.

[0009] Each of the limitations of the present invention may encompass various embodiments of the present invention. Thus, each of the limitations of the present invention involving any one element or combination of elements is expected to be included in each aspect of the present invention. The present invention is not limited in its application to the details of construction or the arrangement of components set forth in the following description. The present invention may be in other embodiments and may be practiced or carried out in various ways. Additionally, the phraseology and terms used herein are for the purpose of explanation and should not be considered as limiting. The use of "including," "comprising," "having," "containing," "involving," and variations thereof herein means to encompass the items listed thereafter and their equivalents as well as additional items. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0010] Lipid nanoparticle (LNP) formulations provide the opportunity to deliver various nucleic acids, such as mRNA vaccines, in vivo for prophylactic and / or therapeutic applications. Critical to the success of LNP formulations, such as mRNA vaccines, including LNPs, is the encapsulation efficiency of the composition. The encapsulation efficiency is the percentage of nucleic acid (e.g., antigen-encoding mRNA) successfully captured within the LNP relative to the total amount of nucleic acid used to prepare the LNP. As used herein, "encapsulation" refers to complete, substantial, or partial surrounding, enclosing, surrounding, or wrapping.

[0011] The present disclosure is based, at least in part, on the surprising discovery that a phenothiazinium dye (e.g., methylene blue) hypochromic shift assay can be used to accurately determine free nucleic acid (e.g., mRNA) and percent encapsulation efficiency (%EE) in a sample that includes an encapsulating agent (e.g., LNP) and nucleic acid (e.g., mRNA). The assay is used to detect free mRNA and assess the percentage of total mRNA encapsulated. Briefly, a phenothiazinium dye (e.g., methylene blue) can be complexed with mRNA in the aqueous phase to cause a hypochromic shift (e.g., as measured by a decrease in absorbance at 665 nm). This decrease is linearly proportional to the amount of nucleic acid, allowing for the calculation of the percentage of free nucleic acid (in the aqueous phase) relative to the total amount of nucleic acid present in the sample. This also allows for the calculation of the %EE. Thus, provided herein is a method for determining the amount of free mRNA and the %EE of a sample.

[0012] pigment As described herein, dyes (e.g., phenothiazinium dyes) can be used in hypochromic shift assays to determine free nucleic acid (e.g., mRNA) and percent encapsulation efficiency (%EE) in a sample containing a mounting agent and nucleic acid. In some embodiments, the dyes are cationic, i.e., the dyes have a net positive charge. In some embodiments, the dyes have high purity, are stable, have a visible absorption spectrum, and / or are intercalating dyes.

[0013] In some embodiments, the dye has high purity. The purity of the dye can be characterized based on the presence of impurities in the dye. Impurities include, for example, metals (e.g., elemental metals) and organic impurities. In some embodiments, a dye is considered to have sufficient purity if less than 10% of the dye contains impurities (e.g., non-dye components). In some embodiments, a dye is considered to have sufficient purity if less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, less than 1%, less than 0.5%, or less than 0.1% of the dye contains impurities (e.g., non-dye components). Purity can be determined by any suitable method known in the art. Non-limiting examples of methods for determining the purity of a dye include melting point determination, boiling point determination, spectroscopy (e.g., UV-VIS spectroscopy), titration, chromatography (e.g., liquid chromatography or gas chromatography), mass spectroscopy, capillary electrophoresis, and optical rotation.

[0014] In some embodiments, the dye is stable (e.g., resistant to degradation). In some embodiments, the stability of the dye is measured in terms of the chemical structure of the dye core structure, the stability of the reactive groups, the photostability of the dye, temperature, color, or a combination thereof. The stability of the dye can be measured by any suitable method known in the art. Non-limiting examples of methods for determining the stability of the dye include spectroscopy, thermal stability assays, and absorbance assays.

[0015] In some embodiments, the dye is an intercalating dye with a visible absorption spectrum. An intercalating dye is one that can intercalate between adjacent nucleotides of double-stranded or single-stranded nucleic acids to produce a detectable color. An absorption spectrum is a plot of absorbance units on the y-axis against frequency (or wavelength) on the x-axis, where the spectral features are a combination of the absorption features of the target species and extinction of the light beam due to scattering by particles and / or aerosols in the light beam. A dye with a visible absorption spectrum is one whose absorption spectrum spans the visible light wavelengths (e.g., 380 nm to 700 nm).

[0016] In some embodiments, the dye is a phenothiazinium dye (e.g., methylene blue). Phenothiazinium dyes are compounds closely related to the thiazine class of heterocyclic compounds and have the basic formula S(C 6 H4) 2 It is a derivative of phenothiazine having an NH group. Non-limiting examples of phenothiazinium dyes and related compounds include methylene blue (also known as urelene blue, provayblue, probeblue, Cl 52015 or basic blue 9), methylene green, thionine, azure A, azure B, azure C, toluidine blue O, safranine O, new methylene blue, acridine orange, proflavine hemisulfate, acriflavine, 1,9-dimethyl-methylene blue, iodomethylene blue, Nile blue A, Nile red, bromophenol blue, brilliant blue G, hematoxylin, natural red, crystal violet, methylene violet, halomethylene violet, phenol red, eosin B, carmine, fluorescein, pyronine Y, and leucomethylene blue (mesylate).

[0017] In some embodiments, the dye comprises methylene blue dye (e.g., methylene blue USP standard, Sigma-Aldrich). Methylene blue (methylthioninium chloride; 16 H 18 ClN 3 S) are thiazine dyes and salts.

[0018] In some embodiments, the dye comprises SYBR® Green dye (Sigma Aldrich).

[0019] Phenothiazinium Dye (e.g., Methylene Blue) Assays and Methods A phenothiazinium dye (e.g., methylene blue) assay can be used to determine free mRNA and / or %EE of a sample containing a nucleic acid (e.g., mRNA) and a mounting medium (e.g., LNP). The nucleic acid can be present in a formulation buffer. Thus, the assay can use a formulation buffer and a phenothiazinium dye (e.g., methylene blue dye).

[0020] According to some embodiments, the formulation buffer comprises an aqueous solution, which is a solution in which the components are dissolved or otherwise dispersed in water.

[0021] In some embodiments, the aqueous solutions disclosed herein have a pH value. In some embodiments, the pH of the aqueous solutions disclosed herein is in the range of about 4.5 to about 8.5. In some embodiments, the pH of the aqueous solutions is in the range of about 5 to about 8, about 6 to about 8, about 7 to about 8, about 6.5 to about 8, about 6.5 to about 7.5, about 6.5 to about 7, about 7.5 to about 8.5, or any range or combination thereof. In some embodiments, the pH of the aqueous solutions is 5 or about 5, 5.5 or about 5.5, 6 or about 6, 6.5 or about 6.5, 7 or about 7, 7.4 or about 7.4, 7.5 or about 7.5, or 8 or about 8.

[0022] In some embodiments, the aqueous solutions disclosed herein include a buffer component, such as Tris (tris(hydroxymethyl)aminomethane) buffer, citrate buffer, phosphate buffer, triethylammonium bicarbonate (TEAB), or histidine buffer. In some embodiments, the buffer is a Tris buffer. In some embodiments, the buffer is a citrate buffer. In some embodiments, the buffer is a phosphate buffer. In some embodiments, the buffer is a TEAB buffer. In some embodiments, the buffer is a histidine buffer.

[0023] In some embodiments, the concentration of the buffer in the formulation is about 1 mM to about 100 mM. In some embodiments, the concentration of the buffer is about 1 mM, about 2 mM, about 3 mM, about 4 mM, about 5 mM, about 6 mM, about 7 mM, about 8 mM, about 9 mM, about 10 mM, about 15 mM, about 20 mM, about 25 mM, about 30 mM, about 35 mM, about 40 mM, about 45 mM, about 50 mM, about 60 mM, about 70 mM, about 80 mM, about 90 mM, or about 100 mM.

[0024] In some embodiments, the composition (e.g., before lyophilization) includes a salt, such as sodium chloride. In some embodiments, the salt concentration in the composition is about 0.1 mM to about 300 mM. Preferably, the salt concentration in the composition before lyophilization is about 50 mM or less, such as about 0 mM to about 50 mM, or about 0.1 mM to about 50 mM. In the reconstitution medium, the salt concentration is preferably from about 25 mM.

[0025] In some embodiments, the formulation buffer comprises 5-15% sucrose, e.g., 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15% or more sucrose. In some embodiments, the formulation buffer has a pH of 7.0-8.0, e.g., 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0 or more. In some embodiments, the formulation buffer comprises 20 mM Tris, 8% sucrose, pH 7.4.

[0026] In some embodiments, the dye is a phenothiazinium dye (e.g., methylene blue). Phenothiazinium dyes are compounds closely related to the thiazine class of heterocyclic compounds and have the basic formula S(C 6 H4) 2It is a derivative of phenothiazine having an NH group. Non-limiting examples of phenothiazinium dyes and related compounds include methylene blue (also known as urelene blue, provayblue, probeblue, Cl 52015 or basic blue 9), methylene green, thionine, azure A, azure B, azure C, toluidine blue O, safranine O, new methylene blue, acridine orange, proflavine hemisulfate, acriflavine, 1,9-dimethyl-methylene blue, iodomethylene blue, Nile blue A, Nile red, bromophenol blue, brilliant blue G, hematoxylin, natural red, crystal violet, methylene violet, halomethylene violet, phenol red, eosin B, carmine, fluorescein, pyronine Y, and leucomethylene blue (mesylate). In some embodiments, performing the assay includes measuring the absorbance of a sample containing mRNA and a phenothiazinium dye (mRNA+phenothiazinium dye absorbance) at one or more wavelengths and comparing the mRNA+phenothiazinium dye absorbance value(s) to the absorbance value of the phenothiazinium dye solution (phenothiazinium dye absorbance). For example, some embodiments include subtracting the mRNA+phenothiazinium dye absorbance from the phenothiazinium dye absorbance at one or more wavelengths. In some embodiments, the phenothiazinium dye absorbance is calculated by subtracting the phenothiazinium dye absorbance at a first wavelength from the phenothiazinium dye absorbance at a second wavelength. In some embodiments, the mRNA+phenothiazinium dye absorbance is calculated by subtracting the mRNA+phenothiazinium dye absorbance at a first wavelength from the mRNA+phenothiazinium dye absorbance at a second wavelength.

[0027] In some embodiments, the dye comprises methylene blue dye. In some embodiments, methylene blue is combined with a formulation buffer to create a methylene blue working solution. In some embodiments, the methylene blue dye and the resulting methylene blue working solution are protected from light exposure (e.g., stored in an amber glass container and / or covered with aluminum foil).

[0028] In some embodiments, performing the assay includes measuring the absorbance of a sample containing mRNA and methylene blue (mRNA+methylene blue absorbance) at one or more wavelengths and comparing the mRNA+methylene blue absorbance value(s) to the absorbance value of the methylene blue solution (methylene blue absorbance). For example, some embodiments include subtracting the mRNA+methylene blue absorbance from the methylene blue absorbance at one or more wavelengths. In some embodiments, the methylene blue absorbance is calculated by subtracting the methylene blue absorbance at a first wavelength (e.g., 760 nm) from the methylene blue absorbance at a second wavelength (e.g., 665 nm). In some embodiments, the mRNA+methylene blue absorbance is calculated by subtracting the mRNA+methylene blue absorbance at a first wavelength (e.g., 760 nm) from the mRNA+methylene blue absorbance at a second wavelength (e.g., 665 nm).

[0029] Some embodiments include considering the effect of the nucleic acid formulation buffer on the absorbance reading. For example, some embodiments include further comparing the phenothiazinium dye absorbance (e.g., methylene blue absorbance) at one or more wavelengths to the absorbance of a sample containing the formulation buffer and the phenothiazinium dye (e.g., methylene blue) (buffer + phenothiazinium dye absorbance). In some embodiments, the phenothiazinium dye absorbance is calculated by subtracting the phenothiazinium dye absorbance at a first wavelength (e.g., 760 nm) from the phenothiazinium dye absorbance at a second wavelength (e.g., 665 nm). In some embodiments, the buffer + phenothiazinium dye absorbance is calculated by subtracting the buffer + phenothiazinium dye absorbance at a first wavelength (e.g., 760 nm) from the buffer + phenothiazinium dye absorbance at a second wavelength (e.g., 665 nm). Some embodiments include accounting for buffer-related effects by subtracting the phenothiazinium dye absorbance from the buffer plus phenothiazinium dye absorbance to determine a buffer-corrected phenothiazinium dye absorbance.

[0030] Some embodiments include determining the amount of free nucleic acid (e.g., mRNA) in a sample by measuring the mRNA plus phenothiazinium dye absorbance, and then subtracting the phenothiazinium dye absorbance and the buffer-corrected phenothiazinium dye absorbance.

[0031] The amount of nucleic acid (e.g., mRNA) in a sample can vary. In some embodiments, a sample contains 0.01 to 1 mg of mRNA (e.g., 0.01 mg, 0.02 mg, 0.03 mg, 0.04 mg, 0.05 mg, 0.06 mg, 0.07 mg, 0.08 mg, 0.09 mg, 0.1 mg, 0.2 mg, 0.3 mg, 0.4 mg, 0.5 mg, 0.6 mg, 0.7 mg, 0.8 mg, 0.9 mg, 1 mg, or more of mRNA.

[0032] In some embodiments, %EE is calculated according to the following formula:

number

[0033] Total mRNA can be calculated as the sample added volume (mL) times the total nucleic acid concentration of the sample (mg / mL). Free mRNA can be calculated as (1 / response factor) times (corrected absorbance of working solution-corrected absorbance of test solution-corrected absorbance of working solution with formulation buffer). The response factor is the ratio of the signal generated by the experimental analyte to the amount of analyte and can be determined experimentally using methods known in the art. In some embodiments, the response factor is 25-35 (e.g., 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35). The corrected absorbance is calculated as (absorbance of working solution / test solution / solution with formulation buffer at a first wavelength)-(absorbance of working solution / test solution / solution with formulation buffer at a second wavelength). In some embodiments, the first wavelength is 665 nm. In some embodiments, the second wavelength is 760 nm.

[0034] In some embodiments, % EE and free mRNA may be determined according to the following table (Note: in the table, methylene blue is used as a non-limiting example of a phenothiazinium dye): [Table 1]

[0035] In some embodiments, the encapsulation efficiency is greater than 60%, greater than 65%, greater than 70%, greater than 75%, greater than 80%, greater than 85%, greater than 90%, greater than 91%, greater than 92%, greater than 93%, greater than 94%, greater than 95%, greater than 96%, greater than 97%, greater than 98%, or greater than 99%. In some embodiments, the encapsulation efficiency is less than 60%, e.g., less than 55%, less than 50%, less than 45%, less than 40%, less than 35%, less than 30%, less than 25%, less than 20%, less than 15%, less than 10%, or less than 5%.

[0036] In some embodiments, measurements are repeated using the same sample; i.e., assays are performed in duplicate, triplicate, quadruplicate, or further replicates. In some embodiments, the average %EE is calculated using the results of the repeated assays on the samples.

[0037] Mounting media and formulations In some embodiments, the sample includes a mounting medium that encapsulates the nucleic acid (e.g., mRNA). Some embodiments include a composition that includes a nucleic acid (e.g., mRNA), a mounting medium (e.g., lipid nanoparticles that encapsulate the nucleic acid), and free nucleic acid (e.g., mRNA), complexed with a phenothiazinium dye (e.g., methylene blue).

[0038] Exemplary encapsulating agents include lipid nanoparticles (LNPs), liposomes (e.g., lipid vesicles), and lipoplexes. Lipid nanoparticles (LNPs) refer to nanoscale constructs (e.g., nanoparticles, typically less than 200 nm in diameter) that comprise lipid molecules preferably arranged in a substantially spherical (e.g., spheroid) geometry, and optionally encapsulate one or more additional molecular species. In some embodiments, the LNPs comprise a bleb region, for example as described in Brader et al., Biophysical Journal 120:1-5 (2021). The LNPs may comprise one or more types of lipids, including, but not limited to, amino lipids (e.g., ionizable amino lipids), neutral lipids, neutral lipids, charged lipids, PEG-modified lipids, phospholipids, structural lipids, and sterols. In some embodiments, the LNPs may further comprise one or more cargo molecules, including, but not limited to, nucleic acids (e.g., mRNA, plasmid DNA, DNA or RNA oligonucleotides, siRNA, shRNA, snRNA, snoRNA, lncRNA, etc.). LNPs may have a unilamellar structure (i.e., have a single lipid layer or lipid bilayer surrounding a central region) or a multilamellar structure (i.e., have multiple lipid layers or lipid bilayers surrounding a central region). In some embodiments, the lipid nanoparticle may be a liposome. A liposome is a nanoparticle that contains lipids arranged in one or more concentric lipid bilayers around a central region. The central region of the liposome may comprise an aqueous solution, suspension, or other aqueous composition.

[0039] In some embodiments, the encapsulating medium comprises a "lipid component" that includes one or more lipids. For example, the lipid component can include one or more cationic / ionizable lipids, PEGylated lipids, structured lipids, or other lipids, such as phospholipids.

[0040] Lipid nanoparticles typically include amino lipids, phospholipids, structural lipids (e.g., sterols), and PEG lipid components along with the nucleic acid cargo of interest. The lipid nanoparticles provided herein can be produced using components, compositions, and methods that are generally known in the art. For example, see PCT / US2016 / 052352, PCT / US2016 / 068300, PCT / US2017 / 037551, PCT / US2015 / 027400, PCT / US2016 / 047406, PCT / US2016 / 000129, PCT / US2016 / 014280, PCT / US2017 / 038426, PCT / US2014 / 010012, PCT / US2015 ... 027077, PCT / US2014 / 055394, PCT / US2016 / 052117, PCT / US2012 / 069610, PCT / US2017 / 027492, PCT / US2016 / 059575, PCT / US2016 / 069491, PCT / US2016 / 069493, and PCT / US2014 / 66242, all of which are incorporated by reference in their entireties.

[0041] Lipid Nanoparticle Compositions In some embodiments, the nucleic acid (e.g., mRNA) is formulated into a lipid nanoparticle (LNP). Lipid nanoparticles typically include ionizable amino (cationic) lipid, neutral lipid, sterol, and PEG lipid components with a nucleic acid cargo of interest. The LNP and nucleic acid form a stabilized composition. As used herein, a stabilized composition comprising an LNP and a nucleic acid can maintain its size (e.g., diameter) and potency (e.g., immunogenicity of mRNA) during storage for a period of time.

[0042] In some embodiments, the stabilized composition is formulated in an aqueous solution. The aqueous solution has water as a dissolution medium or solvent. In some embodiments, the aqueous solution may contain a buffer, such as phosphate buffer, tris buffer, acetate buffer, histidine buffer, citrate buffer, or any combination of buffers. In some embodiments, the pH of the aqueous solution is about 5 to 8, such as about 5.5, about 6, about 6.5, about 7, about 7.5, or about 8.

[0043] The size (e.g., diameter) of LNPs may be measured in some embodiments by suspending synthetic nanocarriers in a liquid (usually aqueous) medium and using dynamic light scattering (DLS) (e.g., using a Brookhaven ZetaPALS instrument). As an example, a suspension of LNPs may be diluted from an aqueous buffer into purified water so that the final synthetic nanocarrier suspension concentration is approximately 0.01-0.5 mg / mL. The diluted suspension may be prepared directly inside a cuvette suitable for DLS analysis or may be transferred to a cuvette. The cuvette is then placed in a DLS machine, equilibrated to a controlled temperature, and then scanned for a sufficient time to obtain a stable and reproducible distribution based on appropriate inputs of the viscosity and refractive index of the sample medium. The effective diameter, or the average of the distribution, is then reported. Determining the effective size of synthetic nanocarriers with high aspect ratios or non-spherical bodies may require supplemental techniques such as electron microscopy to obtain more accurate measurements. By "dimension" or "size" or "diameter" of LNPs, we mean the average of the particle size distribution, e.g., obtained using dynamic light scattering.

