Measuring nucleic acid integrity

Abstract

The present invention relates to a method of measuring nucleic acid integrity in a composition of lipid nanoparticles (LNPs) comprising a cargo of nucleic acids, such as a DNA and / or RNA. In particular, the present invention relates to a method of measuring integrity of RNA in a DNA / RNA cargo of an LNP following digestion of the DNA.

Description

[0001] MEASURING NUCLEIC ACID INTEGRITY

[0002] FIELD OF THE INVENTION

[0003] The present invention relates to a method of measuring nucleic acid integrity in a composition of lipid nanoparticles (LNPs) comprising a cargo of nucleic acids, such as a DNA and / or RNA. In particular, the present invention relates to a method of measuring integrity of RNA in a DNA / RNA cargo of an LNP following digestion of the DNA.

[0004] BACKGROUND TO THE INVENTION

[0005] Lipid nanoparticles (LNPs) have demonstrated huge potential as a delivery technology for therapeutic payloads (e.g. nucleic acid molecules) for treating a wide range of conditions, such as in cancer immunotherapy, gene therapy, and the treatment of infectious diseases.

[0006] These therapeutic payloads can be contained within LNPs as a cargo, and can comprise DNA or RNA alone, or a co-formulated mixture of DNA and RNA, depending on the desired therapeutic application.

[0007] The measurement of nucleic acid integrity, such as RNA integrity, is one of the key parameters to evaluate the quality of the LNPs, which can typically be assessed using capillary gel electrophoresis or classical Agarose gel.

[0008] However, commercially available kits are typically for measuring RNA integrity only or DNA integrity only, creating a challenge for the measurement of RNA integrity and / or DNA integrity within a mixed payload. In particular, when dealing with a mixed cargo of RNA and DNA, the measurement of RNA integrity may be confounded by the presence of DNA, and vice versa.

[0009] An alternative method is needed in order to improve the measurement of the integrity of nucleic acid molecules contained within LNPs. SUMMARY OF THE INVENTION

[0010] The present invention provides a method of measuring nucleic acid integrity in a composition of lipid nanoparticles (LNPs) comprising a cargo of nucleic acids, wherein the method comprises the steps of:

[0011] (i) incubating a sample of the composition with a reagent that releases the cargo from the LNPs;

[0012] (ii) purifying the nucleic acids in the sample;

[0013] (iii) incubating the purified nucleic acids with an enzyme, wherein the enzyme is a DNase or an RNase;

[0014] (iv) incubating the resulting product obtained from step (iii) with proteinase K;

[0015] (v) purifying the resulting product obtained from step (iv); and

[0016] (vi) measuring the nucleic acid integrity in the sample.

[0017] In some embodiments, the reagent in step (i) comprises a detergent.

[0018] In some embodiments, the detergent is selected from the list consisting of: Triton, Zwittergent, and sodium dodecyl sulfate (SDS).

[0019] In some embodiments, the detergent is selected from the list consisting of: 2-[4-(2,4,4- trimethylpentan-2-yl)phenoxy]ethanol, N-tetradecyl-N,N-dimethyl-3-ammonio-1- propanesulfonate, and sodium dodecyl sulfate (SDS).

[0020] In some embodiments, the detergent is Triton. In some embodiments, the Triton is Triton X- 100.

[0021] In some embodiments, the concentration of Triton is 10-40% m / v. In some embodiments, the concentration of Triton is 15-25% m / v. In some embodiments, Triton is 20% m / v.

[0022] In some embodiments, the reagent in step (i) further comprises an alcohol.

[0023] In some embodiments, the alcohol is ethanol.

[0024] In some embodiments, the concentration of the ethanol is 10-40% v / v. In some embodiments, the concentration of the ethanol is 25-35% v / v. In some embodiments, the concentration of the ethanol is 30% v / v. In some embodiments the reagent in step (i) comprises Triton and ethanol.

[0025] In some embodiments the reagent in step (i) comprises Triton X-100 and ethanol.

[0026] In some embodiments, the nucleic acid integrity is RNA integrity and the enzyme in step (iii) is a DNase.

[0027] In some embodiments, the nucleic acid integrity is DNA integrity and the enzyme in step (iii) is an RNase.

[0028] In some embodiments, the DNase in step (iii) is selected from the list consisting of: DNase- H, DNase I, DNase1L1, DNase 1L2, DNase1L3, DNase II a and DNase II [3.

[0029] In some embodiments, the DNase is DNase-l.

[0030] In some embodiments, the initial concentration of the stock DNase added to the purified nucleic acids is 0.1-5 ll / pl. In some embodiments, the initial concentration of the stock DNase is 0.5-2 ll / pl. In some embodiments, the initial concentration of the stock DNase is about 1 ll / pl.

[0031] In some embodiments, the concentration of the DNase in the sample in step (iii) is 0.01-1 LI / pL. In some embodiments, the concentration of the DNase in the sample in step (iii) is 0.01-0.4 LI / pL. In some embodiments, the concentration of the DNase in the sample in step (iii) is about 0.03 LI / pL.

[0032] In some embodiments, all of the DNA in step (iii) is digested by the DNase.

[0033] In some embodiments, the RNase is selected from the list consisting of RNase I, RNase II, RNase III, RNase H, RNase L, RNase P, RNase, PhyM, RNase T1, RNase T2, RNase U2, Rnase V, RNase E, RNase G, and RNase A.

[0034] In some embodiments, the RNase is RNase-A. In some embodiments, the RNaseA is Monarch® RNase A.

[0035] In some embodiments, the initial concentration of the stock RNase added to the purified nucleic acids is 1-100 mg / ml. In some embodiments, the initial concentration of the stock RNase is 10-40 mg / ml. In some embodiments, the initial concentration of the stock RNase is about 20 mg / ml.

[0036] In some embodiments, the concentration of the RNase in the sample in step (iii) is 0.1-2 mg / ml. In some embodiments, the concentration of the RNase in the sample in step (iii) is 0.1-1 mg / ml. In some embodiments, the concentration of the RNase in the sample in step

[0037] (iii) is 0.3-1 mg / ml. In some embodiments, the concentration of the RNase in the sample in step (iii) is about 0.65 mg / ml.

[0038] In some embodiments, all of the RNA in step (iii) is digested by the RNase.

[0039] In some embodiments, all of the enzyme from step (iii) is digested by the proteinase K in step (iv).

[0040] In some embodiments, the initial concentration of the stock proteinase K used in step (iv) is 10-40 U / rnL. In some embodiments, the initial concentration of the stock proteinase K used in step (iv) is 25-35 U / rnL. In some embodiments, the initial concentration of the stock proteinase K used in step (iv) is 30 U / rnL.

[0041] In some embodiments, the concentration of the proteinase K in the sample in step (iv) is 0.1- 2 U / rnL. In some embodiments, the concentration of the proteinase K in the sample in step

[0042] (iv) is about 1 U / rnL.

[0043] In some embodiments, the nucleic acid integrity measured in step (vi) is determined by analysing nucleic acid size and / or nucleic acid size distribution.

[0044] In some embodiments, the nucleic acid integrity is measured using capillary gel electrophoresis or classical gel electrophoresis. In some embodiments, the nucleic acid integrity is measured using capillary gel electrophoresis.

[0045] In some embodiments, the nucleic acid integrity is measured using a Fragment Analyser.

[0046] In some embodiments, the LNPs are functionalised LNPs comprising one or more moieties.

[0047] In some embodiments, the one or more moieties are present on the surface of the LNPs. In some embodiments, the one or more moieties comprise a targeting domain. In some embodiments, the cargo of nucleic acids comprises (i) DNA, (ii) RNA; or (iii) DNA and RNA.

[0048] In some embodiments, the cargo of nucleic acids comprises DNA and RNA.

[0049] In some embodiments, the cargo of nucleic acids comprises DNA.

[0050] In some embodiments, the cargo of nucleic acids comprises RNA.

[0051] In some embodiments, the nucleic acids purified in step (ii) are DNA or RNA.

[0052] In some embodiments, the nucleic acids purified in step (ii) are DNA.

[0053] In some embodiments, the nucleic acids purified in step (ii) are RNA.

[0054] In some embodiments, the nucleic acids purified in step (ii) are DNA and RNA.

[0055] In some embodiments, the DNA is selected from the list consisting of: genomic DNA and cDNA.

[0056] In some embodiments, the RNA is selected from the list consisting of: mRNA, miRNA, siRNA and shRNA.

[0057] The present invention also provides a method of purifying nucleic acid from a composition of LNPs comprising a cargo of nucleic acids, comprising the steps of:

[0058] (i) incubating a sample of the composition with a reagent that releases the cargo from the LNPs;

[0059] (ii) purifying the nucleic acids in the sample;

[0060] (iii) incubating the purified nucleic acids with an enzyme, wherein the enzyme is a DNase or an RNase;

[0061] (iv) incubating the result of step (iii) with proteinase K; and

[0062] (v) purifying the resulting product obtained from step (iv).