[0044] In some embodiments, the lipid nanoparticles contain 1-5%, optionally 1-3 mol%, for example, 1.5-2.5 mol%, 1-2 mol%, 2-3 mol%, 2.5-3.5 mol%, 3-4 mol%, or 4-5 mol% of PEG-lipids. In some embodiments, the lipid nanoparticles contain 0.5-15 mol% of PEG-modified lipids. For example, the lipid nanoparticles may contain 0.5-10 mol%, 0.5-5 mol%, 1-15 mol%, 1-10 mol%, 1-5 mol%, 2-15 mol%, 2-10 mol%, 2-5 mol%, 5-15 mol%, 5-10 mol%, or 10-15 mol%. In some embodiments, the lipid nanoparticles comprise 0.5 mol%, 1 mol%, 2 mol%, 2.5 mol%, 3 mol%, 3.5 mol%, 4 mol%, 4.5 mol%, 5 mol%, 6 mol%, 7 mol%, 8 mol%, 9 mol%, 10 mol%, 11 mol%, 12 mol%, 13 mol%, 14 mol%, or 15 mol% PEG lipids.

[0045] In some embodiments, the lipid nanoparticles contain 5-25 mol% neutral lipid. For example, the lipid nanoparticles may contain 5-20 mol%, 5-15 mol%, 5-10 mol%, 10-25 mol%, 10-20 mol%, 10-25 mol%, 15-25 mol%, 15-20 mol%, or 20-25 mol% neutral lipid. In some embodiments, the lipid nanoparticles contain 5 mol%, 10 mol%, 15 mol%, 20 mol%, or 25 mol% neutral lipid.

[0046] In some embodiments, the lipid nanoparticles comprise 40-50 mol% ionizable amino lipids, optionally 45-50 mol%, e.g., 45-46 mol%, 46-47 mol%, 47-48 mol%, 48-49 mol%, or 49-50 mol%, e.g., about 45 mol%, 45.5 mol%, 46 mol%, 46.5 mol%, 47 mol%, 47.5 mol%, 48 mol%, 48.5 mol%, 49 mol%, or 49.5 mol%.

[0047] In some embodiments, the lipid nanoparticles comprise 30-45 mol% sterol, optionally 35-40 mol%, e.g., 30-31 mol%, 31-32 mol%, 32-33 mol%, 33-34 mol%, 35-35 mol%, 35-36 mol%, 36-37 mol%, 38-38 mol%, 38-39 mol%, or 39-40 mol% sterol. In some embodiments, the lipid nanoparticles comprise 25-55 mol% sterol. For example, the lipid nanoparticles may contain 25-50 mol%, 25-45 mol%, 25-40 mol%, 25-35 mol%, 25-30 mol%, 30-55 mol%, 30-50 mol%, 30-45 mol%, 30-40 mol%, 30-35 mol%, 35-55 mol%, 35-50 mol%, 35-45 mol%, 35-40 mol%, 40-55 mol%, 40-50 mol%, 40-45 mol%, 45-55 mol%, 45-50 mol%, or 50-55 mol% of sterol. In some embodiments, the lipid nanoparticles contain 25 mol%, 30 mol%, 35 mol%, 40 mol%, 45 mol%, 50 mol%, or 55 mol% of sterol.

[0048] Ionizable Amino Lipids In some embodiments, the lipid nanoparticles comprise an ionizable molecule, a polynucleotide, and optional components such as structural lipids, sterols, neutral lipids, phospholipids, and molecules that can reduce particle aggregation (e.g., polyethylene glycol (PEG), PEG-modified lipids), such as one or more of those described above.

[0049] In some embodiments, the LNPs described herein may include one or more ionizable molecules (e.g., amino lipids or ionizable lipids). The ionizable molecules may include charged groups and may have a certain pKa. In certain embodiments, the pKa of the ionizable molecules may be about 6 or more, about 6.2 or more, about 6.5 or more, about 6.8 or more, about 7 or more, about 7.2 or more, about 7.5 or more, about 7.8 or more, about 8 or more. In some embodiments, the pKa of the ionizable molecules may be about 10 or less, about 9.8 or less, about 9.5 or less, about 9.2 or less, about 9.0 or less, about 8.8 or less, or about 8.5 or less. Combinations of the above-mentioned ranges are also possible (e.g., 6 or more and about 8.5 or less). Other ranges are also possible. In embodiments in which more than one type of ionizable molecule is present in the particle, each type of ionizable molecule may independently have a pKa within one or more of the ranges above.

[0050] Generally, an ionizable molecule comprises one or more charged groups. In some embodiments, an ionizable molecule may be positively or negatively charged. For example, an ionizable molecule may be positively charged. For example, an ionizable molecule may comprise an amine group. As used herein, the term "ionizable molecule" has its ordinary meaning in the art and may refer to a molecule or matrix that comprises one or more charged moieties. As used herein, a "charged moiety" is a chemical moiety that has a formal electronic charge, e.g., monovalent (+1 or -1), divalent (+2 or -2), trivalent (+3 or -3), etc. A charged moiety may be anionic (i.e., having a negative charge) or cationic (i.e., having a positive charge). Examples of positively charged moieties include amine groups (e.g., primary, secondary, and / or tertiary amines), ammonium groups, pyridinium groups, guanidine groups, and imidizolium groups. In certain embodiments, the charged moiety comprises an amine group. Examples of negatively charged groups or precursors thereof include carboxylate, sulfonate, sulfate, phosphonate, phosphate, hydroxyl, and the like. The charge of the charged moiety may in some cases be altered by environmental conditions, e.g., a change in pH may change the charge of the moiety and / or make the moiety charged or uncharged. In general, the charge density of the molecule and / or matrix may be selected as desired.

[0051] In some cases, the ionizable molecule (e.g., amino lipid or ionizable lipid) may include one or more precursor moieties that can be converted to a charged moiety. For example, the ionizable molecule may include a neutral moiety that can be hydrolyzed to form a charged moiety such as those described above. As a non-limiting example, the molecule or matrix may include an amide that can be hydrolyzed to form an amine. Those skilled in the art will be able to determine (e.g., by inspection, pH titration, ionic conductivity measurement, etc.) whether a given chemical moiety has a formal electronic charge and / or whether a given chemical moiety can react (e.g., hydrolyzed) to form a chemical moiety with a formal electronic charge.

[0052] Ionizable molecule (e.g., amino lipid or ionizable lipid) can have any suitable molecular weight.In certain embodiments, the molecular weight of ionizable molecule is about 2,500g / mol or less, about 2,000g / mol or less, about 1,500g / mol or less, about 1,250g / mol or less, about 1,000g / mol or less, about 900g / mol or less, about 800g / mol or less, about 700g / mol or less, about 600g / mol or less, about 500g / mol or less, about 400g / mol or less, about 300g / mol or less, about 200g / mol or less, or about 100g / mol or less. In some cases, the molecular weight of the ionizable molecule is about 100 g / mol or more, about 200 g / mol or more, about 300 g / mol or more, about 400 g / mol or more, about 500 g / mol or more, about 600 g / mol or more, about 700 g / mol or more, about 1000 g / mol or more, about 1,250 g / mol or more, about 1,500 g / mol or more, about 1,750 g / mol or more, about 2,000 g / mol or more, or about 2,250 g / mol or more. Combinations of the above ranges are also possible (e.g., at least about 200 g / mol and not more than about 2,500 g / mol). In embodiments in which more than one type of ionizable molecule is present in the particle, each type of ionizable molecule can independently have a molecular weight within one or more of the above ranges.

[0053] In some embodiments, the percentage (e.g., by weight, or by molar) of a single type of ionizable molecule (e.g., amino lipid or ionizable lipid) and / or all ionizable molecules within the particle can be about 15% or more, about 16% or more, about 17% or more, about 18% or more, about 19% or more, about 20% or more, about 21% or more, about 22% or more, about 23% or more, about 24% or more, about 25% or more, about 30% or more, about 35% or more, about 40% or more, about 42% or more, about 45% or more, about 48% or more, about 50% or more, about 52% or more, about 55% or more, about 58% or more, about 60% or more, about 62% or more, about 65% or more, or about 68% or more. In some cases, the percentage (e.g., by weight or by molar basis) may be about 70% or less, about 68% or less, about 65% or less, about 62% or less, about 60% or less, about 58% or less, about 55% or less, about 52% or less, about 50% or less, or about 48% or less. Combinations of the above-mentioned ranges are also possible (e.g., 20% or more and about 60% or less, 40% or more and about 55% or less, etc.). In embodiments in which more than one type of ionizable molecule is present in the particle, each type of ionizable molecule may independently have a percentage (e.g., by weight or by molar basis) within one or more of the ranges above. The percentage (e.g., by weight or by molar basis) may be determined by extracting the ionizable molecule(s) from the dried particle using an organic solvent or the like, and measuring the amount of the material using high pressure liquid chromatography (i.e., HPLC), liquid chromatography mass spectrometry (LC-MS), nuclear magnetic resonance (NMR), or mass spectrometry (MS). Those skilled in the art will be familiar with the techniques for determining the amount of a component using the techniques mentioned above. For example, HPLC can be used to quantify the amount of a component, for example, by comparing the area under the curve of the HPLC chromatogram with a standard curve.

[0054] It should be understood that the term "charge" or "charged moiety" does not refer to a "partial negative charge" or a "partial positive charge" of a molecule. The terms "partial negative charge" and "partial positive charge" are given their ordinary meaning in the art. A "partial negative charge" can arise when a functional group contains a bond that is polar such that electron density is attracted to one of the atoms of the bond, creating a partial negative charge on that atom. Those skilled in the art will generally recognize bonds that can be polar in this way.

[0055] In some embodiments, the lipid nanoparticles comprise at least one ionizable amino lipid, at least one non-cationic lipid, at least one sterol, and / or at least one polyethylene glycol (PEG)-modified lipid.

[0056] In some embodiments, the lipid nanoparticles comprise 40-50 mol% ionizable lipids, optionally 45-50 mol%, e.g., 45-46 mol%, 46-47 mol%, 47-48 mol%, 48-49 mol%, or 49-50 mol%, e.g., about 45 mol%, 45.5 mol%, 46 mol%, 46.5 mol%, 47 mol%, 47.5 mol%, 48 mol%, 48.5 mol%, 49 mol%, or 49.5 mol%.

[0057] In some embodiments, the lipid nanoparticles comprise 20-60 mol% of ionizable amino lipids. For example, the lipid nanoparticles may comprise 20-50 mol%, 20-40 mol%, 20-30 mol%, 30-60 mol%, 30-50 mol%, 30-40 mol%, 40-60 mol%, 40-50 mol%, or 50-60 mol% of ionizable amino lipids. In some embodiments, the lipid nanoparticles comprise 20 mol%, 30 mol%, 40 mol%, 50 mol%, or 60 mol% of ionizable amino lipids. In some embodiments, the lipid nanoparticles comprise 35 mol%, 36 mol%, 37 mol%, 38 mol%, 39 mol%, 40 mol%, 41 mol%, 42 mol%, 43 mol%, 44 mol%, 45 mol%, 46 mol%, 47 mol%, 48 mol%, 49 mol%, 50 mol%, 51 mol%, 52 mol%, 53 mol%, 54 mol%, or 55 mol% ionizable amino lipids.

[0058] In some embodiments, the lipid nanoparticles comprise 45-55 mole percent (mol%) of ionizable amino lipids. For example, the lipid nanoparticles may comprise 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, or 55 mol% of ionizable amino lipids.

[0059] In some embodiments, the ionizable amino lipid has the formula (AI): [ka] or an N-oxide thereof, or a salt or isomer thereof, In the formula, R' a is R' branched where: R' branched teeth, [ka] where: [ka] represents the attachment point, R aα , R aβ , R aγ , and R aδ are each independently H, C 2-12 Alkyl, and C 2-12 alkenyl, R 2 and R 3 are each independently 1-14 Alkyl and C 2-14 alkenyl, R 4 is -(CH 2 ) n OH, where n is selected from the group consisting of 1, 2, 3, 4, and 5, and [ka] (where: [ka] represents the attachment point, R 10 is N(R) 2 and each R is independently C 1-6 Alkyl, C 2-3 alkenyl, and H, and n2 is selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10; Each R 5 is independently 1-3 Alkyl, C 2-3 alkenyl, and H; Each R 6 is independently 1-3 Alkyl, C 2-3 alkenyl, and H; M and M' are each independently selected from the group consisting of -C(O)O- and -OC(O)-; R' is C 1-12 Alkyl or C 2-12 alkenyl, l is selected from the group consisting of 1, 2, 3, 4, and 5; m is selected from the group consisting of 5, 6, 7, 8, 9, 10, 11, 12, and 13.

[0060] In some embodiments of the compound of Formula (AI), R' a is R' branched and R' branched teeth [ka] and [ka] represents the attachment point, and R aα , R aβ , R aγ , and R aδ are H and R 2 and R 3 are C 1-14 is alkyl, R 4 Ha-(CH 2 ) n OH, n is 2, and each R 5 is H, and each R 6 is H, M and M' are each -C(O)O-, and R' is C 1-12 alkyl, l is 5, and m is 7.

[0061] In some embodiments of the compound of Formula (AI), R' a is R' branched and R' branched teeth [ka] and [ka] represents the attachment point, and R aα , R aβ , R aγ , and R aδ are H and R 2 and R 3 are C 1-14 is alkyl, R 4 Ha-(CH 2 )n OH, n is 2, and each R 5 is H, and each R 6 is H, M and M' are each -C(O)O-, and R' is C 1-12 alkyl, l is 3, and m is 7.

[0062] In some embodiments of the compound of Formula (AI), R' a is R' branched and R' branched teeth [ka] and [ka] represents the attachment point, and R aα is C 2-12 is alkyl, R aβ , R aγ , and R aδ are H and R 2 and R 3 are C 1-14 is alkyl, R 4 teeth [ka] and R 10 is NH(C 1-6 alkyl), n2 is 2, and R 5 is H, and each R 6 is H, M and M' are each -C(O)O-, and R' is C 1-12 alkyl, l is 5, and m is 7.

[0063] In some embodiments of the compounds of Formula (I), R' a is R' branched and R' branched teeth [ka] and [ka] represents the attachment point, and R aα , R aβ , and R aδ are H and R aγ is C 2-12 is alkyl, R 2 and R 3 are C 1-14 is alkyl, R 4 Ha-(CH 2 ) n OH, n is 2, and each R 5 is H, and each R 6 is H, M and M' are each -C(O)O-, and R' is C 1-12 alkyl, l is 5, and m is 7.

[0064] In some embodiments, the compound of formula (I) is [ka] is selected from.

[0065] In some embodiments, the ionizable amino lipid has the formula (AIa): [ka] or an N-oxide thereof, or a salt or isomer thereof, In the formula, R' a is R' branched where: R' branched teeth, [ka] where: [ka] represents the attachment point, R aβ , R aγ , and R aδ are each independently H, C 2-12 Alkyl, and C 2-12alkenyl, R 2 and R 3 are each independently 1-14 Alkyl and C 2-14 alkenyl, R 4 is -(CH 2 ) n OH, where n is selected from the group consisting of 1, 2, 3, 4, and 5, and [ka] (where: [ka] represents the attachment point, R 10 is N(R) 2 and each R is independently C 1-6 Alkyl, C 2-3 alkenyl, and H, and n2 is selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10; Each R 5 is independently 1-3 Alkyl, C 2-3 alkenyl, and H; Each R 6 is independently 1-3 Alkyl, C 2-3 alkenyl, and H; M and M' are each independently selected from the group consisting of -C(O)O- and -OC(O)-; R' is C 1-12 Alkyl or C 2-12 alkenyl, l is selected from the group consisting of 1, 2, 3, 4, and 5; m is selected from the group consisting of 5, 6, 7, 8, 9, 10, 11, 12, and 13.

[0066] In some embodiments, the ionizable amino lipid has the formula (AIb): [ka] or an N-oxide thereof, or a salt or isomer thereof, In the formula, R' a is R' branched where: R' branched teeth, [ka] where: [ka] represents the attachment point, R aα , R aβ , R aγ , and R aδ are each independently H, C 2-12 Alkyl, and C 2-12 alkenyl, R 2 and R 3 are each independently 1-14 Alkyl and C 2-14 alkenyl, R 4 is -(CH 2 ) n OH, where n is selected from the group consisting of 1, 2, 3, 4, and 5; Each R 5 is independently 1-3 Alkyl, C 2-3 alkenyl, and H; Each R 6 is independently 1-3 Alkyl, C 2-3 alkenyl, and H; M and M' are each independently selected from the group consisting of -C(O)O- and -OC(O)-; R' is C 1-12 Alkyl or C 2-12 alkenyl, l is selected from the group consisting of 1, 2, 3, 4, and 5; m is selected from the group consisting of 5, 6, 7, 8, 9, 10, 11, 12, and 13.

[0067] In some embodiments of formula (AI) or (AIb), ​​R' a is R' branched and R' branched teeth [ka] and [ka] represents the attachment point, and R aβ , R aγ , and R aδ are H and R 2 and R 3 are C 1-14 is alkyl, R 4 Ha-(CH 2 ) n OH, n is 2, and each R 5 is H, and each R 6 is H, M and M' are each -C(O)O-, and R' is C 1-12 alkyl, l is 5, and m is 7.

[0068] In some embodiments of formula (AI) or (AIb), ​​R' a is R' branched and R' branched teeth [ka] and [ka] represents the attachment point, and R aβ , R aγ , and R aδ are H and R 2 and R 3 are C 1-14 is alkyl, R 4 Ha-(CH 2 ) nOH, n is 2, and each R 5 is H, and each R 6 is H, M and M' are each -C(O)O-, and R' is C 1-12 alkyl, l is 3, and m is 7.

[0069] In some embodiments of formula (AI) or (AIb), ​​R' a is R' branched and R' branched teeth [ka] and [ka] represents the attachment point, and R aβ and R aδ are H and R aγ is C 2-12 is alkyl, R 2 and R 3 are C 1-14 is alkyl, R 4 Ha-(CH 2 ) n OH, n is 2, and each R 5 is H, and each R 6 is H, M and M' are each -C(O)O-, and R' is C 1-12 alkyl, l is 5, and m is 7.

[0070] In some embodiments, the ionizable amino lipid has the formula (AIc): [ka] or an N-oxide thereof, or a salt or isomer thereof, In the formula, R' a is R' branched where: R' branched teeth, [ka] where: [ka] represents the attachment point, R aα , R aβ , R aγ , and R aδ are each independently H, C 2-12 Alkyl, and C 2-12 alkenyl, R 2 and R 3 are each independently 1-14 Alkyl and C 2-14 alkenyl, R 4 teeth, [ka] and Where: [ka] represents the attachment point, and R 10 is N(R) 2 and each R is independently C 1-6 Alkyl, C 2-3 alkenyl, and H, and n2 is selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10; Each R 5 is independently 1-3 Alkyl, C 2-3 alkenyl, and H; Each R 6 is independently 1-3 Alkyl, C 2-3 alkenyl, and H; M and M' are each independently selected from the group consisting of -C(O)O- and -OC(O)-; R' is C 1-12 Alkyl or C 2-12 alkenyl, l is selected from the group consisting of 1, 2, 3, 4, and 5; m is selected from the group consisting of 5, 6, 7, 8, 9, 10, 11, 12, and 13.

[0071] In some embodiments, R' a is R' branched and R' branched teeth, [ka] and [ka] represents the attachment point, and R aβ , R aγ , and R aδ are H and R aα is C 2-12 is alkyl, R 2 and R 3 are respectively, C 1-14 is alkyl, R 4 teeth, [ka] and [ka] represents the attachment point, and R 10 is NH(C 1-6 alkyl), n2 is 2, and each R 5 is H, and each R 6 is H, M and M' are each -C(O)O-, and R' is C 1-12 alkyl, l is 5, and m is 7.

[0072] In some embodiments, the compound of formula (AIc) is [ka] It is.

[0073] In some embodiments, the ionizable amino lipid has formula (AII): [ka] or an N-oxide thereof, or a salt or isomer thereof, In the formula, R' a is R' branched or R' cyclic and R' branched teeth, [ka] and R' cyclic teeth, [ka] and R' b teeth, [ka] and Where: [ka] represents the attachment point, R aγ and R aδ are each independently H, C 1-12 Alkyl, and C 2-12 alkenyl, where R aγ and R aδ At least one of them is C 1-12 Alkyl and C 2-12 alkenyl, R bγ and R bδ are each independently H, C 1-12 Alkyl, and C 2-12 alkenyl, where R bγ and R bδ At least one of them is C 1-12 Alkyl and C 2-12 alkenyl, R 2 and R 3 are each independently 1-14Alkyl and C 2-14 alkenyl, R 4 is -(CH 2 ) n OH, where n is selected from the group consisting of 1, 2, 3, 4, and 5, and [ka] (where: [ka] represents the attachment point, and R 10 is N(R) 2 and each R is independently C 1-6 Alkyl, C 2-3 alkenyl, and H, and n2 is selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10; Each R' is independently C 1-12 Alkyl or C 2-12 alkenyl, Y a is C 3-6 is a carbocyclic ring, R * " a is C 1-15 Alkyl and C 2-15 alkenyl, s is 2 or 3; m is selected from 1, 2, 3, 4, 5, 6, 7, 8, and 9; l is selected from 1, 2, 3, 4, 5, 6, 7, 8, and 9.

[0074] In some embodiments, the ionizable amino lipid has the formula (AII-a): [ka] or an N-oxide thereof, or a salt or isomer thereof, In the formula, R' a is R' branched or R'cyclic and R' branched teeth, [ka] and R' b teeth, [ka] and Where: [ka] represents the attachment point, R aγ and R aδ are each independently H, C 1-12 Alkyl, and C 2-12 alkenyl, where R aγ and R aδ At least one of the 1-12 Alkyl and C 2-12 alkenyl, R bγ and R bδ are each independently H, C 1-12 Alkyl, and C 2-12 alkenyl, where R bγ and R bδ At least one of the 1-12 Alkyl and C 2-12 alkenyl, R 2 and R 3 are each independently 1-14 Alkyl and C 2-14 alkenyl, R 4 is -(CH 2 ) n OH, where n is selected from the group consisting of 1, 2, 3, 4, and 5, and [ka] (where: [ka] represents the attachment point, and R 10 is N(R) 2 and each R is independently C 1-6 Alkyl, C 2-3 alkenyl, and H, and n2 is selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10; Each R' is independently C 1-12 Alkyl or C 2-12 alkenyl, m is selected from 1, 2, 3, 4, 5, 6, 7, 8, and 9; l is selected from 1, 2, 3, 4, 5, 6, 7, 8, and 9.

[0075] In some embodiments, the ionizable amino lipid has the formula (AII-b): [ka] or an N-oxide thereof, or a salt or isomer thereof, In the formula, R' a is R' branched or R' cyclic and R' branched teeth, [ka] and R' b teeth, [ka] and Where: [ka] represents the attachment point, R aγ and R bγ are each independently 1-12 Alkyl and C 2-12 alkenyl, R 2 and R 3 are each independently 1-14 Alkyl and C 2-14 alkenyl, R 4 is -(CH 2 ) n OH, where n is selected from the group consisting of 1, 2, 3, 4, and 5, and [ka] (where: [ka] represents the attachment point, and R 10 is N(R) 2 and each R is independently C 1-6 Alkyl, C 2-3 alkenyl, and H, and n2 is selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10; Each R' is independently C 1-12 Alkyl or C 2-12 alkenyl, m is selected from 1, 2, 3, 4, 5, 6, 7, 8, and 9; l is selected from 1, 2, 3, 4, 5, 6, 7, 8, and 9.

[0076] In some embodiments, the ionizable amino lipid has the formula (AII-c): [ka] or an N-oxide thereof, or a salt or isomer thereof, In the formula, R' a is R' branched or R' cyclic and R' branched teeth, [ka] and R'b teeth, [ka] and Where: [ka] represents the attachment point, R aγ is C 1-12 Alkyl and C 2-12 alkenyl, R 2 and R 3 are each independently 1-14 Alkyl and C 2-14 alkenyl, R 4 is -(CH 2 ) n OH, where n is selected from the group consisting of 1, 2, 3, 4, and 5, and [ka] (where: [ka] represents the attachment point, R 10 is N(R) 2 and each R is independently C 1-6 Alkyl, C 2-3 alkenyl, and H, and n2 is selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10; R' is C 1-12 Alkyl or C 2-12 alkenyl, m is selected from 1, 2, 3, 4, 5, 6, 7, 8, and 9; l is selected from 1, 2, 3, 4, 5, 6, 7, 8, and 9.

[0077] In some embodiments, the ionizable amino lipid has the formula (AII-d): [ka] or an N-oxide thereof, or a salt or isomer thereof, In the formula, R' a is R' branched or R' cyclic and R' branched teeth, [ka] and R' b teeth, [ka] and Where: [ka] represents the attachment point, R aγ and R bγ are each independently 1-12 Alkyl and C 2-12 alkenyl, R 4 is -(CH 2 ) n OH, where n is selected from the group consisting of 1, 2, 3, 4, and 5, and [ka] (where: [ka] represents the attachment point, R 10 is N(R) 2 and each R is independently C 1-6 Alkyl, C 2-3 alkenyl, and H, and n2 is selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10; Each R' is independently C1-12 Alkyl or C 2-12 alkenyl, m is selected from 1, 2, 3, 4, 5, 6, 7, 8, and 9; l is selected from 1, 2, 3, 4, 5, 6, 7, 8, and 9.

[0078] In some embodiments, the ionizable amino lipid has the formula (AII-e): [ka] or an N-oxide thereof, or a salt or isomer thereof, In the formula, R' a is R' branched or R' cyclic and R' branched teeth, [ka] and R' b teeth, [ka] and Where: [ka] represents the attachment point, R aγ is C 1-12 Alkyl and C 2-12 alkenyl, R 2 and R 3 are each independently 1-14 Alkyl and C 2-14 alkenyl, R 4 is -(CH 2 ) n OH, where n is selected from the group consisting of 1, 2, 3, 4, and 5; R' is C 1-12 Alkyl or C 2-12 alkenyl, m is selected from 1, 2, 3, 4, 5, 6, 7, 8, and 9; l is selected from 1, 2, 3, 4, 5, 6, 7, 8, and 9.

[0079] In some embodiments of a compound of Formula (AII), (AII-a), (AII-b), (AII-c), (AII-d), or (AII-e), m and l are each independently selected from 4, 5, and 6. In some embodiments of a compound of Formula (AII), (AII-a), (AII-b), (AII-c), (AII-d), or (AII-e), m and l are each 5.

[0080] In some embodiments of the compound of Formula (AII), (AII-a), (AII-b), (AII-c), (AII-d), or (AII-e), each R' is independently C 1-12 In some embodiments of the compound of Formula (AII), (AII-a), (AII-b), (AII-c), (AII-d), or (AII-e), each R' is independently C 2-5 It is an alkyl.

[0081] In some embodiments of the compound of Formula (AII), (AII-a), (AII-b), (AII-c), (AII-d), or (AII-e), R' b teeth [ka] and R 2 and R 3 are each independently 1-14 In some embodiments of the compound of Formula (AII), (AII-a), (AII-b), (AII-c), (AII-d), or (AII-e), R' is alkyl. b teeth [ka] and R 2 and R 3 are each independently 6-10In some embodiments of the compound of Formula (AII), (AII-a), (AII-b), (AII-c), (AII-d), or (AII-e), R' is alkyl. b teeth [ka] and R 2 and R 3 are respectively, C 8 It is an alkyl.

[0082] In some embodiments of the compound of Formula (AII), (AII-a), (AII-b), (AII-c), (AII-d), or (AII-e), R' branched teeth [ka] and R' b teeth [ka] and R aγ is C 1-12 is alkyl, R 2 and R 3 are each independently 6-10 In some embodiments of the compound of Formula (AII), (AII-a), (AII-b), (AII-c), (AII-d), or (AII-e), R' is alkyl. branched teeth [ka] and R' b teeth [ka] and R aγ is C 2-6 is alkyl, R 2 and R 3 are each independently 6-10In some embodiments of the compound of Formula (AII), (AII-a), (AII-b), (AII-c), (AII-d), or (AII-e), R' is alkyl. branched teeth [ka] and R' b teeth [ka] and R aγ is C 2-6 is alkyl, R 2 and R 3 are respectively, C 8 It is an alkyl.

[0083] In some embodiments of the compound of Formula (AII), (AII-a), (AII-b), (AII-c), (AII-d), or (AII-e), R' branched teeth [ka] and R' b teeth [ka] and R aγ and R bγ are respectively, C 1-12 In some embodiments of the compound of Formula (AII), (AII-a), (AII-b), (AII-c), (AII-d), or (AII-e), R' is alkyl. branched teeth [ka] and R' b teeth [ka] and R aγ and R bγ are respectively, C 2-6 It is an alkyl.

[0084] In some embodiments of the compound of Formula (AII), (AII-a), (AII-b), (AII-c), (AII-d), or (AII-e), m and l are each independently selected from 4, 5, and 6; and each R′ is independently selected from C 1-12 In some embodiments of the compound of Formula (AII), (AII-a), (AII-b), (AII-c), (AII-d), or (AII-e), m and l are each 5 and each R' is independently C 2-5 It is an alkyl.

[0085] In some embodiments of compounds (AII), (AII-a), (AII-b), (AII-c), (AII-d), or (AII-e), R' branched teeth [ka] and R' b teeth [ka] wherein m and l are each independently selected from 4, 5, and 6; and each R′ is independently selected from C 1-12 is alkyl, R aγ and R bγ are respectively, C 1-12 In some embodiments of the compound of Formula (AII), (AII-a), (AII-b), (AII-c), (AII-d), or (AII-e), R' is alkyl. branched teeth [ka] and R' b teeth [ka] m and l are each 5; and each R′ is independently selected from the group consisting of C 2-5 is alkyl, R aγ and R bγ are respectively, C 2-6 It is an alkyl.

[0086] In some embodiments of the compound of Formula (AII), (AII-a), (AII-b), (AII-c), (AII-d), or (AII-e), R' branched teeth [ka] and R' b teeth [ka] m and l are each independently selected from 4, 5 and 6; R′ is C 1-12 is alkyl, R aγ is C 1-12 is alkyl, R 2 and R 3 are each independently 6-10 It is an alkyl.

[0087] In some embodiments of the compound of Formula (AII), (AII-a), (AII-b), (AII-c), (AII-d), or (AII-e), R' branched teeth [ka] and R' b teeth [ka] m and l are each 5; R′ is C 2-5 is alkyl, R aγ is C 2-6 is alkyl, R 2 and R 3 are respectively, C 8 It is an alkyl.

[0088] In some embodiments of compound (AII), (AII-a), (AII-b), (AII-c), (AII-d), or (AII-e), R 4 teeth [ka] where R 10 is NH(C 1-6 In some embodiments of the compound of Formula (AII), (AII-a), (AII-b), (AII-c), (AII-d), or (AII-e), R 4 teeth [ka] where R 10 is NH(CH 3 ) where n2 is 2.

[0089] In some embodiments of the compound of Formula (AII), (AII-a), (AII-b), (AII-c), (AII-d), or (AII-e), R' branched teeth [ka] and R' b teeth [ka] wherein m and l are each independently selected from 4, 5, and 6; and each R′ is independently selected from C 1-12 is alkyl, R aγ and R bγ are respectively, C 1-12 is alkyl, R 4 teeth [ka] where R 10 is NH(C 1-6 In some embodiments of the compound of Formula (AII), (AII-a), (AII-b), (AII-c), (AII-d), or (AII-e), R' branched teeth [ka] and R' b teeth [ka] m and l are each 5; and each R′ is independently selected from the group consisting of C 2-5 is alkyl, R aγ and R bγ are respectively, C 2-6 is alkyl, R 4 teeth [ka] where R 10 is NH(CH 3 ) where n2 is 2.

[0090] In some embodiments of the compound of Formula (AII), (AII-a), (AII-b), (AII-c), (AII-d), or (AII-e), R' branched teeth [ka] and R' b teeth [ka] m and l are each independently selected from 4, 5 and 6; R′ is C 1-12 is alkyl, R 2 and R 3 are each independently 6-10 is alkyl, R aγ is C 1-12 is alkyl, R 4 teeth [ka] where R 10 is NH(C 1-6 In some embodiments of the compound of Formula (AII), (AII-a), (AII-b), (AII-c), (AII-d), or (AII-e), R' branched teeth [ka] and R' b teeth [ka] m and l are each 5; R′ is C 2-5 is alkyl, R aγ is C 2-6 is alkyl, R 2 and R 3 are respectively, C 8 is alkyl, R 4 teeth [ka] where R 10 is NH(CH 3 ) where n2 is 2.

[0091] In some embodiments of the compound of Formula (AII), (AII-a), (AII-b), (AII-c), (AII-d), or (AII-e), R 4 Ha-(CH 2 ) n OH and n is 2, 3, or 4. In some embodiments of the compound of Formula (AII), (AII-a), (AII-b), (AII-c), (AII-d), or (AII-e), R 4 Ha-(CH 2 ) n OH and n is 2.

[0092] In some embodiments of the compound of Formula (AII), (AII-a), (AII-b), (AII-c), (AII-d), or (AII-e), R' branched teeth [ka] and R' b teeth [ka] wherein m and l are each independently selected from 4, 5, and 6; and each R′ is independently selected from C1-12 is alkyl, R aγ and R bγ are respectively, C 1-12 is alkyl, R 4 Ha-(CH 2 ) n OH and n is 2, 3, or 4. In some embodiments of the compound of Formula (AII), (AII-a), (AII-b), (AII-c), (AII-d), or (AII-e), R' branched teeth [ka] and R' b teeth [ka] m and l are each 5; and each R′ is independently selected from the group consisting of C 2-5 is alkyl, R aγ and R bγ are respectively, C 2-6 is alkyl, R 4 Ha-(CH 2 ) n OH and n is 2.

[0093] In some embodiments, the ionizable amino lipid has the formula (AII-f): [ka] or an N-oxide thereof, or a salt or isomer thereof, In the formula, R' a is R' branched or R' cyclic and R' branched teeth, [ka] and R' b teeth, [ka] and Where: [ka] represents the attachment point, R aγ is C 1-12 is alkyl, R 2 and R 3 are each independently 1-14 is alkyl, R 4 is -(CH 2 ) n OH, where n is selected from the group consisting of 1, 2, 3, 4, and 5; R' is C 1-12 is alkyl, m is selected from 4, 5 and 6; l is selected from 4, 5 and 6.

[0094] In some embodiments of the compound of Formula (AII-f), m and l are each 5; and n is 2, 3, or 4.

[0095] In some embodiments of the compound of Formula (AII-f), R′ is C 2-5 is alkyl, R aγ is C 2-6 is alkyl, R 2 and R 3 are respectively, C 6-10 It is an alkyl.

[0096] In some embodiments of the compound of Formula (AII-f), m and l are each 5; n is 2, 3, or 4; and R′ is C 2-5 is alkyl, R aγ is C 2-6 is alkyl, R 2 and R 3 are C 6-10 It is an alkyl.

[0097] In some embodiments, the ionizable amino lipid has the formula (AII-g): [ka] is a compound of the formula R aγ is C 2-6 is alkyl, R' is C 2-5 is alkyl, R 4 is -(CH 2 ) n OH, where n is selected from the group consisting of 3, 4, and 5, and [ka] (where: [ka] represents the attachment point, and R 10 is NH(C 1-6 alkyl), and n2 is selected from the group consisting of 1, 2, and 3.

[0098] In some embodiments, the ionizable amino lipid has the formula (AII-h): [ka] is a compound of the formula R aγ and R bγ are each independently 2-6 is alkyl, Each R' is independently C 2-5 is alkyl, R 4 is -(CH 2 ) n OH, where n is selected from the group consisting of 3, 4, and 5, and [ka] (where: [ka] represents the attachment point, and R 10 is NH(C 1-6alkyl), and n2 is selected from the group consisting of 1, 2, and 3.

[0099] In some embodiments of compounds of Formula (AII-g) or (AII-h), R 4 teeth [ka] where: R 10 is NH(CH 3 ) where n2 is 2.

[0100] In some embodiments of compounds of Formula (AII-g) or (AII-h), R 4 Ha-(CH 2 ) 2 It is OH.

[0101] In some embodiments, the ionizable amino lipid has formula (VI): [ka] or an N-oxide thereof, or a salt or isomer thereof, wherein R 1 is C 5-30 Alkyl, C 5-20 Alkenyl, -R * -YR", -YR", and -R"M'R'; R 2 and R 3 are independently H, C 1-14 Alkyl, C 2-14 Alkenyl, -R * YR”, -YR”, and -R * OR” or R 2 and R 3 form a heterocyclic or carbocyclic ring together with the atom to which they are attached, R 4 is hydrogen, C 3-6 Carbocycle, -(CH 2 ) n Q, -(CH2 ) n CHQR, -CHQR, -CQ(R) 2 , and unsubstituted C 1-6 alkyl, where Q is a carbocycle, a heterocycle, -OR, -O(CH 2 ) n N(R) 2 , -C(O)OR, -OC(O)R, -CX 3 , -CX 2 H, -CXH 2 , -CN, -N(R) 2 , -C(O)N(R) 2 , -N(R)C(O)R, -N(R)S(O) 2 R, -N(R)C(O)N(R) 2 , -N(R)C(S)N(R) 2 , -N(R)R 8 , -N(R)S(O) 2 R 8 , -O(CH 2 ) n OR, -N(R)C(=NR 9 )N(R) 2 , -N(R)C(=CHR 9 )N(R) 2 , -OC(O)N(R) 2 , -N(R)C(O)OR, -N(OR)C(O)R, -N(OR)S(O) 2 R, -N(OR)C(O)OR, -N(OR)C(O)N(R) 2 , -N(OR)C(S)N(R) 2 , -N(OR)C(=NR 9 )N(R) 2 , -N(OR)C(=CHR 9 )N(R) 2 , -C(=NR 9 )N(R) 2 , -C(=NR 9 )R, -C(O)N(R)OR, and -C(R)N(R) 2 C(O)OR, where each n is independently selected from 1, 2, 3, 4, and 5; Each R 5 is independently 1-3 Alkyl, C 2-3 alkenyl, and H; Each R 6 is independently 1-3 Alkyl, C 2-3 alkenyl, and H; M and M' are independently -C(O)O-, -OC(O)-, -OC(O)-M"-C(O)O-, -C(O)N(R')-, -N(R')C(O)-, -C(O)-, -C(S)-, -C(S)S-, -SC(S)-, -CH(OH)-, -P(O)(OR')O-, -S(O) 2 -, -SS-, an aryl group, and a heteroaryl group, where M″ is a bond, C 1-13 Alkyl or C 2-13 alkenyl, R 7 is C 1-3 Alkyl, C 2-3 alkenyl, and H; R 8 is C 3-6 selected from the group consisting of carbocycles and heterocycles; R 9 H, CN, NO 2 , C 1-6 Alkyl, -OR, -S(O) 2 R, -S(O) 2 N(R) 2 , C 2-6 Alkenyl, C 3-6 selected from the group consisting of carbocycles and heterocycles; Each R is independently C 1-3 Alkyl, C 2-3 alkenyl, and H; Each R' is independently C 1-18 Alkyl, C 2-18 Alkenyl, -R * is selected from the group consisting of YR″, -YR″, and H; Each R" is independently 3-15 Alkyl and C 3-15 alkenyl, Each R * is independently 1-12 Alkyl and C 2-12alkenyl, Each Y is independently C 3-6 is a carbocyclic ring, each X is independently selected from the group consisting of F, Cl, Br, and I; m is selected from 5, 6, 7, 8, 9, 10, 11, 12, and 13; R 4 But -(CH 2 ) n Q, -(CH 2 ) n CHQR, -CHQR, or -CQ(R) 2 (i) Q is -N(R) when n is 1, 2, 3, 4, or 5; 2 or (ii) Q is not a 5-, 6-, or 7-membered heterocycloalkyl when n is 1 or 2.