[0063] The present invention also provides a method of manufacturing a composition of lipid nanoparticles (LNPs) comprising a cargo of nucleic acids, wherein the method comprises the steps of: (i) incubating a sample of the composition with a reagent that releases the cargo from the LNPs;

[0064] (ii) purifying the nucleic acids in the sample;

[0065] (iii) incubating the purified nucleic acids with an enzyme, wherein the enzyme is a DNase or an RNase;

[0066] (iv) incubating the resulting product obtained from step (iii) with proteinase K;

[0067] (v) purifying the resulting product obtained from step (iv); and

[0068] (vi) measuring the nucleic acid integrity in the sample.

[0069] BRIEF DESCRIPTION OF THE FIGURES

[0070] Figure 1 - RNA integrity measurement after treatment of RNA with DNase and impact of LNP disruption buffer on RNA integrity after DNase treatment.

[0071] Figure 2 - Workflow for enzymatic sample preparation for RNA quality and / or integrity measurement of RNA from LNPs comprising a RNA-DNA cargo mix.

[0072] Figure 3 - Comparison of RNA integrity after treatment with or without DNase-l. Sample #1 1 #2 = RNA-DNA cargo mix encapsulated in LNPs (LNP buffer: 20 mM Hepes buffer pH 6.0) treated as described in Example 2. Sample #3 / #4 = Non-LNP encapsulated RNA-DNA cargo mix in 20 mM Hepes buffer pH 6.0 and treated such as #1 I #2. Sample #5 / #6 = RNA-DNA cargo mix encapsulated in LNPs treated as described in example 2 without the first purification step after LNP disruption. Sample #7 / #8 = Non-LNP encapsulated RNA-DNA cargo mix in 20 mM Hepes buffer pH 6.0 and treated such as #5 1 #6. Sample #9 = RNA as was used for DNA / RNA cargo samples stored in 10 mM HPESES / EDTA pH 7.1 was used as RNA control sample. Sample #10 = DNA as was used for DNA / RNA cargo samples stored nuclease free water as DNA control sample.

[0073] Figure 4 - Comparison of RNA integrity measurements of RNA released from functionalized LNPs with and without DNase digest and purification as described in example 2.

[0074] Figure 5 - Workflow for enzymatic sample preparation for DNA integrity measurement of DNA from LNPs comprising RNA-DNA cargo mix.

[0075] Figure 6 - Impact of enzymatic sample preparation for DNA or RNA integrity measurement in samples comprising a RNA-DNA cargo mix in LNPs. DETAILED DESCRIPTION OF THE INVENTION

[0076] MEASURING NUCLEIC ACID INTEGRITY

[0077] Lipid nanoparticles (LNPs) can comprise a cargo comprising a mixture of DNA and RNA as the therapeutic payload.

[0078] One challenge that arises when measuring the integrity of DNA and RNA is that the degradation products of each nucleic acid can interfere with the integrity measurement of the other. Indeed, the UV absorbance peaks and / or fluorescence signals of intercalating dyes, measured using capillary electrophoresis, for each of the measured nucleic acids can overlap, which does not allow for accurately distinguishing between the RNA and DNA.

[0079] In order to overcome this issue, samples of LNPs comprising a cargo of a mix of DNA and RNA can be obtained, wherein one sample is used to measure DNA integrity and a separate sample is used to measure RNA integrity.

[0080] When measuring the integrity of DNA, RNase enzyme may be utilised in order to remove any unwanted RNA that may be present in the sample. Likewise, when measuring the integrity of RNA, DNase enzyme may be utilised in order to remove any unwanted DNA that may be present in the sample.

[0081] Proteinase K may also be used to degrade the RNase and DNase enzymes.

[0082] Before measuring the nucleic acid integrity, the DNA and RNA cargo must first be removed from the LNPs using detergents that dissolve the lipid bilayer of the LNPs.

[0083] Although DNases do not typically degrade RNA when in pure solution, it has been found that some RNA degradation does occur when DNases interact with dissolved LNPs and detergent, ultimately affecting the measurement of RNA integrity. Likewise, RNases may also interact with dissolved LNPs and detergent and degrade the DNA, ultimately affecting the measurement of DNA integrity.

[0084] The inventors have surprisingly found that the method of the invention can be used to overcome these issues. Accordingly, the present invention provides a method of measuring nucleic acid integrity in a composition of lipid nanoparticles (LNPs) comprising a cargo of nucleic acids, wherein the method comprises the steps of:

[0085] (i) incubating a sample of the composition with a reagent that releases the cargo from the LNPs;

[0086] (ii) purifying the nucleic acids in the sample;

[0087] (iii) incubating the purified nucleic acids with an enzyme, wherein the enzyme is a DNase or an RNase;

[0088] (iv) incubating the resulting product obtained from step (iii) with proteinase K;

[0089] (v) purifying the resulting product obtained from step (iv); and

[0090] (vi) measuring the nucleic acid integrity in the sample.

[0091] In some embodiments, the method comprises the steps of:

[0092] (i) incubating a sample of the composition with a reagent that releases the cargo from the LNPs;

[0093] (ii) purifying at least some of the nucleic acids in the sample;

[0094] (iii) incubating at least some of the purified nucleic acids with an enzyme, wherein the enzyme is a DNase or an RNase;

[0095] (iv) incubating at least some of the resulting product obtained from step (iii) with proteinase K;

[0096] (v) purifying at least some of the resulting product obtained from step (iv); and

[0097] (vi) measuring the nucleic acid integrity of at least some of the nucleic acids in the sample.

[0098] Step (i)

[0099] Step (i) of the method according to of the invention is a step of incubating a sample of the composition of LNPs with a reagent that releases the cargo from the LNPs.

[0100] In some embodiments, the reagent in step (i) comprises a detergent.

[0101] It will be understood that in order to release the cargo of nucleic acids from the LNPs, the LNPs need to be exposed to a detergent. Without wishing to be bound by theory, it will be understood that the detergent may disrupt the lipid bilayer of the LNP, releasing the nucleic acid cargo from the LNPs.

[0102] Suitable detergents will be known in the art. In some embodiments, the detergent is a surfactant, such as a non-ionic surfactant, amphoteric surfactant or an anionic surfactant.

[0103] In some embodiments, the detergent is selected from the list consisting of: Triton, Zwittergent, and sodium dodecyl sulfate (SDS).

[0104] In some embodiments, the detergent is selected from the list consisting of: 2-[4-(2,4,4- trimethylpentan-2-yl)phenoxy]ethanol, N-tetradecyl-N,N-dimethyl-3-ammonio-1- propanesulfonate, and sodium dodecyl sulfate (SDS).

[0105] In some embodiments, the detergent is Triton. In some embodiments, the Triton is Triton X- 100. In some embodiments, the Triton has the formula: (Ci4H22O(C2H4O)n).

[0106] In some embodiments, the detergent is Zwittergent. In some embodiments, the Zwittergent is Zwittergent 3-14 (N-tetradecyl-N,N-dimethyl-3-ammonio-1 -propanesulfonate).

[0107] In some embodiments, the detergent is sodium dodecyl sulfate (SDS).

[0108] In some embodiments, the concentration of the detergent is 10-40% m / v.

[0109] In some embodiments, the concentration of Triton is 10-40% m / v.

[0110] In some embodiments, the concentration of the detergent is less than 10% m / v.

[0111] In some embodiments, the concentration of Triton is less than 10% m / v.

[0112] In some embodiments, the concentration of the detergent is more than 40% m / v.

[0113] In some embodiments, the concentration of Triton is more than 40% m / v.

[0114] In some embodiments, the concentration of the detergent is selected from the list consisting of: at least 10% m / v, at least 15% m / v, at least 20% m / v, at least 25% m / v, at least 30% m / v, at least 35% m / v, and at least 40% m / v.

[0115] In some embodiments, the concentration of Triton is selected from the list consisting of: at least 10% m / v, at least 15% m / v, at least 20% m / v, at least 25% m / v, at least 30% m / v, at least 35% m / v, and at least 40% m / v. In some embodiments, the concentration of the detergent is selected from the list consisting of: about 10% m / v, about 15% m / v, about 20% m / v, about 25% m / v, about 30% m / v, about 35% m / v, and about 40% m / v.

[0116] In some embodiments, the concentration of Triton is selected from the list consisting of: about 10% m / v, about 15% m / v, about 20% m / v, about 25% m / v, about 30% m / v, about 35% m / v, and about 40% m / v.

[0117] In some embodiments, the concentration of the detergent is selected from the list consisting of: 10-35% m / v, 10-30% m / v, 10-25% m / v, 10-20% m / v, and 10-15% m / v.

[0118] In some embodiments, the concentration of Triton is selected from the list consisting of: 10- 35% m / v, 10-30% m / v, 10-25% m / v, 10-20% m / v, and 10-15% m / v.

[0119] In some embodiments, the concentration of the detergent is selected from the list consisting of: 15-40% m / v, 20-40% m / v, 25-40% m / v, 30-40% m / v, and 35-40% m / v.

[0120] In some embodiments, the concentration of Triton is selected from the list consisting of: 15- 40% m / v, 20-40% m / v, 25-40% m / v, 30-40% m / v, and 35-40% m / v.

[0121] In some embodiments, the concentration of the detergent is 15-25% m / v.

[0122] In some embodiments, the concentration of Triton is 15-25% m / v.

[0123] In some embodiments, the concentration of the detergent is selected from the list consisting of about: 15% m / v, 16% m / v, 17% m / v, 18% m / v, 19% m / v, 20% m / v, 21% m / v, 22% m / v, 23% m / v, 24% m / v, and 25% m / v.

[0124] In some embodiments, the concentration of Triton is selected from the list consisting of about: 15% m / v, 16% m / v, 17% m / v, 18% m / v, 19% m / v, 20% m / v, 21% m / v, 22% m / v, 23% m / v, 24% m / v, and 25% m / v.

[0125] In some embodiments, the concentration of the detergent is about 20% m / v.

[0126] In some embodiments, the concentration of Triton is about 20% m / v. It will be understood that m / v (or mass / volume percent) is defined as the ratio of the mass of a solute relative to the volume of a solution.

[0127] It will be understood that as well as a detergent as described herein, the reagent in step (i) may further comprise other components.

[0128] In some embodiments, the reagent in step (i) further comprises an alcohol.

[0129] In other embodiments, the reagent in step (i) may not comprise an alcohol.

[0130] Suitable alcohols will be known in the art.

[0131] In some embodiments, the alcohol is ethanol.

[0132] In some embodiments, the concentration of the alcohol is 0-40% v / v.

[0133] In some embodiments, the concentration of ethanol is 0-40% v / v.

[0134] In some embodiments, the concentration of the alcohol is 10-40% v / v.

[0135] In some embodiments, the concentration of ethanol is 10-40% v / v.

[0136] In some embodiments, the concentration of the alcohol is less than 10% v / v.

[0137] In some embodiments, the concentration of ethanol is less than 10% v / v.

[0138] In some embodiments, the concentration of the alcohol is 0-10% v / v.

[0139] In some embodiments, the concentration of ethanol is 0-10% v / v.

[0140] In some embodiments, the concentration of the alcohol is 0% v / v.

[0141] In some embodiments, the concentration of ethanol is 0% v / v.

[0142] In some embodiments, the concentration of the alcohol is more than 40% v / v. In some embodiments, the concentration of ethanol is more than 40% v / v.

[0143] In some embodiments, the concentration of the alcohol is selected from the list consisting of: at least 10% v / v, at least 15% v / v, at least 20% v / v, at least 25% v / v, at least 30% v / v, at least 35% v / v, and at least 40% v / v.

[0144] In some embodiments, the concentration of ethanol is selected from the list consisting of: at least 10% v / v, at least 15% v / v, at least 20% v / v, at least 25% v / v, at least 30% v / v, at least 35% v / v, and at least 40% v / v.

[0145] In some embodiments, the concentration of the alcohol is selected from the list consisting of: about 10% v / v, about 15% v / v, about 20% v / v, about 25% v / v, about 30% v / v, about 35% v / v, and about 40% v / v.

[0146] In some embodiments, the concentration of ethanol is selected from the list consisting of: about 10% v / v, about 15% v / v, about 20% v / v, about 25% v / v, about 30% v / v, about 35% v / v, and about 40% v / v.

[0147] In some embodiments, the concentration of the alcohol is selected from the list consisting of: 10-35% v / v, 10-30% v / v, 10-25% v / v, 10-20% v / v, and 10-15% v / v.

[0148] In some embodiments, the concentration of ethanol is selected from the list consisting of: 10- 35% v / v, 10-30% v / v, 10-25% v / v, 10-20% v / v, and 10-15% v / v.

[0149] In some embodiments, the concentration of the alcohol is selected from the list consisting of: 15-40% v / v, 20-40% v / v, 25-40% v / v, 30-40% v / v, and 35-40% v / v.

[0150] In some embodiments, the concentration of ethanol is selected from the list consisting of: 15- 40% v / v, 20-40% v / v, 25-40% v / v, 30-40% v / v, and 35-40% v / v.

[0151] In some embodiments, the concentration of the alcohol is 25-35% v / v.

[0152] In some embodiments, the concentration of ethanol is 25-35% v / v.

[0153] In some embodiments, the concentration of the alcohol is selected from the list consisting of about: 25% v / v, 26% v / v, 27% v / v, 28% v / v, 29% v / v, 30% v / v, 31% v / v, 32% v / v, 33% v / v, 34% v / v, and 35% v / v. In some embodiments, the concentration of ethanol is selected from the list consisting of about: 25% v / v, 26% v / v, 27% v / v, 28% v / v, 29% v / v, 30% v / v, 31% v / v, 32% v / v, 33% v / v, 34% v / v, and 35% v / v.

[0154] In some embodiments, the concentration of the alcohol is about 30% v / v.

[0155] In some embodiments, the concentration of ethanol is about 30% v / v.

[0156] It will be understood that v / v (or volume / volume percent) is defined as the ratio of the concentration of a substance in a solution, expressed relative to the total volume of the solution.

[0157] In some embodiments the reagent in step (i) comprises Triton and ethanol. Within this embodiment the Triton and ethanol may each independently be present in any of the concentrations discussed above.

[0158] In some embodiments the concentration of Triton is about 20% m / v and the concentration of ethanol is about 30% v / v.

[0159] In some embodiments, the incubation in step (i) may be performed at about 18°C to about 50°C.

[0160] In some embodiments, the incubation in step (i) may be performed at about 20°C to about 40°C.

[0161] In some embodiments, the incubation in step (i) may be performed at less than 18°C.

[0162] In some embodiments, the incubation in step (i) may be performed at more than 40°C.

[0163] In some embodiments, the incubation in step (i) may be performed at more than 50°C.

[0164] In some embodiments, the incubation in step (i) may be performed at about 18°C, about 19°C, about 20°C, about 21°C, about 22°C, about 23°C, about 24°C, about 25°C, about 26°C, about 27°C, about 28°C, about 29°C, about 30°C, about 31 °C, about 32°C, about 33°C, about 34°C, about 35°C, about 36°C, about 37°C, about 38°C, about 39°C, about 40°C, about 41 °C, about 42°C, about 43°C, about 44°C, about 45°C, about 46°C, about 47°C, about 48°C, about 49°C, or about 50°C .

[0165] In some embodiments, the incubation in step (i) may be performed at room temperature. It will be understood that room temperature may be considered as a temperature in the range of 18- 25°C, such as about 20°C, about 21 °C or about 22°C.

[0166] In some embodiments, the incubation in step (i) may be performed at 18-20°C.

[0167] In some embodiments, the incubation in step (i) may be performed at 20-50°C. In some embodiments, the incubation in step (i) may be performed at 30-50°C. In some embodiments, the incubation in step (i) may be performed at 40-50°C.

[0168] In some embodiments, the incubation in step (i) may be performed at 20-35°C. In some embodiments, the incubation in step (i) may be performed at 20-30°C. In some embodiments, the incubation in step (i) may be performed at 20-25°C.

[0169] In some embodiments, the incubation in step (i) may be performed at 25-40°C. In some embodiments, the incubation in step (i) may be performed at 30-40°C. In some embodiments, the incubation in step (i) may be performed at 35-40°C.

[0170] In some embodiments, the incubation in step (i) may be performed at about 37°C.

[0171] In some embodiments, the incubation in step (i) may be performed for about 0 minutes to about 60 minutes.

[0172] In some embodiments, the incubation in step (i) may be performed for about 1 minute to about 60 minutes.

[0173] In some embodiments, the incubation in step (i) may be performed for about 1 minute to about 40 minutes.

[0174] In some embodiments, the incubation in step (i) may be performed for more than 60 minutes.

[0175] In some embodiments, the incubation in step (i) may be performed for less than 10 minutes.

[0176] In some embodiments, the incubation in step (i) may be performed for less than 1 minute. In some embodiments, the incubation in step (i) may be performed for about 1 to about 5 minutes, about 5 to about 10 minutes, about 10 to about 15 minutes, about 15 to about 20 minutes, about 20 to about 25 minutes, about 25 to about 30 minutes, about 30 to about 35 minutes, about 35 to about 40 minutes, about 40 to about 45 minutes, about 45 to about 50 minutes, about 50 to about 55 minutes, or about 55 to about 60 minutes.

[0177] In some embodiments, the incubation in step (i) may be performed for about 10 minutes, about 15 minutes, about 20 minutes, about 25 minutes, about 30 minutes, about 35 minutes, about 40 minutes, about 45 minutes, about 50 minutes, or about 55 minutes.

[0178] In some embodiments, the incubation in step (i) may be performed for 0-40 minutes. In some embodiments, the incubation in step may be performed for 5-40 minutes. In some embodiments, the incubation in step may be performed for 10-40 minutes. In some embodiments, the incubation in step may be performed for 15-40 minutes. In some embodiments, the incubation in step may be performed for 20-40 minutes. In some embodiments, the incubation in step may be performed for 25-40 minutes. In some embodiments, the incubation in step may be performed for 30-40 minutes. In some embodiments, the incubation in step (i) may be performed for 35-40 minutes.