[0102] In some embodiments, another subset of compounds of formula (VI) includes R 1 But, C 5-30 Alkyl, C 5-20 Alkenyl, -R * -YR", -YR", and -R"M'R'; R 2 and R 3 However, independently, H, C 1-14 Alkyl, C 2-14 Alkenyl, -R * YR”, -YR”, and -R * OR” or R 2 and R 3 form a heterocyclic or carbocyclic ring together with the atoms to which they are attached, R 4 But, C 3-6 Carbocycle, -(CH 2 ) n Q, -(CH 2 ) n CHQR, -CHQR, -CQ(R) 2 , and unsubstituted C 1-6 alkyl, where Q is selected from the group consisting of C 3-6a 5-14 membered heteroaryl having one or more heteroatoms selected from a carbocycle, N, O, and S; -OR; -O(CH 2 ) n N(R) 2 , -C(O)OR, -OC(O)R, -CX 3 , -CX 2 H, -CXH 2 , -CN, -C(O)N(R) 2 , -N(R)C(O)R, -N(R)S(O) 2 R, -N(R)C(O)N(R) 2 , -N(R)C(S)N(R) 2 ,-CRN(R) 2 C(O)OR, -N(R)R 8 , -O(CH 2 ) n OR, -N(R)C(=NR 9 )N(R) 2 , -N(R)C(=CHR 9 )N(R) 2 , -OC(O)N(R) 2 , -N(R)C(O)OR, -N(OR)C(O)R, -N(OR)S(O) 2 R, -N(OR)C(O)OR, -N(OR)C(O)N(R) 2 , -N(OR)C(S)N(R) 2 , -N(OR)C(=NR 9 )N(R) 2 , -N(OR)C(=CHR 9 )N(R) 2 , -C(=NR 9 )N(R) 2 , -C(=NR 9 )R, -C(O)N(R)OR, as well as oxo(=O), OH, amino, mono- or di-alkylamino, and C 1-3 and each n is independently selected from 1, 2, 3, 4, and 5; Each R 5 But independently, C 1-3 Alkyl, C 2-3alkenyl, and H; Each R 6 But independently, C 1-3 Alkyl, C 2-3 alkenyl, and H; M and M' are independently -C(O)O-, -OC(O)-, -C(O)N(R')-, -N(R')C(O)-, -C(O)-, -C(S)-, -C(S)S-, -SC(S)-, -CH(OH)-, -P(O)(OR')O-, -S(O) 2 -, -SS-, an aryl group, and a heteroaryl group; R 7 But, C 1-3 Alkyl, C 2-3 alkenyl, and H; R 8 But, C 3-6 selected from the group consisting of carbocycles and heterocycles; R 9 H, CN, NO 2 , C 1-6 Alkyl, -OR, -S(O) 2 R, -S(O) 2 N(R) 2 , C 2-6 Alkenyl, C 3-6 selected from the group consisting of carbocycles and heterocycles; Each R is independently C 1-3 Alkyl, C 2-3 alkenyl, and H; Each R' is independently C 1-18 Alkyl, C 2-18 Alkenyl, -R * is selected from the group consisting of YR″, -YR″, and H; Each R” is independently 3-14 Alkyl and C 3-14 alkenyl, Each R * But independently, C 1-12 Alkyl and C 2-12 alkenyl, Each Y is independently 3-6 is a carbocyclic ring, each X is independently selected from the group consisting of F, Cl, Br, and I; m is selected from 5, 6, 7, 8, 9, 10, 11, 12, and 13; or a salt or isomer thereof.

[0103] In some embodiments, another subset of compounds of formula (VI) includes R 1 But, C 5-30 Alkyl, C 5-20 Alkenyl, -R * -YR", -YR", and -R"M'R'; R 2 and R 3 However, independently, H, C 1-14 Alkyl, C 2-14 Alkenyl, -R * YR”, -YR”, and -R * OR” or R 2 and R 3 form a heterocyclic or carbocyclic ring together with the atoms to which they are attached, R 4 But, C 3-6 Carbocycle, -(CH 2 ) n Q, -(CH 2 ) n CHQR, -CHQR, -CQ(R) 2 , and unsubstituted C 1-6 alkyl, where Q is selected from the group consisting of C 3-6 A 5- to 14-membered heterocyclic ring having one or more heteroatoms selected from a carbocycle, N, O, and S, -OR, -O(CH 2 ) n N(R) 2 , -C(O)OR, -OC(O)R, -CX 3 , -CX 2 H, -CXH 2 , -CN, -C(O)N(R) 2 , -N(R)C(O)R, -N(R)S(O) 2 R, -N(R)C(O)N(R) 2 , -N(R)C(S)N(R) 2 , -CRN(R)2 C(O)OR, -N(R)R 8 , -O(CH 2 ) n OR, -N(R)C(=NR 9 )N(R) 2 , -N(R)C(=CHR 9 )N(R) 2 , -OC(O)N(R) 2 , -N(R)C(O)OR, -N(OR)C(O)R, -N(OR)S(O) 2 R, -N(OR)C(O)OR, -N(OR)C(O)N(R) 2 , -N(OR)C(S)N(R) 2 , -N(OR)C(=NR 9 )N(R) 2 , -N(OR)C(=CHR 9 )N(R) 2 , -C(=NR 9 )R, -C(O)N(R)OR, and -C(=NR 9 )N(R) 2 each n is independently selected from 1, 2, 3, 4, and 5; Q is a 5- to 14-membered heterocycle; and (i) R 4 Ga-(CH 2 ) n Q, where n is 1 or 2; or (ii) R 4 Ga-(CH 2 ) n CHQR, where n is 1; or (iii) R 4 -CHQR and -CQ(R) 2 and Q is either a 5- to 14-membered heteroaryl or an 8- to 14-membered heterocycloalkyl; Each R 5 But independently, C 1-3 Alkyl, C 2-3 alkenyl, and H; Each R 6 But independently, C 1-3 Alkyl, C 2-3 alkenyl, and H; M and M' are independently -C(O)O-, -OC(O)-, -C(O)N(R')-, -N(R')C(O)-, -C(O)-, -C(S)-, -C(S)S-, -SC(S)-, -CH(OH)-, -P(O)(OR')O-, -S(O) 2 -, -SS-, an aryl group, and a heteroaryl group; R 7 But, C 1-3 Alkyl, C 2-3 alkenyl, and H; R 8 But, C 3-6 selected from the group consisting of carbocycles and heterocycles; R 9 H, CN, NO 2 , C 1-6 Alkyl, -OR, -S(O) 2 R, -S(O) 2 N(R) 2 , C 2-6 Alkenyl, C 3-6 selected from the group consisting of carbocycles and heterocycles; Each R is independently C 1-3 Alkyl, C 2-3 alkenyl, and H; Each R' is independently C 1-18 Alkyl, C 2-18 Alkenyl, -R * is selected from the group consisting of YR″, -YR″, and H; Each R” is independently 3-14 Alkyl and C 3-14 alkenyl, Each R * But independently, C 1-12 Alkyl and C 2-12 alkenyl, Each Y is independently 3-6 is a carbocyclic ring, each X is independently selected from the group consisting of F, Cl, Br, and I; m is selected from 5, 6, 7, 8, 9, 10, 11, 12, and 13; or a salt or isomer thereof.

[0104] In some embodiments, another subset of compounds of formula (VI) includes R 1 But, C 5-30 Alkyl, C 5-20 Alkenyl, -R * -YR", -YR", and -R"M'R'; R 2 and R 3 However, independently, H, C 1-14 Alkyl, C 2-14 Alkenyl, -R * YR”, -YR”, and -R * OR” or R 2 and R 3 form a heterocyclic or carbocyclic ring together with the atoms to which they are attached, R 4 But, C 3-6 Carbocycle, -(CH 2 ) n Q, -(CH 2 ) n CHQR, -CHQR, -CQ(R) 2 , and unsubstituted C 1-6 alkyl, where Q is selected from the group consisting of C 3-6 Carbocyclic ring, 5-14 membered heteroaryl having one or more heteroatoms selected from N, O, and S, -OR, -O(CH 2 ) n N(R) 2 , -C(O)OR, -OC(O)R, -CX 3 , -CX 2 H, -CXH 2 , -CN, -C(O)N(R) 2 , -N(R)C(O)R, -N(R)S(O) 2 R, -N(R)C(O)N(R) 2 , -N(R)C(S)N(R) 2 , -CRN(R) 2 C(O)OR, -N(R)R 8 , --O(CH 2 ) n OR, -N(R)C(=NR 9 )N(R) 2, -N(R)C(=CHR 9 )N(R) 2 , -OC(O)N(R) 2 , -N(R)C(O)OR, -N(OR)C(O)R, -N(OR)S(O) 2 R, -N(OR)C(O)OR,-N(OR)C(O)N(R) 2 , -N(OR)C(S)N(R) 2 , -N(OR)C(=NR 9 )N(R) 2 , -N(OR)C(=CHR 9 )N(R) 2 , -C(=NR 9 )R, -C(O)N(R)OR, and -C(=NR 9 )N(R) 2 where each n is independently selected from 1, 2, 3, 4, and 5; Each R 5 But independently, C 1-3 Alkyl, C 2-3 alkenyl, and H; Each R 6 But independently, C 1-3 Alkyl, C 2-3 alkenyl, and H; M and M' are independently -C(O)O-, -OC(O)-, -C(O)N(R')-, -N(R')C(O)-, -C(O)-, -C(S)-, -C(S)S-, -SC(S)-, -CH(OH)-, -P(O)(OR')O-, -S(O) 2 -, -SS-, an aryl group, and a heteroaryl group; R 7 But, C 1-3 Alkyl, C 2-3 alkenyl, and H; R 8 But, C 3-6 selected from the group consisting of carbocycles and heterocycles; R 9 H, CN, NO 2 , C 1-6 Alkyl, -OR, -S(O) 2 R, -S(O) 2 N(R) 2 , C2-6 Alkenyl, C 3-6 selected from the group consisting of carbocycles and heterocycles; Each R is independently C 1-3 Alkyl, C 2-3 alkenyl, and H; Each R' is independently C 1-18 Alkyl, C 2-18 Alkenyl, -R * is selected from the group consisting of YR″, -YR″, and H; Each R” is independently 3-14 Alkyl and C 3-14 alkenyl, Each R * But independently, C 1-12 Alkyl and C 2-12 alkenyl, Each Y is independently 3-6 is a carbocyclic ring, each X is independently selected from the group consisting of F, Cl, Br, and I; m is selected from 5, 6, 7, 8, 9, 10, 11, 12, and 13; or a salt or isomer thereof.

[0105] In some embodiments, another subset of compounds of formula (VI) includes R 1 But, C 5-30 Alkyl, C 5-20 Alkenyl, -R * -YR", -YR", and -R"M'R'; R 2 and R 3 However, independently, H, C 2-14 Alkyl, C 2-14 Alkenyl, -R * YR”, -YR”, and -R * OR” or R 2 and R 3 form a heterocyclic or carbocyclic ring together with the atoms to which they are attached, R 4 But -(CH 2) n Q or -(CH 2 ) n CHQR, where Q is -N(R) 2 and n is selected from 3, 4, and 5; Each R 5 But independently, C 1-3 Alkyl, C 2-3 alkenyl, and H; Each R 6 But independently, C 1-3 Alkyl, C 2-3 alkenyl, and H; M and M' are independently -C(O)O-, -OC(O)-, -C(O)N(R')-, -N(R')C(O)-, -C(O)-, -C(S)-, -C(S)S-, -SC(S)-, -CH(OH)-, -P(O)(OR')O-, -S(O) 2 -, -SS-, an aryl group, and a heteroaryl group; R 7 But, C 1-3 Alkyl, C 2-3 alkenyl, and H; Each R is independently C 1-3 Alkyl, C 2-3 alkenyl, and H; Each R' is independently C 1-18 Alkyl, C 2-18 Alkenyl, -R * is selected from the group consisting of YR″, -YR″, and H; Each R” is independently 3-14 Alkyl and C 3-14 alkenyl, Each R * But independently, C 1-12 Alkyl and C 1-12 alkenyl, Each Y is independently 3-6 is a carbocyclic ring, each X is independently selected from the group consisting of F, Cl, Br, and I; m is selected from 5, 6, 7, 8, 9, 10, 11, 12, and 13; or a salt or isomer thereof.

[0106] In some embodiments, another subset of compounds of formula (VI) includes R 1 But, C 5-30 Alkyl, C 5-20 Alkenyl, -R * -YR", -YR", and -R"M'R'; R 2 and R 3 But independently, C 1-14 Alkyl, C 2-14 Alkenyl, -R * YR”, -YR”, and -R * OR” or R 2 and R 3 form a heterocyclic or carbocyclic ring together with the atoms to which they are attached, R 4 But -(CH 2 ) n Q, -(CH 2 ) n CHQR, -CHQR, and -CQ(R) 2 wherein Q is selected from the group consisting of -N(R) 2 and n is selected from 1, 2, 3, 4, and 5; Each R 5 But independently, C 1-3 Alkyl, C 2-3 alkenyl, and H; Each R 6 But independently, C 1-3 Alkyl, C 2-3 alkenyl, and H; M and M' are independently -C(O)O-, -OC(O)-, -C(O)N(R')-, -N(R')C(O)-, -C(O)-, -C(S)-, -C(S)S-, -SC(S)-, -CH(OH)-, -P(O)(OR')O-, -S(O) 2 -, -SS-, an aryl group, and a heteroaryl group; R 7 But, C 1-3 Alkyl, C 2-3 alkenyl, and H; Each R is independently C 1-3 Alkyl, C 2-3 alkenyl, and H; Each R' is independently C 1-18 Alkyl, C 2-18 Alkenyl, -R * is selected from the group consisting of YR″, -YR″, and H; Each R” is independently 3-14 Alkyl and C 3-14 alkenyl, Each R * But independently, C 1-12 Alkyl and C 1-12 alkenyl, Each Y is independently 3-6 is a carbocyclic ring, each X is independently selected from the group consisting of F, Cl, Br, and I; m is selected from 5, 6, 7, 8, 9, 10, 11, 12, and 13; or a salt or isomer thereof.

[0107] In certain embodiments, a subset of compounds of formula (VI) is represented by formula (VI-A): [ka] or an N-oxide thereof, or a salt or isomer thereof, wherein l is selected from 1, 2, 3, 4, and 5; m is selected from 5, 6, 7, 8, and 9; M 1 is a bond or M'; R 4 is hydrogen, unsubstituted C 1-3 Alkyl, or -(CH 2 ) n Q, where Q is OH, -NHC(S)N(R) 2 , -NHC(O)N(R) 2 , -N(R)C(O)R, -N(R)S(O)2 R, -N(R)R 8 , -NHC(=NR 9 )N(R) 2 , -NHC(=CHR 9 )N(R) 2 , -OC(O)N(R) 2 , -N(R)C(O)OR, heteroaryl, or heterocycloalkyl; M and M' are independently selected from -C(O)O-, -OC(O)-, -OC(O)-M"-C(O)O-, -C(O)N(R')-, -P(O)(OR')O-, -SS-, an aryl group, and a heteroaryl group; R 2 and R 3 are independent of each other, H, C 1-14 Alkyl, and C 2-14 For example, m is 5, 7, or 9. For example, Q is selected from the group consisting of OH, -NHC(S)N(R) 2 , or -NHC(O)N(R) 2 For example, Q is -N(R)C(O)R or -N(R)S(O). 2 It's R.

[0108] In certain embodiments, a subset of compounds of formula (VI) is represented by formula (VI-B): [ka] or an N-oxide thereof, or a salt or isomer thereof, wherein all variables are as defined herein. For example, m is selected from 5, 6, 7, 8, and 9; R 4 is hydrogen, unsubstituted C 1-3 Alkyl, or -(CH 2 ) n Q, where Q is H, -NHC(S)N(R) 2 , -NHC(O)N(R) 2 , -N(R)C(O)R, -N(R)S(O) 2 R, -N(R)R 8 , -NHC(=NR 9 )N(R) 2 , -NHC(=CHR 9 )N(R)2 , -OC(O)N(R) 2 , -N(R)C(O)OR, heteroaryl, or heterocycloalkyl; M and M' are independently selected from -C(O)O-, -OC(O)-, -OC(O)-M"-C(O)O-, -C(O)N(R')-, -P(O)(OR')O-, -SS-, an aryl group, and a heteroaryl group; R 2 and R 3 are independent of each other, H, C 1-14 Alkyl, and C 2-14 For example, m is 5, 7, or 9. For example, Q is selected from the group consisting of OH, -NHC(S)N(R) 2 , or -NHC(O)N(R) 2 For example, Q is -N(R)C(O)R or -N(R)S(O). 2 It's R.

[0109] In certain embodiments, a subset of compounds of formula (VI) is represented by formula (VII): [ka] or an N-oxide thereof, or a salt or isomer thereof, wherein l is selected from 1, 2, 3, 4, and 5; 1 is a bond or M', and R 4 is hydrogen, unsubstituted C 1-3 Alkyl, or -(CH 2 ) n Q, where n is 2, 3, or 4, and Q is OH, -NHC(S)N(R) 2 , -NHC(O)N(R) 2 , -N(R)C(O)R, -N(R)S(O) 2 R, -N(R)R 8 , -NHC(=NR 9 )N(R) 2 , -NHC(=CHR 9 )N(R) 2 , -OC(O)N(R) 2, -N(R)C(O)OR, heteroaryl, or heterocycloalkyl; M and M' are independently selected from -C(O)O-, -OC(O)-, -OC(O)-M"-C(O)O-, -C(O)N(R')-, -P(O)(OR')O-, -SS-, an aryl group, and a heteroaryl group; R 2 and R 3 are independent of each other, H, C 1-14 Alkyl, and C 2-14 alkenyl.

[0110] In one embodiment, the compound of formula (VI) has the formula (VIIa) [ka] or an N-oxide thereof, or a salt or isomer thereof, wherein R 4 is as described herein.

[0111] In another embodiment, the compound of formula (VI) has the formula (VIIb) [ka] or an N-oxide thereof, or a salt or isomer thereof, wherein R 4 is as described herein.

[0112] In another embodiment, the compound of formula (VI) has formula (VIIc) or (VIIe): [ka] or an N-oxide thereof, or a salt or isomer thereof, wherein R 4 is as described herein.

[0113] In another embodiment, the compound of formula (VI) has the formula (VIIf): [ka] or an N-oxide thereof, or a salt or isomer thereof, In the formula, M is -C(O)O- or -OC(O)-, and M" is C 1-6 Alkyl or C 2-6 alkenyl, R 2 and R 3 is independent, C 5-14 Alkyl and C 5-14 alkenyl, where n is selected from 2, 3, and 4.

[0114] In a further embodiment, the compound of formula (VI) has the formula (VIId) [ka] or an N-oxide thereof, or a salt or isomer thereof, wherein n is 2, 3, or 4; m, R′, R″, and R 2 ~R 6 is as described herein. For example, R 2 and R 3 Each of the above may be independently selected from the group consisting of C 5-14 Alkyl and C 5-14 alkenyl.

[0115] In some embodiments, the ionizable amino lipids of the present disclosure include compounds having the following structure: [ka]

[0116] In some embodiments, the ionizable amino lipids of the present disclosure include compounds having the following structure: [ka]

[0117] In a further embodiment, the compound of formula (VI) has the formula (VIIg) [ka] or an N-oxide thereof, or a salt or isomer thereof, wherein l is selected from 1, 2, 3, 4, and 5; m is selected from 5, 6, 7, 8, and 9; M 1 is a bond or M', M and M' are independently selected from -C(O)O-, -OC(O)-, -OC(O)-M"-C(O)O-, -C(O)N(R')-, -P(O)(OR')O-, -SS-, an aryl group, and a heteroaryl group; R 2 and R 3 are independent of each other, H, C 1-14 Alkyl, and C 2-14 For example, M" is selected from the group consisting of C 1-6 Alkyl (e.g., C 1-4 Alkyl) or C 2-6 Alkenyl (e.g., C 2-4 alkenyl). For example, R 2 and R 3 is independently 5-14 Alkyl and C 5-14 alkenyl.

[0118] In some embodiments, the ionizable amino lipid is one or more of the compounds described in U.S. Application Nos. 62 / 220,091, 62 / 252,316, 62 / 253,433, 62 / 266,460, 62 / 333,557, 62 / 382,740, 62 / 393,940, 62 / 471,937, 62 / 471,949, 62 / 475,140, ​​and 62 / 475,166, and PCT Application No. PCT / US2016 / 052352.