[0179] In some embodiments, the incubation in step (i) may be performed for 0-35 minutes. In some embodiments, the incubation in step (i) may be performed for 0-30 minutes. In some embodiments, the incubation in step (i) may be performed for 0-25 minutes. In some embodiments, the incubation in step (i) may be performed for 0-20 minutes. In some embodiments, the incubation in step (i) may be performed for 0-15 minutes. In some embodiments, the incubation in step (i) may be performed for 0-10 minutes. In some embodiments, the incubation in step (i) may be performed for 0-5 minutes.

[0180] In some embodiments, the incubation in step (i) may be performed for about 20 minutes to about 40 minutes.

[0181] In some embodiments, the incubation in step (i) may be performed for about 15 minutes to about 30 minutes.

[0182] In some embodiments, the incubation in step (i) may be performed for about 10 minutes to about 20 minutes. In some embodiments, the incubation in step (i) may be performed for about 10 minutes to about 15 minutes. In some embodiments, the incubation in step (i) may be performed for about 15 minutes to about 20 minutes.

[0183] In some embodiments, the incubation in step (i) may be performed for about 20 minutes.

[0184] In some embodiments, the incubation in step (i) may be performed for about 10 minutes.

[0185] In some embodiments, the incubation in step (i) may be performed for

[0186] (a) about 0 minutes to about 40 minutes; and

[0187] (b) at a temperature of about 18°C to about 50°C.

[0188] In some embodiments, the incubation in step (i) may be performed for about 10 minutes at a temperature of about 18°C to about 50°C.

[0189] In some embodiments, the incubation in step (i) may be performed for about 20 minutes at a temperature of about 18°C to about 50°C.

[0190] In some embodiments, the incubation in step (i) may be performed for about 10 minutes to about 20 minutes at a temperature of at 30-40°C.

[0191] In some embodiments, the incubation in step (i) may be performed for about 10 minutes to about 20 minutes at a temperature of about 37°C.

[0192] In some embodiments, the incubation in step (i) may be performed for about 10 minutes at a temperature of at 30-40°C.

[0193] In some embodiments, the incubation in step (i) may be performed for about 10 minutes at a temperature of about 37°C.

[0194] In some embodiments, the incubation in step (i) may be performed for about 20 minutes at a temperature of at 30-40°C.

[0195] In some embodiments, the incubation in step (i) may be performed for about 20 minutes at a temperature of about 37°C. It will be understood that the amount of reagent, duration, and temperature selected for step (i) may be modified as necessary, which will be within the remit of the person skilled in the art of releasing cargo from an LNP. Indeed, the person skilled in the art will know that e.g. a lower amount of reagent may require a longer incubation duration and / or higher temperature.

[0196] Step (ii)

[0197] Step (ii) of the method according to the invention is a step of purifying the nucleic acids in the sample.

[0198] In some embodiments, step (ii) comprises a step of purifying at least some of the nucleic acids in the sample. In some embodiments, step (ii) comprises a step of purifying all of the nucleic acids in the sample.

[0199] Suitable methods for purifying nucleic acids will be known in the art.

[0200] In some embodiments, the purification in step (ii) is performed using a commercially available RNA isolation kit according to the manufacturer’s instructions. In some embodiments, the RNA isolation kit is the RNeasy Mini Kit (QIAGEN GmbH). In some embodiments, the RNA isolation kit is the RNeasy Midi Kit (QIAGEN GmbH). In some embodiments, the RNA isolation kit is RNeasy Maxi Kit (QIAGEN GmbH).

[0201] It will also be understood that the protocols for using the RNeasy Kits described above (QIAGEN GmbH) may be modified as necessary, which will be within the remit of the person skilled in the art.

[0202] It will also be understood that the person skilled in the art is well aware that there are other commercially available nucleic acid isolation kits for the same purpose available from other manufacturers, which can be used to perform the present invention.

[0203] The person skilled in the art is also aware that there are alternative methods for performing RNA isolation, for example by using automised column based solutions or bead-based nucleic acid extraction methods.

[0204] In some embodiments, the sample volume is 10 pL - 15000 pL.

[0205] In some embodiments, the purified nucleic acids are eluted in nuclease free water. In some embodiments, the purified nucleic acids are eluted in RNase free water.

[0206] In some embodiments, the purified nucleic acids are eluted in DNase free water.

[0207] In some embodiments, the purification step of step (ii) may be repeated prior to step (iii). It will be understood that repeating the purification step may increase the purity of the purified nucleic acids.

[0208] In some embodiments, the purification step of step (ii) may be repeated two, three, four, five or more times.

[0209] Step (iii)

[0210] Step (iii) of the method according to the invention is a step of incubating the purified nucleic acids with an enzyme, wherein the enzyme is a DNase or an RNase.

[0211] In some embodiments, the incubation step of step (iii) may comprise incubating the purified nucleic acids and the enzyme in the presence of a reaction buffer, such as a buffer comprising bivalent cations.

[0212] In some embodiments, the reaction buffer may comprise magnesium chloride (MgCh).

[0213] In some embodiments, the reaction buffer may comprise TRIS buffer.

[0214] In some embodiments, the reaction buffer may comprise 10x TRIS MgCh.

[0215] In some embodiments, step (iii) may comprise a step of incubating at least some of the purified nucleic acids obtained from step (ii) with a DNase or an RNase. In some embodiments, step (iii) may comprise a step of incubating all of the purified nucleic acids obtained from step (ii) with a DNase or an RNase.

[0216] It will be understood that the enzyme used in step (iii) will depend on the particular nucleic acid for which integrity is to be measured.

[0217] In some embodiments, the nucleic acid integrity to be measured is RNA integrity and the enzyme in step (iii) is a DNase.

[0218] In some embodiments the enzyme in step (iii) is a DNase. In some embodiments where the nucleic acid integrity to be measured is RNA integrity, the method is performed in the presence of an RNase inhibitor.

[0219] In some embodiments, the RNase inhibitor is a non protein-based RNase inhibitor.

[0220] In some embodiments the method is performed in the presence of an RNase inhibitor.

[0221] In some embodiments where the nucleic acid integrity to be measured is RNA integrity, the method is performed in RNase-free conditions.

[0222] In some embodiments the method is performed in RNase-free conditions.

[0223] In some embodiments, the DNase in step (iii) is selected from the list consisting of: DNase-H, DNase I, DNase1 L1 , DNase 1L2, DNase1L3, DNase II a and DNase II [3.

[0224] In some embodiments, the DNase in step (iii) is DNase-l.

[0225] In some embodiments, the initial concentration of the stock DNase used in step (iii) is 0.1-5 LI / pL. In some embodiments, the initial concentration of the stock DNase used in step (iii) is 0.5-2 U / pL. In some embodiments, the initial concentration of the stock DNase used in step (iii) is about 1 LI / pL.

[0226] In some embodiments, the final concentration of the DNase in the sample in step (iii) is 0.01- 1 LI / pL. In some embodiments, the final concentration of the DNase in the sample in step (iii) is 0.01-0.1 LI / pL. In some embodiments, the final concentration of the DNase in the sample in step (iii) is 0.1-1 LI / pL.

[0227] In some embodiments, the final concentration of the DNase in the sample in step (iii) is 0.01- 0.4 U / pL.

[0228] In some embodiments, the final concentration of the DNase in the sample in step (iii) is about 0.03 U / pL.

[0229] In some embodiments, at least some of the DNA in step (iii) is digested by the DNase.

[0230] In some embodiments, all of the DNA in step (iii) is digested by the DNase. In some other embodiments, the nucleic acid integrity to be measured is DNA integrity and the enzyme in step (iii) is an RNase.

[0231] In some embodiments the enzyme in step (iii) is an RNase.

[0232] In some embodiments where the nucleic acid integrity to be measured is DNA integrity, the method is performed in the presence of a DNase inhibitor.

[0233] In some embodiments the method is performed in the presence of a DNase inhibitor.

[0234] In some embodiments where the nucleic acid integrity to be measured is DNA integrity, the method is performed in DNase-free conditions.

[0235] In some embodiments the method is performed in DNase-free conditions.

[0236] In some embodiments, the RNase in step (iii) is selected from the list consisting of RNase I, RNase II, RNase III, RNase H, RNase L, RNase P, RNase, PhyM, RNase T1 , RNase T2, RNase U2, Rnase V, RNase E, RNase G, RNase A and Monarch RNase A.

[0237] In some embodiments, the RNase in step (iii) is an RNase A. In some embodiments, the RNase in step (iii) is Monarch RNase A.

[0238] In some embodiments, the initial concentration of the stock RNase used in step (iii) is 1-100 mg / ml. In some embodiments, the initial concentration of the stock RNase used in step (iii) is- 10-40 mg / ml. In some embodiments, the initial concentration of the stock RNase used in step (iii) is about 20 mg / ml.