[0119] The central amine moiety of lipids according to formula (VI), (VI-A), (VI-B), (VII), (VIIa), (VIIb), (VIIc), (VIId), (VIIe), (VIIf), or (VIIg) can be protonated at physiological pH. Thus, the lipids can have a positive charge or a partial positive charge at physiological pH. Such amino lipids may be referred to as cationic lipids, ionizable lipids, cationic amino lipids, or ionizable amino lipids. Amino lipids can also be zwitterionic, i.e., neutral molecules that have both positive and negative charges.

[0120] In some embodiments, the ionizable amino lipid of the present disclosure has formula (VIII): [ka] or a salt or isomer thereof, wherein W is [ka] and Ring A is [ka] and t is 1 or 2; A 1 and A 2 are each independently selected from CH or N; Z is CH 2 or absent, where Z is CH 2 when Z is absent, then dashed lines (1) and (2) are both absent; R 1 , R 2 , R 3 , R 4 , and R 5 is independently 5-20 Alkyl, C 5-20 Alkenyl, -R"MR', -R * YR”, -YR”, and -R *OR" R X1 and R X2 are each independently H or C 1 - 3 is alkyl, Each M is independently -C(O)O-, -OC(O)-, -OC(O)O-, -C(O)N(R')-, -N(R')C(O)-, -C(O)-, -C(S)-, -C(S)S-, -SC(S)-, -CH(OH)-, -P(O)(OR')O-, -S(O) 2 is selected from the group consisting of -, -C(O)S-, -SC(O)-, an aryl group, and a heteroaryl group; M * is C 1 -C 6 is alkyl, W 1 and W 2 are each independently -O- and -N(R 6 )-, Each R 6 are independently H and C 1-5 is selected from the group consisting of alkyl, X 1 , X 2 , and X 3 are independently a bond, -CH 2 -, -(CH 2 ) 2 -, -CHR-, -CHY-, -C(O)-, -C(O)O-, -OC(O)-, -(CH 2 ) n -C(O)-, -C(O)-(CH 2 ) n -, -(CH 2 ) n -C(O)O-, -OC(O)-(CH 2 ) n -, -(CH 2 ) n -OC(O)-, -C(O)O-(CH 2 ) n is selected from the group consisting of -, -CH(OH)-, -C(S)-, and -CH(SH)-; Each Y is independently C 3-6 is a carbocyclic ring, Each R* is independently 1-12 Alkyl and C 2-12 alkenyl, Each R is independently C 1-3 Alkyl and C 3-6 is selected from the group consisting of carbocycles; Each R' is independently C 1-12 Alkyl, C 2-12 alkenyl, and H; Each R" is independently 3-12 Alkyl, C 3-12 Alkenyl, and -R * MR′, n is an integer from 1 to 6, Here, ring A is [ka] in the case of, i)X 1 , X 2 , and X 3 At least one of the groups is -CH 2 -Not and / or ii) R 1 , R 2 , R 3 , R 4 , and R 5 At least one of the is -R"MR'.

[0121] In some embodiments, the compound has the formula (VIIIa1)-(VIIIa8): [ka] [ka] It is either of the following.

[0122] In some embodiments, the ionizable amino lipid is [ka] or a salt thereof.

[0123] The central amine moiety of a lipid according to formula (VIII), (VIIIa1), (VIIIa2), (VIIIa3), (VIIIa4), (VIIIa5), (VIIIa6), (VIIIa7), or (VIIIa8) can be protonated at physiological pH. Thus, the lipid can have a positive charge or a partial positive charge at physiological pH.

[0124] In some embodiments, the lipid nanoparticle has the following structure: [ka] or a pharma- ceutically acceptable salt, tautomer, or stereoisomer thereof, wherein R 1 is optionally replaced by C 1 -C 24 Alkyl or optionally substituted C 2 -C 24 alkenyl, R 2 and R 3 each independently represents an optionally substituted C 1 -C 36 is alkyl, R 4 and R 5 each independently represents an optionally substituted C 1 -C 6 alkyl or R 4 and R 5 together with the N to which they are attached form a heterocyclyl or heteroaryl; L 1 , L 2 and L 3 each independently represents an optionally substituted C 1 -C I8 is alkylene, G 1 is a direct bond, -(CH 2 ) n O(C=O)-, -(CH 2 ) n(C=O)O-, or -(C=O)-; G 2 and G 3 is independently -(C=O)O- or -0(C=O)-, and n is an integer greater than 0.

[0125] In some embodiments, the lipid nanoparticle has the following structure: [ka] or a pharma- ceutically acceptable salt, tautomer, or stereoisomer thereof, wherein G 1 is -N(R 3 )R 4 -OR 5 and R 1 is an optionally substituted branched, saturated or unsaturated C 12 -C 36 is alkyl, R 2 When L is -C(=O)-, optionally substituted branched or unbranched, saturated or unsaturated C 12 -C 36 alkyl or R 2 L is C 6 -C 12 Alkylene, C 6 -C 12 Alkenylene or C 2 -C 6 When alkynylene, it is an optionally substituted branched or unbranched, saturated or unsaturated C 4 -C 36 is alkyl, R 3 and R 4 each independently represents H, optionally substituted branched or unbranched, saturated or unsaturated C 1 -C 6 alkyl or R 3 and R 4 are each independently 6 -C 12 Alkylene, C6 -C 12 Alkenylene or C 2 -C 6 When alkynylene, it is an optionally substituted branched or unbranched, saturated or unsaturated C 1 -C 6 alkyl or R 3 and R 4 together with the nitrogen to which they are attached form a heterocyclyl, R 5 is H or optionally substituted C 1 -C 6 is alkyl, L is -C(=O)-, C 6 -C 12 Alkylene, C 6 -C 12 Alkenylene, or C 2 -C 6 is alkynylene, n is an integer from 1 to 12.

[0126] In some embodiments, the lipid nanoparticle has the following structure: [ka] or a pharma- ceutically acceptable salt thereof, wherein Each R la are independently hydrogen, R lc , or R ld and Each R lb are independently lc or R ld and Each R 1c are independently -[CH 2 ] 2 C(O)X 1 R 3 and Each R ld are independently -C(O)R 4 and Each R 2 are independently -[C(R 2a ) 2 ]c R 2b and Each R 2a are independently hydrogen or C 1 -C 6 is alkyl, R 2b is -N(L 1 -B) 2 , -(OCH 2 CH 2 ) 6 OH, or -(OCH 2 CH 2 ) b OCH 3 and Each R 3 and R 4 is independently 6 -C 30 It is aliphatic, Each I. 3 is independently 1 -C 10 is alkylene, each B is independently hydrogen or an ionizable nitrogen-containing group; each X 1 are independently a covalent bond or O; each a is independently an integer from 1 to 10; each b is independently an integer from 1 to 10; Each c is independently an integer from 1 to 10.

[0127] In some embodiments, the lipid nanoparticle has the following structure: [ka] or a pharma- ceutically acceptable salt, prodrug or stereoisomer thereof, wherein X is N and Y is absent, or X is CR and Y is NR; L 1 is -O(CO)R 1 , -(C=O)OR 1 , -C(=O)R 1 , -OR 1 , -S(O) x R1 , -S-SR 1 , -C(=O)SR 1 , -SC(=O)R 1 , -NR a C(=O)R 1 , -C(=O)NR b R c , -NR a C(=O)NR b R c , -OC(=O)NR b R c , or -NR a C(=O)OR 1 and L 2 is -O(C=O)R 2 , -(C=O)OR 2 , -C(=O)R 2 , -OR 2 , -S(O) x R 2 , -S-SR 2 , -C(=O)SR 2 , -SC(=O)R 2 , -NR d C(=O)R 2 , -C(=O)NR e R f , -NR d C(=O)NR e R f , -OC(=O)NR e R f , -NR d C(=O)OR 2 , or R 2 is a direct bond to L 3 is -O(C=O)R 3 OR - (C=O)OR 3 and G 1 and G 2 are each independently 2 -C 12 Alkylene or C 2 -C 12 alkenylene, G 3 If X is CR and Y is NR, then C 1 -C 24 Alkylene, C2 -C 24 Alkenylene, C 1 -C 24 Heteroalkylene, or C 2 -C 24 heteroalkenylene, G 3 If X is N and Y is absent, then C 1 -C 24 Heteroalkylene or C 2 -C 24 heteroalkenylene, R a , R b , R d and R e are each independently H or C 1 -C 12 Alkyl, or C 1 -C 12 alkenyl, R c and R f are each independently 1 -C 12 Alkyl or C 2 -C 12 alkenyl, Each R is independently H or C 1 -C 12 is alkyl, R 1 , R 2 , and R 3 are each independently 1 -C 24 Alkyl or C 2 -C 24 alkenyl, x is 0, 1, or 2; Each alkyl, alkenyl, alkylene, alkenylene, heteroalkylene, and heteroalkenylene is independently substituted or unsubstituted, unless otherwise specified.

[0128] In some embodiments, the lipid nanoparticle has the following structure: [ka] or a pharma- ceutically acceptable salt, tautomer, prodrug or stereoisomer thereof, wherein L 1 and L 2 are each independently -0(C=0)-, -(C=0)0-, -C(=0)-, -0-, -S(0)x- s -SS-, -C(=0)S-, -SC(=0)-, -NR a C(=0)-, -C(=0)NR a -, -NR a C(=0)NR a -, -OC(=0)NR a -, -NR a C(=0)0- or a direct bond; G 1 is C,-C 2 Alkylene, -(C=0)-, -0(C=0)-, -SC(=0)-, -NR a C(=0)- or a direct bond; G 2 are -C(0)-, -(CO)O-, -C(=0)S-, -C(=0)NR a - or a direct bond, G 3 is C 1 -C 6 is alkylene, R a is H or C 1 -C 12 is alkyl, R la and R lb is, independently in each occurrence: (a) H or C 1 -C 12 alkyl, or (b) R la is H or C 1 -C 12 is alkyl, R lb together with the carbon atom to which it is attached, the adjacent R Ib and together with the carbon atom to which it is attached form a carbon-carbon double bond; R 2a and R 2b is, independently in each occurrence: (a) H or C 1-C 12 alkyl, or (b) R 2a is H or C 1 -C 12 is alkyl, R 2b together with the carbon atom to which it is attached, the adjacent R 2b and together with the carbon atom to which it is attached form a carbon-carbon double bond; R 3a and R 3b is, independently in each occurrence: (a) H or C 1 -C 12 alkyl, or (b) R 3a is H or C 1 -C 12 is alkyl, R 3b together with the carbon atom to which it is attached, forms a carbon-carbon double bond with the adjacent R and the carbon atom to which it is attached; R 4A and R 4B is, independently in each occurrence: (a) H or C 1 -C 12 alkyl, or (b) R 4A is H or C 1 -C 12 is alkyl, R 4B together with the carbon atom to which it is attached, the adjacent R 4B and together with the carbon atom to which it is attached form a carbon-carbon double bond; R 5 and R 6 are each independently H or methyl; R 7 is H or C,-C 20 is alkyl, R 8 OH, -N(R 9 )(C=0)R 10 , -(C=0)NR 9 R 10 , -NR 9 R 10 , -(C=0)0R” 1or -0(C=0)R”, where R 8 Ga-NR 9 R 10 If G 3 is C 4 -C 6 is alkylene, R 9 and R 10 are each independently H or C 1 -C 12 is alkyl, R" is aralkyl; a, b, c, and d each independently represent an integer of 1 to 24; x represents 0, 1, or 2; Each alkyl, alkylene, and aralkyl is optionally substituted.

[0129] In some embodiments, the lipid nanoparticle has the following structure: [ka] or a pharma- ceutically acceptable salt, prodrug or stereoisomer thereof, wherein X and X′ are each independently N or CR; Y and Y′ are each independently absent, —O(C═O)—, —(C═O)O—, or NR, with the proviso that a) if X is N then Y is absent; b) if X' is N then Y' is absent; c) when X is CR, Y is -O(C=O)-, -(C=O)O-, or NR; d) when X' is CR, Y' is -O(C=O)-, -(C=O)O-, or NR; L 1 and L 1’ are each independently -O(C=O)R', -(C=O)OR', -C(=O)R', -OR 1 , -S(O) z R', -S-SR 1 , -C(=O)SR', -SC(=O)R', -NR aC(=O)R', -C(=O)NR b R c , -NR a C(=O)NR b R c , -OC(=O)NR b R c , or -NR a C(=O)OR', L 2 and L 2’ are each independently -O(C=O)R 2 , -(C=O)OR 2 , -C(=O)R 2 , -OR 2 , -S(O) z R 2 , -S-SR 2 , -C(=O)SR 2 , -SC(=O)R 2 , -NR d C(=O)R 2 , -C(=O)NR e R f , -NR d C(=O)NR e R f , -OC(=O)NR e R f , -NR d C(=O)OR 2 , or R 2 is a direct bond to G 1 , G 1’ , G 2 , and G 2’ are each independently 2 -Ci 2 Alkylene or C 2 -C 12 alkenylene, G is C 2 -C 24 Heteroalkylene or C 2 -C 24 heteroalkenylene, R a , R b , R d , and R e independently in each occurrence: H, C 1 -C12 Alkyl or C 2 -C 12 alkenyl, R c and R f independently in each occurrence, C 1 -C 12 Alkyl or C 2 -C 12 alkenyl, R is independently in each occurrence H or C 1 -C 12 is alkyl, R 1 and R 2 independently at each occurrence, a branched C 6 -C 24 Alkyl or branched C 6 -C 24 alkenyl, z is 0, 1, or 2, and each alkyl, alkenyl, alkylene, alkenylene, heteroalkylene, and heteroalkenylene is independently substituted or unsubstituted, unless otherwise specified.

[0130] In some embodiments, the lipid nanoparticle has the following structure: [ka] or a pharma- ceutically acceptable salt, prodrug or stereoisomer thereof, wherein L 1 is -O(C=O)R 1 , -(C=O)OR 1 , -C(=O)R 1 , -OR 1 , -S(O) x R 1 , -S-SR 1 , -C(=O)SR 1 , -SC(=O)R 1 , -NR a C(=O)R 1 , -C(=O)NR b R c , -NR a C(=O)NR b R c, -OC(=O)NR b R c ,or -NR a C(=O)OR 1 and L 2 is -O(C=O)R 2 , -(C=O)OR 2 , -C(=O)R 2 , -OR 2 , -S(O) x R 2 , -S-SR 2 , -C(=O)SR 2 , -SC(=O)R 2 , -NR d C(=O)R 2 , -C(=O)NR e R f , -NR d C(=O)NR e R f , -OC(=O)NR e R f , -NR d C(=O)OR 2 , or R 2 is a direct bond to G 1 and G 2 are each independently 2 -C 12 Alkylene or C 2 -C 12 alkenylene, G 3 is C 1 -C 24 Alkylene, C 2 -C 24 Alkenylene, C 3 -C 8 Cycloalkylene, or C 3 -C 8 is cycloalkenylene, R a , R b , R d and R e are each independently H or C 1 -C 12 Alkyl, or C 1 -C 12 alkenyl, R c and R f are each independently 1 -C 12 Alkyl or C 2 -C 12 alkenyl, R 1 and R 2 each independently represents a branched C 6 -C 24 Alkyl or branched C 6 -C 24 alkenyl, R 3 is -N(R 4 )R 5 and R 4 is C 1 -C 12 is alkyl, R 5 is a substitution C 1 -C 12 is alkyl, x is 0, 1 or 2; Each alkyl, alkenyl, alkylene, alkenylene, cycloalkylene, cycloalkenylene, aryl, and aralkyl is independently substituted or unsubstituted, unless otherwise specified.

[0131] In some embodiments, the lipid nanoparticle has the following structure: [ka] or a pharma- ceutically acceptable salt, prodrug or stereoisomer thereof, wherein L 1 is -O(C=O)R 1 , -(C=O)OR 1 , -C(=O)R 1 , -OR 1 , -S(O) x R 1 , -S-SR 1 , -C(=O)SR 1 , -SC(=O)R 1 , -NR a C(=O)R 1, -C(=O)NR b R c , -NR a C(=O)NR b R c , -OC(=O)NR b R c ,or -NR a C(=O)OR 1 and L 2 is -O(C=O)R 2 , -(C=O)OR 2 , -C(=O)R 2 , -OR 2 , -S(O) x R 2 , -S-SR 2 , -C(=O)SR 2 , -SC(=O)R 2 , -NR d C(=O)R 2 , -C(=O)NR e R f , -NR d C(=O)NR e R f , -OC(=O)NR e R f ;-NR d C(=O)OR 2 , or R 2 is a direct bond to G 1a and G 2b are each independently 2 -C 12 Alkylene or C 2 -C 12 alkenylene, G 1b and G 2b are each independently 1 -C 12 Alkylene or C 2 -C 12 alkenylene, G 3 is C 1 -C 24 Alkylene, C 2 -C 24 Alkenylene, C 3 -C 8Cycloalkylene, or C 3 -C 8 is cycloalkenylene, R a , R b , R d and R e are each independently H or C 1 -C 12 Alkyl, or C 2 -C 12 alkenyl, R c and R f are each independently 1 -C 12 Alkyl or C 2 -C 12 alkenyl, R 1 and R 2 each independently represents a branched C 6 -C 24 Alkyl or branched C 6 -C 24 alkenyl, R 3a is -C(=O)N(R 4a )R 5a OR -C(=O)OR 6 and R 3b -NR 4b C(=O)R 5b and R 4a is C 1 -C 12 is alkyl, R 4b , H, C 1 -C 12 Alkyl or C 2 -C 12 alkenyl, R 5a , H, C 1 -C 8 Alkyl or C 2 -C 8 alkenyl, R 4b If H, then R 5b is C 2 -C 12 Alkyl or C2 -C 12 alkenyl or R 4b C 1 -C 12 Alkyl or C 2 -C 12 When R is alkenyl, 5b is C 1 -C 12 Alkyl or C 2 -C 12 alkenyl, R 6 is H, aryl, or aralkyl; x is 0, 1 or 2; Each alkyl, alkenyl, alkylene, alkenylene, cycloalkylene, cycloalkenylene, aryl, and aralkyl is independently substituted or unsubstituted.

[0132] In some embodiments, the lipid nanoparticle has the following structure: [ka] or a pharma- ceutically acceptable salt, prodrug or stereoisomer thereof, wherein G 1 -OH, -R 3 R 4 , -(C=0)R 5 , or -R 3 (C=0)R 5 and G 2 -CH 2 - or -(C=0)-, R is independently in each occurrence H or OH; R 1 and R 2 each independently represents an optionally substituted branched, saturated or unsaturated C 12 -C 36 is alkyl, R 3 and R 4 are each independently H or an optionally substituted linear or branched, saturated or unsaturated Ci-C6 is alkyl, R 5 is an optionally substituted linear or branched, saturated or unsaturated Ci-C 6 is alkyl, n is an integer from 2 to 6.

[0133] In some embodiments, the lipid nanoparticle has the following structure: [ka] or a pharma- ceutically acceptable salt, prodrug or stereoisomer thereof, wherein G 1 or G 2 is selected from the group consisting of, at each occurrence, -O(C=O)-, -(C=O)O-, -C(=O)-, -O-, -S(O), -SS-, -C(=O)S-, SC(=O)-, -N(R a )C(=O)-, -C(=O)N(R a )-, -N(R a )C(=O)N(R a )-, -OC(=O)N(R a )-, or -N(R a )C(=O)O-, and G 1 or G 2 The other of each occurrence is -O(C=O)-, -(C=O)O-, -C(=O)-, -O-, -S(O), -SS-, -C(=O)S-, -SC(=O)-, -N(R a )C(=O)-, -C(=O)N(R a )-, -N(R a )C(=O)N(R a )-, -OC(=O)N(R a )- or -N(R a )C(=O)O-, or a direct bond; L, in each occurrence, is ~O(C=O)-, where ~ represents a covalent bond to X; X is CR a and Z is an alkyl, cycloalkyl, or monovalent moiety containing at least one polar functional group when n is 1, or Z is an alkylene, cycloalkylene, or polyvalent moiety containing at least one polar functional group when n is greater than 1; R a independently in each occurrence: H, C 1 -C 12 Alkyl, C 1 -C 12 Hydroxyl alkyl, C 1 -C 12 Aminoalkyl, C 1 -C 12 Alkylaminylalkyl, C 1 -C 12 Alkoxyalkyl, C 1 -C 12 Alkoxycarbonyl, C 1 -C 12 Alkylcarbonyloxy, C 1 -C 12 Alkylcarbonyloxyalkyl, or C 1 -C 12 is an alkylcarbonyl; R, in each occurrence, is independently: (a) H or C 1 -C 12 alkyl, or (b) R, together with the carbon atom to which it is attached, forms a carbon-carbon double bond with an adjacent R and the carbon atom to which it is attached; R 1 and R 2 Each occurrence of [ka] a 1 and a 2 is independently in each occurrence an integer from 3 to 12; 1 and b 2 is independently 0 or 1 in each occurrence, c 1 and c 2 is independently in each occurrence an integer from 5 to 10; d1 and d 2 is independently in each occurrence an integer from 5 to 10; y is independently in each occurrence an integer from 0 to 2; and n is an integer from 1 to 6; Each alkyl, alkylene, hydroxylalkyl, aminoalkyl, alkylaminylalkyl, alkoxyalkyl, alkoxycarbonyl, alkylcarbonyloxy, alkylcarbonyloxyalkyl, and alkylcarbonyl is optionally substituted with one or more substituents.