[0239] In some embodiments, the final concentration of the RNase in the sample in step (iii) is 0.1-2 mg / ml. In some embodiments, the final concentration of the RNase in the sample in step (iii) is 0.1-1 mg / ml.

[0240] In some embodiments, the final concentration of the RNase in the sample in step (iii) is 0.1- 0.8 mg / ml. In some embodiments, the final concentration of the RNase in the sample in step (iii) is 0.5-1 mg / ml.

[0241] In some embodiments, the final concentration of the RNase in the sample in step (iii) is about 0.65 mg / ml. In some embodiments, at least some of the RNA in step (iii) is digested by the RNase.

[0242] In some embodiments, all of the RNA in step (iii) is digested by the RNase.

[0243] In some embodiments, the incubation in step (iii) may be performed at about 18°C to about 50°C.

[0244] In some embodiments, the incubation in step (iii) may be performed at about 20°C to about 40°C.

[0245] In some embodiments, the incubation in step (iii) may be performed at less than 18°C.

[0246] In some embodiments, the incubation in step (iii) may be performed at more than 40°C.

[0247] In some embodiments, the incubation in step (iii) may be performed at more than 50°C.

[0248] In some embodiments, the incubation in step (iii) may be performed at about 18°C, about 19°C, about 20°C, about 21°C, about 22°C, about 23°C, about 24°C, about 25°C, about 26°C, about 27°C, about 28°C, about 29°C, about 30°C, about 31 °C, about 32°C, about 33°C, about 34°C, about 35°C, about 36°C, about 37°C, about 38°C, about 39°C, about 40°C, about 41°C, about 42°C, about 43°C, about 44°C, about 45°C, about 46°C, about 47°C, about 48°C, about 49°C, or about 50°C.

[0249] In some embodiments, the incubation in step (iii) may be performed at room temperature. It will be understood that room temperature may be considered as a temperature in the range of 18-25°C, such as about 20°C, about 21°C or about 22°C.

[0250] In some embodiments, the incubation in step (iii) may be performed at 18-20°C.

[0251] In some embodiments, the incubation in step (iii) may be performed at 20-50°C. In some embodiments, the incubation in step (iii) may be performed at 30-50°C. In some embodiments, the incubation in step (iii) may be performed at 40-50°C.

[0252] In some embodiments, the incubation in step (iii) may be performed at 20-35°C. In some embodiments, the incubation in step (iii) may be performed at 20-30°C. In some embodiments, the incubation in step (iii) may be performed at 20-25°C. In some embodiments, the incubation in step (iii) may be performed at 25-40°C. In some embodiments, the incubation in step (iii) may be performed at 30-40°C. In some embodiments, the incubation in step (iii) may be performed at 35-40°C.

[0253] In some embodiments, the incubation in step (iii) may be performed at 37°C.

[0254] In some embodiments, the incubation in step (iii) may be performed for about 0 minutes to about 60 minutes.

[0255] In some embodiments, the incubation in step (iii) may be performed for about 1 minute to about 60 minutes.

[0256] In some embodiments, the incubation in step (iii) may be performed for about 1 minute to about 40 minutes.

[0257] In some embodiments, the incubation in step (iii) may be performed for more than 60 minutes.

[0258] In some embodiments, the incubation in step (iii) may be performed for less than 10 minutes.

[0259] In some embodiments, the incubation in step (iii) may be performed for less than 1 minute.

[0260] In some embodiments, the incubation in step (iii) may be performed for about 1 to about 5 minutes, about 5 to about 10 minutes, about 10 to about 15 minutes, about 15 to about 20 minutes, about 20 to about 25 minutes, about 25 to about 30 minutes, about 30 to about 35 minutes, about 35 to about 40 minutes, about 40 to about 45 minutes, about 45 to about 50 minutes, about 50 to about 55 minutes, or about 55 to about 60 minutes.

[0261] In some embodiments, the incubation in step (iii) may be performed for about 10 minutes, about 15 minutes, about 20 minutes, about 25 minutes, about 30 minutes, about 35 minutes, about 40 minutes, about 45 minutes, about 50 minutes, or about 55 minutes.

[0262] In some embodiments, the incubation in step (iii) may be performed for 0-40 minutes. In some embodiments, the incubation in step (iii) may be performed for 5-40 minutes. In some embodiments, the incubation in step (iii) may be performed for 10-40 minutes. In some embodiments, the incubation in step (iii) may be performed for 15-40 minutes. In some embodiments, the incubation in step (iii) may be performed for 20-40 minutes. In some embodiments, the incubation in step (iii) may be performed for 25-40 minutes. In some embodiments, the incubation in step (iii) may be performed for 30-40 minutes. In some embodiments, the incubation in step (iii) may be performed for 35-40 minutes.

[0263] In some embodiments, the incubation in step (iii) may be performed for 0-35 minutes. In some embodiments, the incubation in step may be performed for 0-30 minutes. In some embodiments, the incubation in step may be performed for 0-25 minutes. In some embodiments, the incubation in step may be performed for 0-20 minutes. In some embodiments, the incubation in step may be performed for 0-15 minutes. In some embodiments, the incubation in step may be performed for 0-10 minutes. In some embodiments, the incubation in step (iii) may be performed for 0-5 minutes.

[0264] In some embodiments, the incubation in step (iii) may be performed for about 20 minutes to about 40 minutes.

[0265] In some embodiments, the incubation in step (iii) may be performed for about 15 minutes to about 30 minutes.

[0266] In some embodiments, the incubation in step (iii) may be performed for about 10 minutes to about 20 minutes.

[0267] In some embodiments, the incubation in step (iii) may be performed for about 10 minutes to about 15 minutes. In some embodiments, the incubation in step (iii) may be performed for about 15 minutes to about 20 minutes.

[0268] In some embodiments, the incubation in step (iii) may be performed for about 20 minutes.

[0269] In some embodiments, the incubation in step (iii) may be performed for about 10 minutes.

[0270] In some embodiments, the incubation in step (iii) may be performed for

[0271] (a) about 10 minutes to about 40 minutes; and

[0272] (b) at a temperature of about 18°C to about 50°C.

[0273] In some embodiments, the incubation in step (iii) may be performed for about 10 minutes at a temperature of about 18°C to about 50°C. In some embodiments, the incubation in step (iii) may be performed for about 20 minutes at a temperature of about 18°C to about 50°C.

[0274] In some embodiments, the incubation in step (iii) may be performed for about 10 minutes to about 20 minutes at a temperature of at 30-40°C.

[0275] In some embodiments, the incubation in step (iii) may be performed for about 10 minutes to about 20 minutes at a temperature of about 37°C.

[0276] In some embodiments, the incubation in step (iii) may be performed for about 10 minutes at a temperature of at 30-40°C.

[0277] In some embodiments, the incubation in step (iii) may be performed for about 10 minutes at a temperature of about 37°C.

[0278] In some embodiments, the incubation in step (iii) may be performed for about 20 minutes at a temperature of at 30-40°C.

[0279] In some embodiments, the incubation in step (iii) may be performed for about 20 minutes at a temperature of about 37°C.

[0280] It will be understood that the enzyme concentration, duration, and temperature selected for step (iii) may be modified as necessary, which will be within the remit of the person skilled in the art of nucleic acid degradation. Indeed, the person skilled in the art will know that e.g. a lower enzyme concentration may require a longer incubation duration and / or higher temperature.

[0281] Step (iv)

[0282] Step (iv) of the method according to of the invention is a step of incubating the resulting product obtained from step (iii) with proteinase K.

[0283] It will be understood that the proteinase K will digest the DNase or RNase from step (iii).

[0284] In some embodiments, the proteinase K will digest the DNase from step (iii).

[0285] In some embodiments, the proteinase K will digest the RNase from step (iii). It will be also understood that the proteinase K may digest proteins present as part of the LNPs, such as proteins present on the surface of the LNPs (e.g. functionalised LNPs).

[0286] In some embodiments, step (iv) comprises a step of incubating at least some of the resulting product obtained from step (iii) with proteinase K.

[0287] In some embodiments, step (iv) comprises a step of incubating all of the resulting product obtained from step (iii) with proteinase K.

[0288] In some embodiments, at least some of the DNase enzyme from step (iii) is digested by the proteinase K in step (iv).

[0289] In some embodiments, all of the DNase enzyme from step (iii) is digested by the proteinase K in step (iv).

[0290] In some embodiments, at least some of the RNase enzyme from step (iii) is digested by the proteinase K in step (iv).

[0291] In some embodiments, all of the RNase enzyme from step (iii) is digested by the proteinase K in step (iv).

[0292] In some embodiments, the initial concentration of the stock proteinase K used in step (iv) is 10-40 U / rnL. In some embodiments, the initial concentration of the stock proteinase K used in step (iv) is 25-35 U / rnL. In some embodiments, the initial concentration of the stock proteinase K used in step (iv) is about 30 U / mL.

[0293] In some embodiments, the final concentration of the proteinase K in the sample in step (iv) is 0.1-5 U / mL.