[0134] In some embodiments, the lipid nanoparticle has the following structure: [ka] or a pharma- ceutically acceptable salt, prodrug or stereoisomer thereof, wherein L 1 or L 2 One of the groups is -O(C=O)-, -(C=O)O-, -C(=O)-, -O-, -S(O) x -, -SS-, -C(=O)S-, -SC(=O)-, -R a C(=O)-, -C(=O)R a -, R a C(=O)R a -, -OC(=O)R a -, or -R a C(=O)O-, L 1 or L 2 The other of the above is -O(C=O)-, -(C=O)O-, -C(=O)-, -O-, -S(O) x -, -SS-, -C(=O)S-, SC(=O)-, -R a C(=O)-, -C(=O)R a -, R a C(=O)R a -, -OC(=O)R a - or -NR a C(=O)O- or a direct bond; G 1 and G 2 are each independently an unsubstituted C 1 -C12 Alkylene or C 1 -C 12 alkenylene, G 3 is C 1 -C 24 Alkylene, C 1 -C 24 Alkenylene, C 3 -C 8 Cycloalkylene, C 3 -C 8 is cycloalkenylene, R a is H or C 1 -C 12 is alkyl, R 1 and R 2 are each independently 6 -C 24 Alkyl or C 6 -C 24 alkenyl, R 3 H, OR 5 , CN, -C(=O)OR 4 , -OC(=O)R 4 , or -R 5 C(=O)R 4 and R 4 is C 1 -C 12 is alkyl, R 5 is H or C 1 -C 6 is alkyl, x is 0, 1, or 2.

[0135] In some embodiments, the lipid nanoparticle has the following structure: [ka] or a pharma- ceutically acceptable salt, tautomer, prodrug or stereoisomer thereof, wherein L 1 and L 2are each independently -0(C=0)-, -(C=0)0-, -C(=0)-, -0-, -S(0) x -, -SS-, -C(=0)S-, -SC(=0)-, -R a C(=0)-, -C(=0)R a -, -R a C(=0)R a -, -OC(=0)R a -, -R a C(=0)0- or a direct bond; G 1 is Ci-C 2 Alkylene, -(C=0)-, -0(C=0)-, -SC(=0)-, -R a C(=0)- or a direct bond; G 2 are -C(=0)-, -(C=0)0-, -C(=0)S-, -C(=0)NR a - or a direct bond, G 3 is C 1 -C 6 is alkylene, R a is H or C 1 -C 12 is alkyl, R la and R lb is, independently in each occurrence: (a) H or C 1 -C 12 alkyl, or (b) R la is H or C 1 -C 12 is alkyl, R lb together with the carbon atom to which it is attached, the adjacent R lb and together with the carbon atom to which it is attached form a carbon-carbon double bond; R 2a and R 2b is, independently in each occurrence: (a) H or C 1 -C 12 alkyl, or (b) R 2a is H or C 1 -C 12 is alkyl, R 2btogether with the carbon atom to which it is attached, the adjacent R 2b and together with the carbon atom to which it is attached form a carbon-carbon double bond; R 3a and R 3b is, independently in each occurrence: (a) H or C 1 -C 12 alkyl, or (b) R 3a is H or C 1 -C 12 is alkyl, R 3b together with the carbon atom to which it is attached, forms a carbon-carbon double bond with the adjacent R and the carbon atom to which it is attached; R 4a and R 4b is, independently in each occurrence: (a) H or C 1 -C 12 alkyl, or (b) R 4a is H or C 1 -C 12 is alkyl, R 4b together with the carbon atom to which it is attached, the adjacent R 4b and together with the carbon atom to which it is attached form a carbon-carbon double bond; R 5 and R 6 are each independently H or methyl; R 7 is C 4 -C 20 is alkyl, R 8 and R 9 are each independently 1 -C 12 alkyl or R 8 and R 9 form together with the nitrogen atom to which they are attached a 5-, 6-, or 7-membered heterocyclic ring; a, b, c, and d each independently represent an integer of 1 to 24; x is 0, 1, or 2.

[0136] In some embodiments, the lipid nanoparticle has the following structure: [ka] or a pharma- ceutically acceptable salt, tautomer, prodrug or stereoisomer thereof, wherein L 1 and L 2 are each independently -0(C=0)-, -(C=0)0-, or a carbon-carbon double bond; R la and R lb is, independently in each occurrence: (a) H or C 1 -C 12 alkyl, or (b) R la is H or C 1 -C 12 is alkyl, R lb together with the carbon atom to which it is attached, the adjacent R lb and together with the carbon atom to which it is attached form a carbon-carbon double bond; R 2a and R 2b is, independently in each occurrence: (a) H or C 1 -C 12 alkyl, or (b) R 2a is H or C 1 -C 12 is alkyl, R 2b together with the carbon atom to which it is attached, the adjacent R 2b and together with the carbon atom to which it is attached form a carbon-carbon double bond; R 3a and R 3b is, independently in each occurrence: (a) H or C 1 -C 12 alkyl, or (b) R 3a is H or C 1 -C 12 is alkyl, R 3b together with the carbon atom to which it is attached, the adjacent R 3band together with the carbon atom to which it is attached form a carbon-carbon double bond; R 4a and R 4b is, independently in each occurrence: (a) H or C 1 -C 12 alkyl, or (b) R 4a is H or C 1 -C 12 is alkyl, R 4b together with the carbon atom to which it is attached, the adjacent R 4b and together with the carbon atom to which it is attached form a carbon-carbon double bond; R 5 and R 6 are each independently methyl or cycloalkyl; R 7 is independently in each occurrence H or C 1 -C 12 is alkyl, R 8 and R 9 are each independently an unsubstituted C 1 -C 12 alkyl or R 8 and R 9 form together with the nitrogen atom to which they are attached a 5-, 6-, or 7-membered heterocyclic ring containing one nitrogen atom; a and d are each independently an integer of 0 to 24, b and c are each independently an integer of 1 to 24, and e is 1 or 2. (however, R la , R 2a , R 3a , or R 4a At least one of them is C 1 -C 12 alkyl or L 1 Or L 2 At least one of is -0(C=0)- or -(C=0)0-; R la and R lb is not isopropyl when a is 6 or n-butyl when a is 8).

[0137] In some embodiments, the lipid nanoparticle has the following structure: [ka] or a pharma- ceutically acceptable salt thereof, wherein R 1 and R 2 are the same or different, each being a straight or branched alkyl having 1 to 9 carbon atoms, or an alkenyl or alkynyl having 2 to 11 carbon atoms; L 1 and L 2 are the same or different, each of which is a linear alkyl group having 5 to 18 carbon atoms or which together with N form a heterocycle; X 1 is a bond or -CG-G- to L2-CO-OR 2 is formed, X 2 is S or O, L 3 is a bond or lower alkyl, or together with N forms a heterocycle; R 3 is lower alkyl, R 4 and R 5 are the same or different and each is lower alkyl.

[0138] In some embodiments, the lipid nanoparticle has the following structure: [ka] or a pharma- ceutically acceptable salt thereof.

[0139] In some embodiments, the lipid nanoparticle has the following structure: [ka] or a pharma- ceutically acceptable salt thereof.

[0140] In some embodiments, the lipid nanoparticle has the following structure: [ka] or a pharma- ceutically acceptable salt thereof.

[0141] In some embodiments, the lipid nanoparticle has the following structure: [ka] or a pharma- ceutically acceptable salt thereof.

[0142] In some embodiments, the lipid nanoparticle has the following structure: [ka] or a pharma- ceutically acceptable salt thereof.

[0143] In some embodiments, the lipid nanoparticle has the following structure: [ka] or a pharma- ceutically acceptable salt thereof.

[0144] In some embodiments, the lipid nanoparticle has the following structure: [ka] or a pharma- ceutically acceptable salt thereof.

[0145] In some embodiments, the lipid nanoparticle has the following structure: [ka] or a pharma- ceutically acceptable salt thereof.

[0146] In some embodiments, the lipid nanoparticle has the following structure: [ka] or a pharma- ceutically acceptable salt thereof.

[0147] In some embodiments, the lipid nanoparticle has the following structure: [ka] or a pharma- ceutically acceptable salt thereof.

[0148] In some embodiments, the lipid nanoparticle has the following structure: [ka] or a pharma- ceutically acceptable salt thereof.

[0149] Non-cationic lipids In certain embodiments, the lipid nanoparticles provided herein comprise one or more non-cationic lipids. The non-cationic lipids may be phospholipids.

[0150] In some embodiments, the lipid nanoparticles contain 5-25 mol% non-cationic lipid. For example, the lipid nanoparticles may contain 5-20 mol%, 5-15 mol%, 5-10 mol%, 10-25 mol%, 10-20 mol%, 10-25 mol%, 15-25 mol%, 15-20 mol%, or 20-25 mol% non-cationic lipid. In some embodiments, the lipid nanoparticles contain 5 mol%, 10 mol%, 15 mol%, 20 mol%, or 25 mol% non-cationic lipid.

[0151] In some embodiments, the non-cationic lipid is 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dipalmitoyl-sn-glycero 1,2-Diundecanoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-Diundecanoyl-sn-glycero-phosphocholine (DUPC), 1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-Di-O-Octadecenyl-sn-glycero-3-phosphocholine (18:0 diether PC), 1-Oleoyl-2-cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine (OChemsPC), 1-Hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC), 1,2-dilinoleoyl-sn-glycero-3-phosphocholine, 1,2-diarachidonoyl-sn-glycero-3-phosphocholine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphocholine, 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (ME16.0PE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine -phosphoethanolamine, 1,2-dilinolenoyl-sn-glycero-3-phosphoethanolamine, 1,2-diarachidonoyl-sn-glycero-3-phosphoethanolamine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphoethanolamine, 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt (DOPG), sphingomyelin, and mixtures thereof.

[0152] In some embodiments, the lipid nanoparticles contain 5-15 mol%, 5-10 mol%, or 10-15 mol% DSPC. For example, the lipid nanoparticles may contain 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 mol% DSPC.

[0153] In certain embodiments, the lipid composition of the lipid nanoparticle composition disclosed herein may comprise one or more phospholipids, such as one or more saturated or (poly)unsaturated phospholipids or combinations thereof. Generally, a phospholipid comprises a phospholipid moiety and one or more fatty acid moieties.

[0154] The phospholipid moiety can be selected from the non-limiting group consisting of, for example, phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylserine, phosphatidic acid, 2-lysophosphatidylcholine, and sphingomyelin.

[0155] The fatty acid moiety may be selected from the non-limiting group consisting of, for example, lauric acid, myristic acid, myristoleic acid, palmitic acid, palmitoleic acid, stearic acid, oleic acid, linoleic acid, alpha-linolenic acid, erucic acid, phytanic acid, arachidic acid, arachidonic acid, eicosapentaenoic acid, behenic acid, docosapentaenoic acid, and docosahexaenoic acid.

[0156] Certain phospholipids can promote fusion to membrane.For example, cationic phospholipids can interact with one or more negatively charged phospholipids of membrane (for example, cell membrane or intracellular membrane).Fusion of phospholipid to membrane can allow one or more elements (for example, therapeutic agent) of lipid-containing composition (for example, LNP) to pass through membrane, for example, deliver one or more elements to target tissue.

[0157] Non-natural phospholipid species are also contemplated, including natural species with modifications and substitutions, including branching, oxidation, cyclization, and alkynes. For example, phospholipids can be functionalized or crosslinked with one or more alkynes (e.g., alkenyl groups with one or more double bonds replaced with triple bonds). Under appropriate reaction conditions, alkyne groups can undergo copper-catalyzed cycloaddition when exposed to azides. Such reactions can be useful for functionalizing the lipid bilayer of nanoparticle compositions to facilitate membrane penetration or cell recognition, or for conjugating nanoparticle compositions to useful components, such as targeting or imaging moieties (e.g., dyes).

[0158] Phospholipids include, but are not limited to, glycerophospholipids, such as phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidylglycerol, and phosphatidic acid. Phospholipids also include sphingophospholipids, such as sphingomyelin.

[0159] In some embodiments, the phospholipid is 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1 ,2-Dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-Diundecanoyl-sn-glycero-phosphocholine (DUPC), 1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-Di-O-Octadecenyl-sn-glycero-3-phosphocholine (18:0 diether PC), 1-Oleoyl-2-cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine (OChemsPC), 1-Hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC), 1,2-dilinoleoyl-sn-glycero-3-phosphocholine, 1,2-diarachidonoyl-sn-glycero-3-phosphocholine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphocholine, 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (ME16.0PE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine -phosphoethanolamine, 1,2-dilinolenoyl-sn-glycero-3-phosphoethanolamine, 1,2-diarachidonoyl-sn-glycero-3-phosphoethanolamine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphoethanolamine, 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt (DOPG), sphingomyelin, and mixtures thereof.

[0160] In certain embodiments, the phospholipid is an analog or variant of DSPC. In certain embodiments, the phospholipid has formula (IX): [ka] or a salt thereof, wherein Each R 1 are independently optionally substituted alkyl; or optionally, two R 1 together with the intervening atoms form an optionally substituted monocyclic carbocyclyl or an optionally substituted monocyclic heterocyclyl; or optionally, three R 1 together with the intervening atoms form an optionally substituted bicyclic carbocyclyl or an optionally substituted bicyclic heterocyclyl; n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; m is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; A is a group represented by the formula: [ka] The L 2 Each instance of is independently a bond or an optionally substituted C 1-6 alkylene, wherein optionally substituted C 1-6 One methylene unit of the alkylene is optionally O, N(R N ), S, C(O), C(O)N(R N ), N.R. N C(O), C(O)O, OC(O), OC(O)O, OC(O)N(R N ), -NR N C(O)O, or NR N C(O)N(R N ), R 2 Each instance of is independently an optionally substituted C 1-30 Alkyl, optionally substituted C 1-30 alkenyl, or optionally substituted C 1-30 alkynyl; optionally, R 2one or more methylene units of are independently selected from optionally substituted carbocyclylene, optionally substituted heterocyclylene, optionally substituted arylene, optionally substituted heteroarylene, N(R N ), O, S, C(O), C(O)N(R N ), N.R. N C(O), NR N C(O)N(R N ), C(O)O, OC(O), -OC(O)O, OC(O)N(R N ), N.R. N C(O)O, C(O)S, SC(O), C(=NR N ), C(=NR N )N(R N ), N.R. N C(=NR N ), -NR N C(=NR N )N(R N ), C(S), C(S)N(R N ), N.R. N C(S), NR N C(S)N(R N ), S(O), OS(O), S(O)O, -OS(O)O, OS(O) 2 , S(O) 2 O, OS(O) 2 O,N(R N )S(O), S(O)N(R N ), N(R N )S(O)N(R N ), OS(O)N(R N ), -N(R N )S(O)O, S(O) 2 , N(R N )S(O) 2 , S(O) 2 N(R N ), N(R N )S(O) 2 N(R N ), OS(O) 2 N(R N ), or -N(R N )S(O) 2 It has been replaced by O. R Neach instance of is independently hydrogen, an optionally substituted alkyl, or a nitrogen protecting group; Ring B is an optionally substituted carbocyclyl, an optionally substituted heterocyclyl, an optionally substituted aryl, or an optionally substituted heteroaryl; p is 1 or 2, However, this compound has the formula: [ka] (In the formula, R 2 Each instance of is independently unsubstituted alkyl, unsubstituted alkenyl, or unsubstituted alkynyl.

[0161] In some embodiments, the phospholipid can be one or more of the phospholipids described in PCT Application No. PCT / US2018 / 037922.

[0162] In some embodiments, the lipid nanoparticles contain 5-25% molar phospholipids relative to other lipid components. For example, the lipid nanoparticles may contain 5-30%, 5-15%, 5-10%, 10-25%, 10-20%, 10-25%, 15-25%, 15-20%, 20-25%, or 25-30% molar phospholipids. In some embodiments, the lipid nanoparticles contain 5%, 10%, 15%, 20%, 25%, or 30% molar non-cationic lipids.

[0163] In some embodiments, the lipid nanoparticles contain 25-55% molar structured lipid relative to other lipid components. For example, the lipid nanoparticles may contain 10-55%, 25-50%, 25-45%, 25-40%, 25-35%, 25-30%, 30-55%, 30-50%, 30-45%, 30-40%, 30-35%, 35-55%, 35-50%, 35-45%, 35-40%, 40-55%, 40-50%, 40-45%, 45-55%, 45-50%, or 50-55% structured lipid. In some embodiments, the lipid nanoparticles contain 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, or 55% structured lipid.

[0164] In some embodiments, the lipid nanoparticles contain PEG lipids at a molar ratio of 0.5-15% relative to other lipid components. For example, the lipid nanoparticles may contain PEG lipids at a molar ratio of 0.5-10%, 0.5-5%, 1-15%, 1-10%, 1-5%, 2-15%, 2-10%, 2-5%, 5-15%, 5-10%, or 10-15%. In some embodiments, the lipid nanoparticles contain PEG lipids at a molar ratio of 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, or 15%.

[0165] In some embodiments, the lipid nanoparticles comprise a molar ratio of 20-60% amino lipid, 5-25% phospholipid, 25-55% structural lipid, and 0.5-15% PEG lipid.

[0166] In some embodiments, the lipid nanoparticles comprise a molar ratio of 20-60% amino lipid, 5-30% phospholipid, 10-55% structural lipid, and 0.5-15% PEG lipid.

[0167] structured lipids The lipid composition of the pharmaceutical compositions provided herein may comprise one or more structured lipids. As used herein, the term "structured lipid" refers to sterols and also to lipids that contain a sterol moiety.

[0168] The incorporation of structured lipids into the lipid nanoparticles may help reduce the aggregation of other lipids within the particle. The structured lipid may be selected from the group including, but not limited to, cholesterol, fecosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatidine, tomatine, ursolic acid, alpha-tocopherol, hopanoids, phytosterols, steroids, and mixtures thereof. In some embodiments, the structured lipid is a sterol. As used herein, "sterol" is a subgroup of steroids consisting of steroid alcohols. In certain embodiments, the structured lipid is a steroid. In certain embodiments, the structured lipid is cholesterol. In certain embodiments, the structured lipid is an analog of cholesterol. In certain embodiments, the structured lipid is alpha-tocopherol.

[0169] In some embodiments, the structured lipids can be one or more of the structured lipids described in US Application Serial No. 16 / 493,814.

[0170] In some embodiments, the lipid nanoparticles comprise 30-45 mol% sterol, optionally 35-40 mol%, e.g., 30-31 mol%, 31-32 mol%, 32-33 mol%, 33-34 mol%, 35-35 mol%, 35-36 mol%, 36-37 mol%, 38-38 mol%, 38-39 mol%, or 39-40 mol% sterol. In some embodiments, the lipid nanoparticles comprise 25-55 mol% sterol. For example, the lipid nanoparticles may contain 25-50 mol%, 25-45 mol%, 25-40 mol%, 25-35 mol%, 25-30 mol%, 30-55 mol%, 30-50 mol%, 30-45 mol%, 30-40 mol%, 30-35 mol%, 35-55 mol%, 35-50 mol%, 35-45 mol%, 35-40 mol%, 40-55 mol%, 40-50 mol%, 40-45 mol%, 45-55 mol%, 45-50 mol%, or 50-55 mol% of sterol. In some embodiments, the lipid nanoparticles contain 25 mol%, 30 mol%, 35 mol%, 40 mol%, 45 mol%, 50 mol%, or 55 mol% of sterol.