[0294] In some embodiments, the final concentration of the proteinase K in the sample in step (iv) is 0.1-2 U / mL.

[0295] In some embodiments, the final concentration of the proteinase K in the sample in step (iv) is 0.5-2 U / mL.

[0296] In some embodiments, the final concentration of the proteinase K in the sample in step (iv) is about 1 U / mL. In some embodiments, the incubation in step (iv) may be performed at about 18°C to about 50°C.

[0297] In some embodiments, the incubation in step (iv) may be performed at about 20°C to about 40°C.

[0298] In some embodiments, the incubation in step (iv) may be performed at less than 18°C.

[0299] In some embodiments, the incubation in step (iv) may be performed at more than 40°C.

[0300] In some embodiments, the incubation in step (iv) may be performed at more than 50°C.

[0301] In some embodiments, the incubation in step (iv) may be performed at about 18°C, about

[0302] 19°C, about 20°C, about 21°C, about 22°C, about 23°C, about 24°C, about 25°C, about 26°C, about 27°C, about 28°C, about 29°C, about 30°C, about 31 °C, about 32°C, about 33°C, about 34°C, about 35°C, about 36°C, about 37°C, about 38°C, about 39°C, about 40°C, about 41°C, about 42°C, about 43°C, about 44°C, about 45°C, about 46°C, about 47°C, about 48°C, about 49°C, or about 50°C.

[0303] In some embodiments, the incubation in step (iv) may be performed at room temperature. It will be understood that room temperature may be considered as a temperature in the range of 18-25°C, such as about 20°C, about 21°C or about 22°C.

[0304] In some embodiments, the incubation in step (iv) may be performed at 18-20°C.

[0305] In some embodiments, the incubation in step (iv) may be performed at 20-50°C. In some embodiments, the incubation in step (iv) may be performed at 30-50°C. In some embodiments, the incubation in step (iv) may be performed at 40-50°C.

[0306] In some embodiments, the incubation in step (iv) may be performed at 20-35°C. In some embodiments, the incubation in step (iv) may be performed at 20-30°C. In some embodiments, the incubation in step (iv) may be performed at 20-25°C.

[0307] In some embodiments, the incubation in step (iv) may be performed at 25-40°C. In some embodiments, the incubation in step (iv) may be performed at 30-40°C. In some embodiments, the incubation in step (iv) may be performed at 35-40°C. In some embodiments, the incubation in step (iv) may be performed at 37°C.

[0308] In some embodiments, the incubation in step (iv) may be performed for about 0 minutes to about 60 minutes.

[0309] In some embodiments, the incubation in step (iv) may be performed for about 1 minute to about 60 minutes.

[0310] In some embodiments, the incubation in step (iv) may be performed for about 1 minute to about 40 minutes.

[0311] In some embodiments, the incubation in step (iv) may be performed for more than 60 minutes.

[0312] In some embodiments, the incubation in step (iv) may be performed for less than 10 minutes.

[0313] In some embodiments, the incubation in step (iv) may be performed for less than 1 minute.

[0314] In some embodiments, the incubation in step (iv) may be performed for about 1 to about 5 minutes, about 5 to about 10 minutes, about 10 to about 15 minutes, about 15 to about 20 minutes, about 20 to about 25 minutes, about 25 to about 30 minutes, about 30 to about 35 minutes, about 35 to about 40 minutes, about 40 to about 45 minutes, about 45 to about 50 minutes, about 50 to about 55 minutes, or about 55 to about 60 minutes.

[0315] In some embodiments, the incubation in step (iv) may be performed for about 10 minutes, about 15 minutes, about 20 minutes, about 25 minutes, about 30 minutes, about 35 minutes, about 40 minutes, about 45 minutes, about 50 minutes, or about 55 minutes.

[0316] In some embodiments, the incubation in step (iv) may be performed for 0-40 minutes. In some embodiments, the incubation in step (iv) may be performed for 5-40 minutes. In some embodiments, the incubation in step (iv) may be performed for 10-40 minutes. In some embodiments, the incubation in step (iv) may be performed for 15-40 minutes. In some embodiments, the incubation in step (iv) may be performed for 20-40 minutes. In some embodiments, the incubation in step (iv) may be performed for 25-40 minutes. In some embodiments, the incubation in step (iv) may be performed for 30-40 minutes. In some embodiments, the incubation in step (iv) may be performed for 35-40 minutes. In some embodiments, the incubation in step (iv) may be performed for 0-35 minutes. In some embodiments, the incubation in step (iv) may be performed for 0-30 minutes. In some embodiments, the incubation in step (iv) may be performed for 0-25 minutes. In some embodiments, the incubation in step (iv) may be performed for 0-20 minutes. In some embodiments, the incubation in step (iv) may be performed for 0-15 minutes. In some embodiments, the incubation in step (iv) may be performed for 0-10 minutes. In some embodiments, the incubation in step (iv) may be performed for 0-5 minutes.

[0317] In some embodiments, the incubation in step (iv) may be performed for about 20 minutes to about 40 minutes.

[0318] In some embodiments, the incubation in step (iv) may be performed for about 15 minutes to about 30 minutes.

[0319] In some embodiments, the incubation in step (iv) may be performed for about 10 minutes to about 20 minutes.

[0320] In some embodiments, the incubation in step (iv) may be performed for about 10 minutes to about 15 minutes. In some embodiments, the incubation in step (iv) may be performed for about 15 minutes to about 20 minutes.

[0321] In some embodiments, the incubation in step (iv) may be performed for about 20 minutes.

[0322] In some embodiments, the incubation in step (iv) may be performed for about 10 minutes.

[0323] In some embodiments, the incubation in step (iv) may be performed for

[0324] (a) about 10 minutes to about 40 minutes; and

[0325] (b) at a temperature of about 18°C to about 50°C.

[0326] In some embodiments, the incubation in step (iv) may be performed for about 10 minutes at a temperature of about 18°C to about 50°C.

[0327] In some embodiments, the incubation in step (iv) may be performed for about 20 minutes at a temperature of about 18°C to about 50°C.

[0328] In some embodiments, the incubation in step (iv) may be performed for about 10 minutes to about 20 minutes at a temperature of at 30-40°C. In some embodiments, the incubation in step (iv) may be performed for about 10 minutes to about 20 minutes at a temperature of about 37°C.

[0329] In some embodiments, the incubation in step (iv) may be performed for about 10 minutes at a temperature of at 30-40°C.

[0330] In some embodiments, the incubation in step (iv) may be performed for about 10 minutes at a temperature of about 37°C.

[0331] In some embodiments, the incubation in step (iv) may be performed for about 20 minutes at a temperature of at 30-40°C.

[0332] In some embodiments, the incubation in step (iv) may be performed for about 20 minutes at a temperature of about 37°C.

[0333] It will be understood that the proteinase K concentration, duration, and temperature selected for step (iv) may be modified as necessary, which will be within the remit of the person skilled in the art. Indeed, the person skilled in the art will know that e.g. a lower proteinase K concentration may require a longer incubation duration and / or higher temperature.

[0334] Step (v)

[0335] Step (v) of the method according to of the invention is a step of purifying the resulting product obtained from step (iv).

[0336] It will be understood that step (v) is a purification step that is in addition to the purification step of step (ii).

[0337] Step (ii) and step (v) are independent such that the parameters of these steps may be independently selected.

[0338] In some embodiments, step (v) comprises a step of purifying at least some of the resulting product obtained from step (iv).

[0339] In some embodiments, step (v) comprises a step of purifying all of the resulting product obtained from step (iv). Suitable methods for purifying nucleic acids will be known in the art.

[0340] In some embodiments, the purification in step (v) is performed using a commercially available RNA isolation kit according to the manufacturer’s instructions. In some embodiments, the RNA isolation kit is the RNeasy Mini Kit (QIAGEN GmbH). In some embodiments, the RNA isolation kit is the RNeasy Midi Kit (QIAGEN GmbH). In some embodiments, the RNA isolation kit is RNeasy Maxi Kit (QIAGEN GmbH).

[0341] It will also be understood that the protocols for using the RNeasy Kits described above (QIAGEN GmbH) may be modified as necessary, which will be within the remit of the person skilled in the art.

[0342] It will also be understood that the person skilled in the art is well aware that there are other commercially available nucleic acid isolation kits for the same purpose available from other manufacturers, which can be used to perform the present invention.

[0343] The person skilled in the art is also aware that there are alternative methods for performing RNA isolation, for example by using automised column based solutions or bead-based nucleic acid extraction methods.

[0344] In some embodiments, the sample volume is 10 pL - 15000 pL.

[0345] In some embodiments, the purified nucleic acids are eluted in nuclease free water.

[0346] In some embodiments, the purified nucleic acids are eluted in RNase free water.

[0347] In some embodiments, the purified nucleic acids are eluted in DNase free water.

[0348] In some embodiments, the purification step of step (v) may be repeated prior to step (vi). It will be understood that repeating the purification step may increase the purity of the purified nucleic acids.

[0349] In some embodiments, the purification step of step (v) may be repeated two, three, four, five or more times.

[0350] Step (vi) Step (vi) of the method according to of the invention is a step of measuring the nucleic acid integrity in the sample.

[0351] In some embodiments, step (vi) comprises a step of measuring the nucleic acid integrity of at least some of the nucleic acids in the sample.

[0352] In some embodiments, step (vi) comprises a step of measuring the nucleic acid integrity of all of the nucleic acids in the sample.

[0353] In some embodiments, the nucleic acid integrity measured in step (vi) is determined by analysing nucleic acid size and / or nucleic acid size distribution.

[0354] In some embodiments, the nucleic acid integrity measured in step (vi) is determined by analysing the nucleic acid profile.

[0355] In some embodiments, the nucleic acid integrity is measured using capillary gel electrophoresis or classical gel electrophoresis.

[0356] In some embodiments, the nucleic acid integrity is measured using classical gel electrophoresis.

[0357] In some embodiments, the nucleic acid integrity is measured using capillary gel electrophoresis

[0358] In some embodiments, the nucleic acid integrity is measured using a Fragment Analyser.

[0359] It will be understood that the nucleic acid integrity can be measured using a Fragment Analyser according to the manufacturer’s instructions.

[0360] It will also be understood that the protocol for using the Fragment Analyser may be modified as necessary, which will be within the remit of the person skilled in the art of nucleic acid integrity measurement. The protocol for using the Fragment Analyser may be performed as outlined in the Examples.

[0361] In some embodiments, the concentration of RNA to be measured is 0.001-1000 pg / mL. In some embodiments, the concentration of RNA to be measured is 0.01-1000 pg / mL. In some embodiments, the concentration of RNA to be measured is 0.1-1000 pg / mL. In some embodiments, the concentration of RNA to be measured is 1-200 pg / mL.

[0362] In some embodiments, the concentration of RNA to be measured is less than 1 pg / mL.

[0363] In some embodiments, the concentration of RNA to be measured is more than 200 pg / mL.

[0364] In some embodiments, the concentration of RNA to be measured is 0.001-0.01 pg / mL. In some embodiments, the concentration of RNA to be measured is 0.01-0.1 pg / mL. In some embodiments, the concentration of RNA to be measured is 0.1-1 pg / mL. In some embodiments, the concentration of RNA to be measured is 1-10 pg / mL. In some embodiments, the concentration of RNA to be measured is 1-100 pg / mL.

[0365] In some embodiments, the concentration of RNA to be measured is 0.01-0.025 pg / mL.

[0366] In some embodiments, the concentration of DNA to be measured is 0.001-1000 pg / mL. In some embodiments, the concentration of DNA to be measured is 0.01-1000 pg / mL. In some embodiments, the concentration of DNA to be measured is 0.1-1000 pg / mL.

[0367] In some embodiments, the concentration of DNA to be measured is 1-200 pg / mL.

[0368] In some embodiments, the concentration of DNA to be measured is less than 1 pg / mL.

[0369] In some embodiments, the concentration of DNA to be measured is more than 200 pg / mL.

[0370] In some embodiments, the concentration of DNA to be measured is 0.001-0.01 pg / mL. In some embodiments, the concentration of DNA to be measured is 0.01-0.1 pg / mL. In some embodiments, the concentration of DNA to be measured is 0.1-1 pg / mL. In some embodiments, the concentration of DNA to be measured is 1-10 pg / mL. In some embodiments, the concentration of DNA to be measured is 1-100 pg / mL.

[0371] In some embodiments, the concentration of DNA to be measured is 0.01-0.025 pg / mL.

[0372] NUCLEIC ACID It will be understood that the cargo of nucleic acids may be considered as a nucleic acid payload.

[0373] As used herein, the terms “cargo of nucleic acids”, “nucleic acid payload”, and “nucleic acid cargo” may be used interchangeably. The terms “cargo”, “payload” and “therapeutic payload” may also be used interchangeably.

[0374] The nucleic acid payload may be delivered to target cells to genetically modify the target cells and / or enable the target cells to express a biomolecule, e.g., a peptide or a protein encoded by the nucleic acid(s).

[0375] In some embodiments, the cargo of nucleic acids comprises one or more nucleic acid molecules.

[0376] The term "nucleic acid" comprises deoxyribonucleic acid (DNA), ribonucleic acid (RNA), combinations thereof, and modified forms thereof. The term comprises genomic DNA, cDNA, mRNA, recombinantly produced and chemically synthesized nucleic acid molecules.

[0377] A nucleic acid may be present as a single-stranded or double-stranded molecule. A nucleic acid may be present as a linear or covalently circularly closed molecule. A nucleic acid can be isolated. The term "isolated nucleic acid" means, according to the present disclosure, that the nucleic acid (i) was amplified in vitro, for example via polymerase chain reaction (PCR) for DNA or in vitro transcription (using, e.g., an RNA polymerase) for RNA, (ii) was produced recombinantly by cloning, (iii) was purified, for example, by cleavage and separation by gel electrophoresis, or (iv) was synthesized, for example, by chemical synthesis.

[0378] According to the present disclosure, the term "RNA" means a nucleic acid molecule which includes ribonucleotide residues. RNA typically comprises adenosine (A), uridine (II), cytidine (C) and guanosine (G). RNA encompasses without limitation, double stranded RNA, single stranded RNA, isolated RNA such as partially purified RNA, essentially pure RNA, synthetic RNA, recombinantly produced RNA, as well as modified RNA that differs from naturally occurring RNA by the addition, deletion, substitution and / or alteration of one or more nucleotides. Such alterations may refer to addition of non-nucleotide material to internal RNA nucleotides or to the end(s) of RNA. It is also contemplated herein that nucleotides in RNA may be non-standard nucleotides, such as chemically synthesized nucleotides or deoxynucleotides. For the present disclosure, these altered / modified nucleotides (or modified nucleosides) can be referred to as analogs of naturally occurring nucleotides (nucleosides), and the corresponding RNAs containing such altered / modified nucleotides or nucleosides ( / .e., altered / modified RNAs) can be referred to as analogs of naturally occurring RNAs.

[0379] According to the present disclosure, the term "DNA" relates to a nucleic acid molecule which includes deoxyribonucleotide residues. DNA typically comprises adenosine (dA), thymidine (dT), cytidine (dC) and guanosine (dG) ("d" represents "deoxy"). In preferred embodiments, the DNA contains all or a majority of deoxyribonucleotide residues. DNA encompasses without limitation, double stranded DNA, single stranded DNA, isolated DNA such as partially purified DNA, essentially pure DNA, synthetic DNA, recombinantly produced DNA, as well as modified DNA that differs from naturally occurring DNA by the addition, deletion, substitution and / or alteration of one or more nucleotides. Such alterations may refer to addition of non-nucleotide material to internal DNA nucleotides or to the end(s) of DNA. It is also contemplated herein that nucleotides in DNA may be non-standard nucleotides, such as chemically synthesized nucleotides or ribonucleotides. For the present disclosure, these altered DNAs are considered analogs of naturally-occurring DNA. DNA may be recombinant DNA and may be obtained by cloning of a nucleic acid, in particular cDNA. The cDNA may be obtained by reverse transcription of RNA. The DNA may comprise a plasmid, a nanoplasmid, a minicircle, a transposon, or linear DNA such as doggybone DNA.

[0380] In some embodiments, the cargo of nucleic acids comprises nucleic acid molecules comprising (i) DNA, (ii) RNA; or (iii) DNA and RNA.

[0381] In some embodiments, the nucleic acid molecules are RNA.

[0382] In some embodiments, the nucleic acid molecules are mRNA.

[0383] In some embodiments, the nucleic acid molecules are DNA.

[0384] In some embodiments, the nucleic acid molecules are a mixture of RNA and DNA.

[0385] In some embodiments, the nucleic acid molecules are a mixture of mRNA and DNA.

[0386] In some embodiments, the nucleic acid molecules encode one or more protein(s).

[0387] In some embodiments, the nucleic acid molecules encode one or more peptide(s). In some embodiments, the DNA is selected from the list consisting of: genomic DNA and cDNA, a plasmid, a nanoplasmid, a minicircle, a transposon, or linear DNA.

[0388] In some embodiments, the DNA is selected from the list consisting of: genomic DNA and cDNA.

[0389] In some embodiments, the DNA may encode a protein or a peptide.

[0390] In some embodiments, the DNA encodes one or more protein(s).

[0391] In some embodiments, the DNA encodes one or more peptide(s).

[0392] In some embodiments, the DNA comprises DNA molecules comprising a length of 15 nucleotides to 15,000, preferably 20 to 12,000, in particular 100 to 10,000, 150 to 8,000, 200 to 7,000, 250 to 6,000, 300 to 5,000 nucleotides, such as 15 to 2,000, 20 to 1 ,000, 25 to 800, 30 to 600, 35 to 500, 40 to 400, 45 to 300, 50 to 250, 55 to 200, 60 to 150, or 65 to 100 nucleotides.