[0171] In some embodiments, the lipid nanoparticles contain 35-40 mol% cholesterol. For example, the lipid nanoparticles may contain 35, 35.5, 36, 36.5, 37, 37.5, 38, 38.5, 39, 39.5, or 40 mol% cholesterol.

[0172] Polyethylene glycol (PEG) lipids Effective in vivo delivery of nucleic acids represents an ongoing medical challenge. Exogenous nucleic acids (i.e., those that originate outside a cell or organism) are easily degraded in the body, for example, by the immune system. Thus, the use of particulate carriers (e.g., lipid nanoparticles) is often necessary to effectively deliver nucleic acids to cells. Particulate carriers should be formulated to minimize particle aggregation, be relatively stable prior to intracellular delivery, effectively deliver nucleic acids into cells, and elicit no or minimal immune responses. To achieve minimal particle aggregation and pre-delivery stability, many conventional particulate carriers rely on the presence and / or concentration of certain components (e.g., PEG lipids). However, it has been discovered that certain components can reduce the stability of encapsulated nucleic acids (e.g., mRNA molecules). Reduced stability can limit the broad applicability of particulate carriers. Thus, there remains a need for methods to improve the stability of nucleic acids (e.g., mRNA) encapsulated within lipid nanoparticles.

[0173] The lipid composition of the pharmaceutical composition disclosed herein may include one or more polyethylene glycol (PEG) lipids. As used herein, the term "PEG lipid" or "PEG-modified lipid" refers to a polyethylene glycol (PEG)-modified lipid. Non-limiting examples of PEG lipids include PEG-modified phosphatidylethanolamine and phosphatidic acid, PEG-ceramide conjugates (e.g., PEG-CerC14 or PEG-CerC20), PEG-modified dialkylamines, and PEG-modified 1,2-diacyloxypropan-3-amines. Such lipids are also referred to as PEGylated lipids. For example, the PEG lipid may be a PEG-c-DOMG, PEG-DMG, PEG-DLPE, PEG-DMPE, PEG-DPPC, or PEG-DSPE lipid.

[0174] In some embodiments, the PEG lipid includes, but is not limited to, 1,2-dimyristoyl-sn-glycerol methoxypolyethylene glycol (PEG-DMG), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)] (PEG-DSPE), PEG-disteryl glycerol (PEG-DSG), PEG-dipalmetoleyl, PEG-dioleyl, PEG-distearyl, PEG-diacylglycamide (PEG-DAG), PEG-dipalmitoylphosphatidylethanolamine (PEG-DPPE), or PEG-1,2-dimyristyloxlpropyl-3-amine (PEG-c-DMA).

[0175] In some embodiments, the PEG-lipid is selected from the group consisting of PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, PEG-modified dialkylglycerol, and mixtures thereof. In some embodiments, the PEG-modified lipid is PEG-DMG, PEG-c-DOMG (also known as PEG-DOMG), PEG-DSG, and / or PEG-DPG.

[0176] In some embodiments, the lipid portion of the PEG-lipid has a molecular weight of about C 14 ~About C 22 , preferably about C 14 ~About C 16 In some embodiments, the PEG moiety, e.g., mPEG-NH 2 In some embodiments, the PEG lipid has a size of about 1000, 2000, 5000, 10,000, 15,000, or 20,000 daltons. 2k -DMG.

[0177] In some embodiments, the lipid nanoparticles described herein can include a PEG lipid that is a non-diffusible PEG. Non-limiting examples of non-diffusible PEG include PEG-DSG and PEG-DSPE.

[0178] PEG lipids are known in the art, such as those described in U.S. Pat. No. 8,158,601 and International Publication No. WO2015 / 130584 A2, which are incorporated by reference in their entireties.

[0179] Generally, some of the other lipid components (e.g., PEG lipids) of the various formulas described herein can be synthesized as described in International Patent Application No. PCT / US2016 / 000129, entitled "Compositions and Methods for Delivery of Therapeutic Agents," filed December 10, 2016, which is incorporated by reference in its entirety.

[0180] The lipid component of the lipid nanoparticle composition may include one or more molecules including polyethylene glycol, e.g., PEG or PEG-modified lipid. Such species may alternatively be referred to as PEGylated lipid. PEG lipid is a lipid modified with polyethylene glycol. PEG lipid may be selected from the non-limiting group including PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, PEG-modified dialkylglycerol, and mixtures thereof. For example, PEG lipid may be PEG-c-DOMG, PEG-DMG, PEG-DLPE, PEG-DMPE, PEG-DPPC, or PEG-DSPE lipid.

[0181] In some embodiments, the PEG-modified lipid is a modified PEG DMG. PEG-DMG has the following structure: [ka]

[0182] In some embodiments, the PEG lipid may be a PEGylated lipid described in International Publication No. WO2012 / 099755, the contents of which are incorporated herein by reference in their entirety. Any of the exemplary PEG lipids may be modified to include hydroxyl groups on the PEG chain. In certain embodiments, the PEG lipid is a PEG-OH lipid. As broadly defined herein, a "PEG-OH lipid" (also referred to herein as a "hydroxy-PEGylated lipid") is a PEGylated lipid having one or more hydroxyl (-OH) groups on the lipid. In certain embodiments, the PEG-OH lipid comprises one or more hydroxyl groups on the PEG chain. In certain embodiments, the PEG-OH lipid, i.e., the hydroxy-PEGylated lipid, comprises an -OH group at the end of the PEG chain. Each possibility represents a separate embodiment.

[0183] In certain embodiments, the PEG lipid has the formula (X): [ka] or a salt thereof, wherein R 3 -OR O and R O is hydrogen, an optionally substituted alkyl, or an oxygen protecting group; r is an integer between 1 and 100, inclusive; L 1 is optionally replaced by C 1-10 alkylene, wherein optionally substituted C 1-10 At least one methylene of the alkylene is independently an optionally substituted carbocyclylene, an optionally substituted heterocyclylene, an optionally substituted arylene, an optionally substituted heteroarylene, O, N(R N ), S, C(O), C(O)N(R N ), N.R. N C(O), C(O)O, OC(O), OC(O)O, -OC(O)N(R N ), N.R. NC(O)O, or NR N C(O)N(R N ), D is a moiety obtained by click chemistry or cleavable under physiological conditions; m is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; A is a group represented by the formula: [ka] The L 2 Each instance of is independently a bond or an optionally substituted C 1-6 alkylene, wherein optionally substituted C 1-6 One methylene unit of the alkylene is optionally O, N(R N ), S, C(O), C(O)N(R N ), N.R. N C(O), C(O)O, OC(O), OC(O)O, OC(O)N(R N ), -NR N C(O)O, or NR N C(O)N(R N ), R 2 Each instance of is independently an optionally substituted C 1-30 Alkyl, optionally substituted C 1-30 alkenyl, or optionally substituted C 1-30 alkynyl; optionally, R 2 one or more methylene units of are independently selected from optionally substituted carbocyclylene, optionally substituted heterocyclylene, optionally substituted arylene, optionally substituted heteroarylene, N(R N ), O, S, C(O), C(O)N(R N ), N.R. N C(O), NR N C(O)N(R N ), C(O)O, OC(O), -OC(O)O, OC(O)N(R N ), N.R. NC(O)O, C(O)S, SC(O), C(=NR N ), C(=NR N )N(R N ), N.R. N C(=NR N ), -NR N C(=NR N )N(R N ), C(S), C(S)N(R N ), N.R. N C(S), NR N C(S)N(R N ), S(O), OS(O), S(O)O, -OS(O)O, OS(O) 2 , S(O) 2 O, OS(O) 2 O,N(R N )S(O), S(O)N(R N ), N(R N )S(O)N(R N ), OS(O)N(R N ), -N(R N )S(O)O, S(O) 2 , N(R N )S(O) 2 , S(O) 2 N(R N ), N(R N )S(O) 2 N(R N ), OS(O) 2 N(R N ), or -N(R N )S(O) 2 It has been replaced by O. R N each instance of is independently hydrogen, an optionally substituted alkyl, or a nitrogen protecting group; Ring B is an optionally substituted carbocyclyl, an optionally substituted heterocyclyl, an optionally substituted aryl, or an optionally substituted heteroaryl; p is 1 or 2.

[0184] In certain embodiments, the compound of formula (X) is a PEG-OH lipid (i.e., R 3 -OR O and RO is hydrogen).

[0185] In certain embodiments, the compound of formula (X) has the formula (X-OH): [ka] or a salt thereof.

[0186] In certain embodiments, the PEG lipid is a PEGylated fatty acid. In certain embodiments, the PEG lipid is a compound of formula (XI). As used herein, formula (XI): [ka] or a salt thereof, wherein R 3 -OR O and R O is hydrogen, an optionally substituted alkyl, or an oxygen protecting group; r is an integer between 1 and 100, inclusive; R 5 is optionally replaced by C 10-40 Alkyl, optionally substituted C 10-40 alkenyl, or optionally substituted C 10-40 alkynyl, and optionally R 5 One or more methylene groups in the formula (I) are optionally substituted carbocyclylene, optionally substituted heterocyclylene, optionally substituted arylene, optionally substituted heteroarylene, N(R N ), O, S, C(O), -C(O)N(R N ), N.R. N C(O), NR N C(O)N(R N ), C(O)O, OC(O), OC(O)O, OC(O)N(R N ), N.R. N C(O)O, -C(O)S, SC(O), C(=NR N ), C(=NR N )N(R N), N.R. N C(=NR N ), N.R. N C(=NR N )N(R N ), C(S), -C(S)N(R N ), N.R. N C(S), NR N C(S)N(R N ), S(O), OS(O), S(O)O, OS(O)O, OS(O) 2 , S(O) 2 O, -OS(O) 2 O,N(R N )S(O), S(O)N(R N ), N(R N )S(O)N(R N ), OS(O)N(R N ), N(R N )S(O)O, S(O) 2 , -N(R N )S(O) 2 , S(O) 2 N(R N ), N(R N )S(O) 2 N(R N ), OS(O) 2 N(R N ), or N(R N )S(O) 2 It has been replaced by O. R N Each instance of is independently hydrogen, an optionally substituted alkyl, or a nitrogen protecting group.

[0187] In certain embodiments, the compound of formula (XI) has the formula (XI-OH): [ka] or a salt thereof. In some embodiments, r is 40-50.

[0188] In yet another embodiment, the compound of formula (XI) is [ka] or a salt thereof.

[0189] In one embodiment, the compound of formula (XI) is [ka] It is.

[0190] In some embodiments, the lipid composition of the pharmaceutical compositions disclosed herein does not comprise a PEG lipid.

[0191] In some embodiments, the PEG lipid can be one or more of the PEG lipids described in US Application No. US15 / 674,872.

[0192] In some embodiments, the lipid nanoparticles contain 1-5%, optionally 1-3 mol%, for example, 1.5-2.5 mol%, 1-2 mol%, 2-3 mol%, 3-4 mol%, or 4-5 mol% of PEG-modified lipids. In some embodiments, the lipid nanoparticles contain 0.5-15 mol% of PEG-modified lipids. For example, the lipid nanoparticles may contain 0.5-10 mol%, 0.5-5 mol%, 1-15 mol%, 1-10 mol%, 1-5 mol%, 2-15 mol%, 2-10 mol%, 2-5 mol%, 5-15 mol%, 5-10 mol%, or 10-15 mol%. In some embodiments, the lipid nanoparticles comprise 0.5 mol%, 1 mol%, 2 mol%, 3 mol%, 4 mol%, 5 mol%, 6 mol%, 7 mol%, 8 mol%, 9 mol%, 10 mol%, 11 mol%, 12 mol%, 13 mol%, 14 mol%, or 15 mol% PEG-modified lipids.

[0193] In some embodiments, the lipid nanoparticles comprise 20-60 mol % ionizable amino lipid, 5-25 mol % non-cationic lipid, 25-55 mol % sterol, and 0.5-15 mol % PEG-modified lipid.

[0194] In some embodiments, the LNP comprises an ionizable amino lipid of Compound 1, where the non-cationic lipid is DSPC, the structural lipid is cholesterol, and the PEG lipid is DMG-PEG.

[0195] In some embodiments, the LNP comprises an ionizable amino lipid of any of formulas VI, VII, or VIIII, a phospholipid comprising DSPC, a structured lipid, and a PEG lipid comprising PEG-DMG.

[0196] In some embodiments, the LNP comprises an ionizable amino lipid of any of formulas VI, VII, or VIII, a phospholipid comprising DSPC, a structured lipid, and a PEG lipid comprising a compound having formula XI.

[0197] In some embodiments, the LNP comprises an ionizable amino lipid of formula VI, VII, or VIII, a phospholipid comprising a compound having formula VIII, a structured lipid, and a PEG lipid comprising a compound having formula X or XI.

[0198] In some embodiments, the LNP comprises an ionizable amino lipid of formula VI, VII, or VIII, a phospholipid comprising a compound having formula IX, a structured lipid, and a PEG lipid comprising a compound having formula X or XI.

[0199] In some embodiments, the LNP comprises an ionizable amino lipid of formula VI, VII, or VIII, a phospholipid having formula IX, a structured lipid, and a PEG lipid comprising a compound having formula XI.

[0200] In some embodiments, the lipid nanoparticles comprise 49 mol% ionizable amino lipid, 10 mol% DSPC, 38.5 mol% cholesterol, and 2.5 mol% DMG-PEG.

[0201] In some embodiments, the lipid nanoparticles comprise 49 mol% ionizable amino lipid, 11 mol% DSPC, 38.5 mol% cholesterol, and 1.5 mol% DMG-PEG.

[0202] In some embodiments, the lipid nanoparticles comprise 48 mol% ionizable amino lipid, 11 mol% DSPC, 38.5 mol% cholesterol, and 2.5 mol% DMG-PEG.

[0203] Nucleic Acids and Antigens Nucleic acids are also provided. The term "nucleic acid" refers to a molecule that includes a plurality of nucleotides (i.e., a sugar (e.g., ribose or deoxyribose) linked to a phosphate group and an exchangeable organic base that is either a substituted pyrimidine (e.g., cytosine (C), thymine (T) or uracil (U)) or a substituted purine (e.g., adenine (A) or guanine (G)). As used herein, the term "nucleic acid" refers to polyribonucleotides and polydeoxyribonucleotides. The term nucleic acid also includes polynucleosides (i.e., polynucleotides minus the phosphates) and any other organic base-containing polymers. Non-limiting examples of nucleic acids include chromosomes, genomic loci, genes or gene segments that encode polynucleotides or polypeptides, coding sequences of genes, non-coding sequences (e.g., introns, 5'-UTR, or 3'-UTR), pri-mRNA, pre-mRNA, cDNA, mRNA, etc. In some embodiments, the nucleic acid is an mRNA. The nucleic acid may include substitutions and / or modifications. In some embodiments, the substitutions and / or modifications are in one or more bases and / or sugars. For example, in some embodiments, the nucleic acid comprises a nucleic acid having a backbone sugar covalently attached to a low molecular weight organic group other than a hydroxyl group at the 2' position and other than a phosphate or hydroxy group at the 5' position. Thus, in some embodiments, the substituted or modified nucleic acid comprises a 2'-O-alkylated ribose group. In some embodiments, the modified nucleic acid comprises a sugar such as hexose, 2'-F hexose, 2'-amino ribose, constrained ethyl (cEt), locked nucleic acid (LNA), arabinose or 2'-fluoroarabinose instead of ribose. Thus, in some embodiments, the nucleic acid is heterogeneous in backbone composition, thereby comprising all possible combinations of polymer units linked together, such as peptide-nucleic acids (those with an amino acid backbone that includes nucleobases).

[0204] As used herein, "modified" means non-natural. In some embodiments, RNA can be modified RNA. That is, RNA can contain one or more nucleobases, nucleosides, nucleotides, or linkers that are non-naturally occurring. In this specification, "modified" species may also be referred to as "altered" species. Species can be chemically, structurally, or functionally modified or altered. In some embodiments, modified nucleobase species can contain one or more substitutions that are not naturally occurring.

[0205] In some embodiments, the nucleic acid is DNA, RNA, PNA, cEt, LNA, ENA, or a hybrid containing any chemical or natural modification thereof. Chemical and natural modifications are well known in the art. Non-limiting examples of modifications include modifications designed to increase the translation of nucleic acid, modifications designed to increase the cellular penetration or subcellular distribution of nucleic acid, modifications designed to stabilize nucleic acid against nucleases and other enzymes that degrade or interfere with the structure or activity of nucleic acid, and modifications designed to improve the pharmacokinetic properties of nucleic acid.

[0206] As used herein, the term "polypeptide" or "polypeptide of interest" refers to a polymer of amino acid residues, typically linked by peptide bonds, which may be produced naturally (e.g., isolated or purified) or produced synthetically.

[0207] As used herein, "RNA" refers to ribonucleic acid, which may be naturally occurring or non-naturally occurring. In some embodiments, the RNA may include modified and / or non-naturally occurring components, such as one or more nucleobases, nucleosides, nucleotides, or linkers. The RNA may include a cap structure, a chain terminating nucleoside, a stem loop, a polyA sequence, and / or a polyadenylation signal. The RNA may have a nucleotide sequence that encodes a polypeptide of interest. In some embodiments, the RNA may be a messenger RNA (mRNA). Translation of an mRNA that encodes a particular polypeptide, for example, in vivo translation of the mRNA inside a mammalian cell, may produce the encoded polypeptide. The RNA may be selected from the non-limiting group consisting of small interfering RNA (siRNA), asymmetric interfering RNA (aiRNA), microRNA (miRNA), dicer substrate RNA (dsRNA), short hairpin RNA (shRNA), mRNA, long non-coding RNA (lncRNA), and mixtures thereof.

[0208] In some embodiments, the composition comprises an RNA having an open reading frame (ORF) encoding a polypeptide. In some embodiments, the RNA is a messenger RNA (mRNA). In some embodiments, the RNA (e.g., an mRNA) further comprises a 5' UTR, a 3' UTR, a poly(A) tail, and / or a 5' cap analog.

[0209] Messenger RNA (mRNA) is any RNA that codes for (at least one) protein (e.g., a naturally occurring, non-naturally occurring, or modified polymer of amino acids) and can be translated in vitro, in vivo, in situ, or ex vivo to produce the encoded protein. As used herein, the term "in vitro" refers to events that occur not in an organism (e.g., an animal, plant, or microorganism) but in an artificial environment, e.g., in a test tube or reaction vessel, in a cell culture, in a petri dish, etc. As used herein, the term "in vivo" refers to events that occur in an organism (e.g., an animal, plant, or microorganism, or cells or tissues thereof). As used herein, the term "ex vivo" refers to events that occur outside of an organism (e.g., an animal, plant, or microorganism, or cells or tissues thereof). Ex vivo events can occur in an environment that is minimally modified from the natural (e.g., in vivo) environment.

[0210] Unless otherwise indicated, the nucleic acid sequences described in this application may describe a "T" in a representative DNA sequence, but it will be understood by one of skill in the art that where the sequence corresponds to RNA (e.g., mRNA), "U" is replaced with "T." Thus, any DNA disclosed herein and identified by a particular sequence identification number also discloses the corresponding RNA (e.g., mRNA) sequence complementary to that DNA, in which case each "T" in the DNA sequence is replaced with a "U."

[0211] An open reading frame (ORF) is a continuous stretch of DNA or RNA that begins with a start codon (e.g., a methionine (ATG or AUG)) and ends with a stop codon (e.g., TAA, TAG, or TGA, or UAA, UAG, or UGA). ORFs typically encode proteins. The sequences disclosed herein may further comprise additional elements (e.g., 5' and 3' UTRs), although it will be understood that these elements, unlike ORFs, need not necessarily be present in an RNA polynucleotide.

[0212] Naturally occurring eukaryotic mRNA molecules may contain stabilizing elements, including but not limited to untranslated regions (UTRs) at their 5' ends (5'UTRs) and / or untranslated regions (UTRs) at their 3' ends (3'UTRs), in addition to other structural features such as a 5'-cap structure or a 3'-poly(A) tail. Both 5'UTRs and 3'UTRs are typically transcribed from genomic DNA and are elements of the premature mRNA. Structural features characteristic of mature mRNAs, such as the 5'-cap and 3'-poly(A) tail, are usually added to the transcribed (premature) mRNA during mRNA processing.

[0213] In some embodiments, the composition comprises an RNA polynucleotide having an open reading frame encoding at least one polypeptide having at least one modification, at least one 5' end cap, and is formulated in a lipid nanoparticle with a stabilizing compound.

[0214] The 5'-end cap may comprise an endogenous cap or a cap analog. According to the present invention, the 5'-end cap may comprise a guanine analog. Useful guanine analogs include, but are not limited to, inosine, N1-methyl-guanosine, 2'fluoro-guanosine, 7-deaza-guanosine, 8-oxo-guanosine, 2-amino-guanosine, LNA-guanosine, and 2-azido-guanosine.

[0215] Also provided herein are exemplary caps, including those that can be used in co-transcriptional capping methods for ribonucleic acid (RNA) synthesis using an RNA polymerase, such as a wild-type RNA polymerase or a variant thereof, such as a variant described herein. In some embodiments, the cap can be added when the RNA is produced in a "one-pot" reaction, without the need for a separate capping reaction. Thus, the method, in some embodiments, includes reacting a polynucleotide template with an RNA polymerase variant, a nucleoside triphosphate, and a cap analog under in vitro transcription reaction conditions to produce an RNA transcript.

[0216] In some embodiments, a cap analog is attached to a polynucleotide template that includes a promoter region that includes a transcription initiation site having a first nucleotide at nucleotide position +1, a second nucleotide at nucleotide position +2, and a third nucleotide at nucleotide position +3. In some embodiments, the cap analog hybridizes to the polynucleotide template at least at nucleotide position +1, e.g., at positions +1 and +2, or at positions +1, +2, and +3.

[0217] The cap analog may be, for example, a dinucleotide cap, a trinucleotide cap, or a tetranucleotide cap. In some embodiments, the cap analog is a dinucleotide cap. In some embodiments, the cap analog is a trinucleotide cap. In some embodiments, the cap analog is a tetranucleotide cap. As used herein, the term "cap" includes an inverted G nucleotide and can include additional nucleotides 3' to the inverted G, e.g., one, two, or more nucleotides 3' to the inverted G and 5' to the 5'UTR.

[0218] A 3'-poly(A) tail is typically a stretch of adenine nucleotides added to the 3' end of a transcribed mRNA and can, in some cases, contain up to about 400 adenine nucleotides. In some embodiments, the length of the 3'-poly(A) tail can be an essential factor for the stability of an individual mRNA.

[0219] In some embodiments, the composition includes an RNA (e.g., an mRNA) having an ORF encoding a signal peptide fused to the expressed polypeptide. Signal peptides usually comprise 15-60 amino acids on the N-terminus of a protein and are typically required for translocation across membranes in the secretory pathway, thus universally controlling entry of most proteins into the secretory pathway in both eukaryotes and prokaryotes. Signal peptides can have a length of 15-60 amino acids.

[0220] In some embodiments, the ORF encoding the polypeptide is codon-optimized. Methods of codon optimization are known in the art. For example, any one or more ORFs of the sequences provided herein may be codon-optimized. Codon optimization may be used in some embodiments to match codon frequencies in target and host organisms to ensure proper folding, to bias GC content to enhance mRNA stability or reduce secondary structures, to minimize runs of tandem repeat codons or bases that may impair gene organization or expression, to customize transcriptional and translational regulatory regions, to insert or remove protein trafficking sequences, to remove / add post-translational modification sites (e.g., glycosylation sites) of the encoded protein, to add, remove, or swap protein domains, to insert or delete restriction enzyme recognition sites, to modify ribosome binding sites and mRNA degradation sites, to adjust the translation rate and correctly fold various domains of the protein, or to reduce or remove problematic secondary structures within the polynucleotide. Codon optimization tools, algorithms and services are known in the art, non-limiting examples include services from GeneArt (Life Technologies), DNA2.0 (Menlo Park CA), and / or proprietary methods. In some embodiments, open reading frame (ORF) sequences are optimized using an optimization algorithm.

[0221] In some embodiments, the RNA (e.g., mRNA) is not chemically modified and comprises standard ribonucleotides consisting of adenosine, guanosine, cytosine, and uridine. In some embodiments, the nucleotides and nucleosides comprise standard nucleoside residues (e.g., A, G, C, or U) as present in transcribed RNA. In some embodiments, the nucleotides and nucleosides comprise standard deoxyribonucleosides (e.g., dA, dG, dC, or dT) as present in DNA.

[0222] The composition, in some embodiments, can include an RNA having an open reading frame encoding a polypeptide, the nucleic acid comprising nucleotides and / or nucleosides that can be standard (unmodified) or modified as known in the art. In some embodiments, the nucleotides and nucleosides comprise modified nucleotides or nucleosides. Such modified nucleotides and nucleosides can be naturally occurring modified nucleotides and nucleosides or non-naturally occurring modified nucleotides and nucleosides. Such modifications can include modifications at the sugar, backbone, or nucleobase moieties of the nucleotides and / or nucleosides that are recognized in the art.

[0223] In some embodiments, the naturally occurring modified nucleotide or nucleotides are those commonly known or recognized in the art. Non-limiting examples of such naturally occurring modified nucleotides and nucleotides can be found, inter alia, in the widely recognized MODOMICS database.

[0224] Modified nucleosides and nucleotides of nucleic acids (e.g., RNA nucleic acids, e.g., mRNA nucleic acids) are also provided. A "nucleoside" refers to a compound that includes a sugar molecule (e.g., pentose or ribose) or a derivative thereof in combination with an organic base (e.g., a purine or pyrimidine) or a derivative thereof (also referred to herein as a "nucleobase"). A "nucleotide" refers to a nucleoside that includes a phosphate group. Modified nucleotides can be synthesized in any useful manner, e.g., chemically, enzymatically, or recombinantly, to include one or more modified or non-natural nucleosides. A nucleic acid can include a region or regions of linked nucleosides. Such regions may have variable backbone linkages. The linkages can be standard phosphodiester linkages, in which case the nucleic acid includes a region of nucleotides.

[0225] In some embodiments, modified nucleobases in a nucleic acid (e.g., an RNA nucleic acid, e.g., an mRNA nucleic acid) include 1-methyl-pseudouridine (m1ψ), 1-ethyl-pseudouridine (e1ψ), 5-methoxy-uridine (mo5U), 5-methyl-cytidine (m5C), and / or pseudouridine (ψ). In some embodiments, modified nucleobases in a nucleic acid (e.g., an RNA nucleic acid, such as an mRNA nucleic acid) include 5-methoxymethyluridine, 5-methylthiouridine, 1-methoxymethyl-pseudouridine, 5-methylcytidine, and / or 5-methoxycytidine. In some embodiments, a polyribonucleotide includes a combination of at least two (e.g., two, three, four, or more) of any of the foregoing modified nucleobases (including, but not limited to, chemical modifications).

[0226] In some embodiments, the mRNA comprises 1-methyl-pseudouridine (m1ψ) substitutions at one or more or all uridine positions in the nucleic acid.

[0227] In some embodiments, the mRNA comprises 1-methyl-pseudouridine (m1ψ) substitutions at one or more or all of the uridine positions in the nucleic acid and 5-methylcytidine substitutions at one or more or all of the cytidine positions in the nucleic acid.

[0228] In some embodiments, the mRNA contains pseudouridine (ψ) substitutions at one or more or all uridine positions in the nucleic acid.

[0229] In some embodiments, the mRNA comprises pseudouridine (ψ) substitutions at one or more or all of the uridine positions in the nucleic acid and 5-methylcytidine substitutions at one or more or all of the cytidine positions in the nucleic acid.

[0230] In some embodiments, the mRNA comprises a uridine at one or more or all uridine positions of the nucleic acid.

[0231] In some embodiments, the mRNA is uniformly modified for a particular modification (e.g., completely modified, modified throughout the entire sequence). For example, the nucleic acid can be uniformly modified with 1-methyl-pseudouridine. This means that all uridine residues in the mRNA sequence are replaced with 1-methyl-pseudouridine. Similarly, the nucleic acid can be uniformly modified by replacing any type of nucleoside residue present in the sequence with a modified residue such as those described above.

[0232] A nucleic acid may be partially or completely modified along the entire length of the molecule. For example, one or more or all or a given type of nucleotide (e.g., purines or pyrimidines, or any one or more or all of A, G, U, C) may be uniformly modified within the nucleic acid or within a defined sequence region thereof (e.g., within an mRNA, including or excluding a poly(A) tail). In some embodiments, all nucleotides X in the nucleic acid (or sequence region thereof) are modified nucleotides, where X may be any one of nucleotides A, G, U, C, or any one of the combinations A+G, A+U, A+C, G+U, G+C, U+C, A+G+U, A+G+C, G+U+C, or A+G+C.

[0233] An mRNA may contain one or more regions or parts that act or function as untranslated regions. If the mRNA is designed to code for at least one polypeptide of interest, the nucleic acid may contain one or more of these untranslated regions (UTRs). The wild-type untranslated regions of a nucleic acid are transcribed but not translated. In an mRNA, the 5'UTR starts at the transcription initiation site and continues to the start codon but does not include the start codon. On the other hand, the 3'UTR starts immediately after the stop codon and continues to the transcription termination signal. Regulatory functions of UTRs can be incorporated into polynucleotides to, among other things, improve the stability of the molecule. Also, specific functions can be incorporated to ensure that the downregulation of the transcript is controlled in case the transcript is misdirected to an undesired organ site. A variety of 5'UTR and 3'UTR sequences are known and available in the art.

[0234] Further embodiments 1. 1. A method for measuring free nucleic acid and / or encapsulation efficiency (%EE) of a sample containing nucleic acid and an encapsulating agent, comprising: (a) measuring the absorbance of the formulation buffer at 665 nm and 760 nm; (b) measuring the absorbance of the methylene blue working solution at 665 nm and 760 nm; (c) contacting the sample with the methylene blue working solution, thereby forming a test solution; (d) measuring the absorbance of the test solution at 665 nm and 760 nm; (e) The following formula:

number

[0235] 2. 2. The method of paragraph 1, wherein the encapsulating agent comprises a lipid nanoparticle (LNP).

[0236] 3. 3. The method of paragraph 2, wherein the LNP comprises an amino lipid, a neutral lipid, a PEG-modified lipid, and a sterol.

[0237] 4. 4. The method of any one of paragraphs 1 to 3, wherein the polynucleotide is mRNA.

[0238] 5. 5. The method of any one of paragraphs 1 to 4, wherein the methylene blue working solution comprises a formulation buffer and methylene blue dye.

[0239] 6. 6. The method of any one of paragraphs 1 to 5, wherein the formulation buffer comprises Tris and sucrose.

[0240] 7. 7. The method of paragraph 6, wherein the formulation buffer has a pH of 7.2 to 7.6.

[0241] 8. 8. The method of paragraph 7, wherein the pH of the formulation buffer is 7.4.

[0242] 9. 9. The method of any one of paragraphs 1 to 8, wherein the response factor is the ratio of the signal generated by an experimental analyte to the amount of the analyte.

[0243] 10. 10. The method of any one of paragraphs 1-9, wherein steps (b)-(e) are repeated one, two or three times.

[0244] 11. 11. The method of paragraph 10, comprising calculating an average %EE for the samples.

[0245] 12. 1. A method for measuring free nucleic acid and / or encapsulation efficiency (%EE) of a sample containing nucleic acid and an encapsulating agent, comprising: (a) measuring absorbance of a formulation buffer at a first wavelength and at a second wavelength; (b) measuring the absorbance of the phenothiazinium dye working solution at the first wavelength and at the second wavelength; (c) contacting the sample with the phenothiazinium dye working solution, thereby forming a test solution; (d) measuring the absorbance of the test solution at the first wavelength and at the second wavelength; (e) The following formula:

number

[0246] 13. 13. The method of paragraph 12, wherein the encapsulating agent comprises a lipid nanoparticle (LNP).

[0247] 14. 14. The method of paragraph 13, wherein the LNP comprises an amino lipid, a neutral lipid, a PEG-modified lipid, and a sterol.

[0248] 15. 15. The method of any one of paragraphs 12 to 14, wherein the polynucleotide is mRNA.

[0249] 16. 16. The method of any one of paragraphs 12 to 15, wherein the phenothiazinium dye working solution comprises a formulation buffer and a phenothiazinium dye.

[0250] 17. 17. The method of any one of paragraphs 12 to 16, wherein the formulation buffer comprises Tris and sucrose.

[0251] 18. 18. The method of paragraph 17, wherein the formulation buffer has a pH of 7.2 to 7.6.

[0252] 19. 19. The method of paragraph 18, wherein the pH of the formulation buffer is 7.4.

[0253] 20. 20. The method of any one of paragraphs 12 to 19, wherein the response factor is the ratio of the signal generated by an experimental analyte to the amount of the analyte.

[0254] twenty one. 21. The method of any one of paragraphs 12-20, wherein steps (b)-(e) are repeated one, two or three times.

[0255] twenty two. 22. The method of paragraph 21, comprising calculating an average %EE for the samples.

[0256] Equivalent While several embodiments of the present invention have been described and illustrated herein, it is believed that those skilled in the art will readily envision a variety of other means and / or structures for performing the functions and / or obtaining one or more of the results and / or advantages described herein, and each such variation and / or modification is deemed to be within the scope of the present invention. More broadly, those skilled in the art will readily appreciate that all parameters, dimensions, materials, and configurations described herein are intended to be exemplary, and that the actual parameters, dimensions, materials, and / or configurations will depend on the particular application(s) for which the teachings of the present invention are used. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. It is therefore to be understood that the foregoing embodiments are presented by way of example only, and that the invention may be practiced otherwise than as specifically described and claimed, within the scope of the appended claims and equivalents thereto. The present invention is directed to each individual feature, system, article, material, and / or method described herein. Furthermore, any combination of two or more of such features, systems, articles, materials, and / or methods is included within the scope of the present invention, if such features, systems, articles, materials, and / or methods are not mutually inconsistent.

[0257] As used herein, the indefinite articles "a" and "an," as used in the specification and the claims, unless clearly indicated to the contrary, should be understood to mean "at least one."

[0258] The term "and / or," as used in the specification and claims, should be understood to mean "either or both" of the elements so conjoined, i.e., elements that are conjunctive in some cases and disjunctive in other cases. Unless clearly indicated to the contrary, other elements other than the elements specifically identified by the "and / or" clause may optionally be present, whether related or unrelated to the elements specifically identified. Thus, as a non-limiting example, a reference to "A and / or B," when used in conjunction with open-ended language such as "comprising," may in some embodiments refer to A without B (optionally including elements other than B), in other embodiments may refer to B without A (optionally including elements other than A), in yet other embodiments may refer to both A and B (optionally including other elements), and so forth.

[0259] "Or" as used herein and in the claims shall be understood to have the same meaning as "and / or" as defined above. For example, when separating items in a list, "or" or "and / or" shall be interpreted as being inclusive, i.e., the inclusion of at least one of a plurality of elements or a list of elements, but including more than one of them, and optionally including additional items not in the list. Only terms clearly indicating the opposite, such as "only one" or "only one" or, when used in the claims, "consisting of", shall refer to the inclusion of only one element of a plurality of elements or a list of elements. In general, the term "or" as used herein shall be interpreted to indicate exclusive alternatives (i.e., "one or the other but not both") only when preceded by a term indicating exclusivity, such as "either," "one," "only one," or "only one." "Consisting essentially of," when used in the claims, shall have its ordinary meaning as used in the field of patent law.

[0260] As used herein and in the claims, the phrase "at least one" in reference to a list of one or more elements should be understood to mean at least one element selected from any one or more elements in the list of elements, but does not necessarily include at least one of each and every element specifically listed in the list of elements, and does not exclude any combinations of elements in the list of elements. This definition also allows that elements other than those specifically identified in the list of elements to which the phrase "at least one" refers may optionally be present, whether related or unrelated to the elements specifically identified. Thus, as a non-limiting example, "at least one of A and B" (or, equivalently, "at least one of A or B" or, equivalently, "at least one of A and / or B") can in some embodiments refer to the presence of at least one (optionally including multiple) A and no B (and optionally including elements other than B), in other embodiments it can refer to the presence of at least one (optionally including multiple) B and no A (and optionally including elements other than A), in yet other embodiments it can refer to the presence of at least one (optionally including multiple) A and at least one (optionally including multiple) B (and optionally including other elements), and so forth.

[0261] In the claims and the above specification, all transitional phrases such as "comprising," "including," "carrying," "having," "containing," "involving," "holding," and the like, are to be understood to be open-ended, i.e., meaning including but not limited to. Only the transitional phrases "consisting of" and "consisting essentially of" shall be closed or semi-closed transitional phrases, respectively, as set forth in the United States Patent Office Manual of Patent Examining Procedures, Section 2111.03.

[0262] The use of ordinal terms such as "first," "second," "third," etc. in the claims to modify claim elements does not, of itself, imply a priority, ranking, or order of one claim element relative to another, or the temporal order in which method actions are performed, but is merely used as a marker to distinguish one claim element having a particular name from other elements having the same name (but using different ordinal terms).

Claims

1. 1. A method for measuring free nucleic acid and / or encapsulation efficiency (% EE) of a sample containing nucleic acid and an encapsulating agent, comprising: contacting the sample containing the nucleic acid and the mounting medium with a phenothiazinium dye; measuring the absorbance of a solution containing the nucleic acid, the mounting medium, and the phenothiazinium dye; determining the amount of free nucleic acid and / or the % EE based on the absorbance value; The method comprising:

2. A method for determining the amount of free nucleic acid and / or encapsulation efficiency (% EE) of a sample containing nucleic acid and an encapsulating agent, the method comprising measuring a hypocolor shift in absorbance of the sample resulting from an interaction between the nucleic acid and a phenothiazinium dye.

3. 2. The method of any one of the preceding claims, wherein the nucleic acid is DNA or RNA.

4. 4. The method of claim 3, wherein the RNA is an mRNA, siRNA, shRNA, snRNA, snoRNA, or lncRNA.

5. The method of claim 4, wherein the RNA is mRNA.

6. 2. The method of any one of the preceding claims, wherein the encapsulating agent comprises a lipid nanoparticle (LNP), a lipoplex, or a liposome.

7. The method of claim 6 , wherein the encapsulating agent comprises an ionizable amino lipid.

8. The method of claim 6 or 7, wherein the encapsulating agent further comprises a PEG lipid.

9. The method of any one of claims 6 to 8, wherein the encapsulating medium further comprises a structured lipid.

10. The method of any one of claims 6 to 9, wherein the encapsulating medium further comprises a phospholipid.

11. The method of any one of claims 6 to 10, wherein the encapsulating agent comprises an ionizable amino lipid, a PEG lipid, a structured lipid, and a phospholipid.

12. 12. The method of claim 11, wherein the encapsulating medium comprises a ratio of 20-60% ionizable amino lipid, 5-30% phospholipid, 10-55% structured lipid, and 0.5-15% PEG-modified lipid.

13. 12. The method of claim 11, wherein the encapsulating medium comprises a ratio of 20-60% ionizable amino lipid, 5-25% phospholipid, 25-55% structured lipid, and 0.5-15% PEG-modified lipid.

14. The method of any one of claims 1 to 13, wherein the encapsulating agent comprises a lipid nanoparticle (LNP).

15. The method of any one of claims 1 to 13, wherein the encapsulating agent comprises a liposome.

16. The method of any one of claims 1 to 13, wherein the encapsulating agent comprises a lipoplex.

17. The method of any one of claims 1 to 16, wherein the sample is formulated in an aqueous solution.

18. 18. The method of claim 17, wherein the aqueous solution has a pH of 5 to 8 or about 5 to 8, including a pH of about 5, 5.5, 6, 6.5, 7, 7.5, or 8.

19. 19. The method of claim 17 or 18, wherein the aqueous solution comprises a phosphate buffer, a tris buffer, an acetate buffer, a histidine buffer, or a citrate buffer.

20. The method of any one of claims 1 to 19, wherein the phenothiazinium dye is methylene blue.

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