[0393] In some embodiments, the DNA comprises DNA molecules comprising a length of at least 45 nucleotides, at least 60, at least 90, at least 100, at least 200, at least 300, at least 400, at least 500, at least 600, at least 700, at least 800, at least 900, at least 1 ,000, at least 1 ,500, at least 2,000, at least 2,500, at least 3,000, at least 3,500, at least 4,000, at least 4,500, at least 5,000, at least 6,000, at least 7,000, at least 8,000, at least 9,000 nucleotides, preferably up to 15,000, such as up to 14,000, up to 13,000, up to 12,000 nucleotides, up to 11 ,000 nucleotides or up to 10,000 nucleotides.

[0394] In some embodiments, the RNA is selected from the list consisting of: mRNA, tRNA, ribosomal RNA (rRNA), small nuclear RNA (snRNA), self-amplifying RNA (saRNA), trans-amplifying RNA (taRNA), single-stranded RNA (ssRNA), dsRNA, inhibitory RNA, interfering RNA, antisense ssRNA, small interfering RNA (siRNA), microRNA (miRNA), activating RNA, small activating RNA, and immunostimulatory RNA (isRNA).

[0395] In some embodiments, the RNA is selected from the list consisting of: mRNA, miRNA, siRNA and shRNA.

[0396] In some embodiments, the RNA is an interfering RNA. An interfering RNA may be understood as RNA that may elicit RNA interference (RNAi) and produce a gene silencing effect, for example by hybridising under physiological conditions to DNA comprising a particular gene or to mRNA encoding said gene, thereby inhibiting transcription of said gene and / or translation of said mRNA.

[0397] In some embodiments, the RNA is a siRNA, a miRNA or a shRNA.

[0398] In some embodiments, the RNA may be in a form selected from an mRNA, a circular RNA, a self-replicating RNA (saRNA), a trans-amplifying RNA (taRNA), a replicon, or mixtures thereof.

[0399] In some embodiments, the RNA is mRNA.

[0400] In some embodiments, the RNA may encode a protein or a peptide.

[0401] In some embodiments, the RNA encodes one or more protein(s).

[0402] In some embodiments, the RNA encodes one or more peptide(s).

[0403] In some embodiments, the RNA comprises RNA molecules comprising a length of 15 nucleotides to 15,000, preferably 20 to 12,000, in particular 100 to 10,000, 150 to 8,000, 200 to 7,000, 250 to 6,000, 300 to 5,000 nucleotides, such as 15 to 2,000, 20 to 1 ,000, 25 to 800, 30 to 600, 35 to 500, 40 to 400, 45 to 300, 50 to 250, 55 to 200, 60 to 150, or 65 to 100 nucleotides.

[0404] In some embodiments, the RNA comprises RNA molecules comprising a length of at least 45 nucleotides, at least 60, at least 90, at least 100, at least 200, at least 300, at least 400, at least 500, at least 600, at least 700, at least 800, at least 900, at least 1 ,000, at least 1 ,500, at least 2,000, at least 2,500, at least 3,000, at least 3,500, at least 4,000, at least 4,500, at least 5,000, at least 6,000, at least 7,000, at least 8,000, at least 9,000 nucleotides, preferably up to 15,000, such as up to 14,000, up to 13,000, up to 12,000 nucleotides, up to 11 ,000 nucleotides or up to 10,000 nucleotides.

[0405] In some embodiments, the cargo of nucleic acids is a mixture of a DNA nanoplasmid and a mRNA.

[0406] In some embodiments, the cargo of nucleic acids is a mixture of a DNA transposon and an mRNA encoding a transposase. In some embodiments, the nucleic acids purified in step (ii) are DNA or RNA.

[0407] In some embodiments, the nucleic acids purified in step (ii) are DNA.

[0408] In some embodiments, the nucleic acids purified in step (ii) are RNA.

[0409] In some embodiments, the nucleic acids purified in step (ii) are DNA and RNA.

[0410] In some embodiments, the nucleic acid molecules in the cargo of nucleic acids may be a mixture of RNA and DNA, and the nucleic acids purified in step (ii) are DNA.

[0411] In some embodiments, the nucleic acid molecules in the cargo of nucleic acids may be a mixture of RNA and DNA, and the nucleic acids purified in step (ii) are RNA.

[0412] In some embodiments, the nucleic acid molecules in the cargo of nucleic acids may be a mixture of RNA and DNA, and the nucleic acids purified in step (ii) are DNA and RNA.

[0413] In some embodiments, the nucleic acid molecules in the cargo of nucleic acids may be a mixture of mRNA and DNA, and the nucleic acids purified in step (ii) are DNA.

[0414] In some embodiments, the nucleic acid molecules in the cargo of nucleic acids may be a mixture of mRNA and DNA, and the nucleic acids purified in step (ii) are mRNA.

[0415] In some embodiments, the nucleic acid molecules in the cargo of nucleic acids may be a mixture of mRNA and DNA, and the nucleic acids purified in step (ii) are DNA and mRNA.

[0416] The method described herein can be applied on various lipid-based nanoparticle systems (e.g. lipid nanoparticles (LNPs), lipoplexes (LPX), liposomes or any other lipid-based nanoparticle systems comprising different lipids (e.g. ionizable, non-ionizable and permanent charged), buffer matrices (e.g. sugar (sucrose, trehalose), HEPES, Tris, PBS, EDTA), nucleic acid formats and a broad concentration range. Finally, the method is compatible with a wide range of standard downstream analyses / assays (e.g. nuclei acid integrity measurements, 5' -capping detection of pharmaceutical mRNA constructs, mRNA identity and / or ratio by PCR-based techniques, spectroscopy techniques , sequencing techniques or any other techniques to analyse nucleic acids, its sequence and / or its physical parameters). LIPID NANOPARTICLES (LNPs)

[0417] It will be understood that lipid nanoparticles (LNPs) are particles that provide delivery of one or more payload(s) (e.g. cargo) to target cells, in particular delivery of payloads into the cytosol of target cells.

[0418] Indeed, LNPs enable the payload (e.g. cargo of nucleic acids) to be administered by functioning as delivery vehicles that protect the payload from degradation, maximize delivery to on-target cells and minimize exposure to off-target cells. Indeed, in use the LNPs act to stabilise and encapsulate the payload to enable it to be delivered into a cell while facilitating its uptake into the cell and release into the cytosol.

[0419] The present invention relates to LNPs comprising a cargo of nucleic acids as a payload. LNPs may complex or encapsulate the payload. In particular, the LNPs described herein may encapsulate the payload.

[0420] The nucleic acid of the present disclosure may be formulated in (e.g., encapsulated in) a lipid nanoparticle, as further described herein. In some embodiments, the lipid nanoparticle comprises a nucleic acid (e.g., DNA and / or RNA), and a cationic lipid, a cationically ionizable lipid, or a cationic polymer or a pharmaceutically acceptable salt thereof, where n’ is an integer from about 45 to about 50.

[0421] In some embodiments, the PEG-lipid is represented by: wherein n has a mean value ranging from 30 to 60. In some embodiments, n is 50. In one embodiment, the PEG-conjugated lipid (pegylated lipid) is PEG2000-C-DMA which preferably refers to 3-N-[(w-methoxy poly(ethylene glycol)2000)carbamoyl]-1 ,2-dimyristyloxy- propylamine (MPEG-(2 kDa)-C-DMA) or methoxy-polyethylene glycol-2,3-bis(tetradecyloxy) propylcarbamate (2000).

[0422] In some embodiments, a PEG-lipid is selected from PEG-DAG, PEG-PE, PEG-S-DAG, PEG2000-DMG, ALC-159, PEG2000-C-DMA PEG-S-DMG, PEG-cer, and combinations thereof. In some embodiments, a PEG-lipid is ALC-0159 or PEG2000-DMG. In some embodiments, a PEG-lipid is ALC-0159. In some embodiments, a PEG-lipid is PEG2000- DMG. In some embodiments, a polymer-conjugated lipid is a polysarcosine-conjugated lipid, also referred to herein as sarcosinylated lipid or pSar-lipid. The term “sarcosinylated lipid” refers to a molecule comprising both a lipid portion and a polysarcosine (poly(N-methylglycine) portion.

[0423] In some embodiments, a polymer-conjugated lipid is one described in WO2024 / 028325, which is incorporated herein by reference in its entirety. In some embodiments, a polymer- conjugated lipid is represented by formula PCL-II: or a pharmaceutically acceptable salt thereof, wherein, as applied to formula PCL-II: X2and X1taken together are optionally substituted amide, optionally substituted thioamide, ester, or thioester; Y is -CH2-, -(CH2)2-, or-(CH2)3-; z is 2 to 24; and n is 1 to 100. In some embodiments of formula PCL-II: (i) when X1is -C(O)- then X2is -NR1-; (ii) when X1is -NR1- then X2is -C(O)- ; (iii) when X1is -C(S)- then X2is -NR1-; (iv) when X1is -NR1- then X2is -C(S)-; (v) when X1is -C(O)- then X2is -O-; (vi) when X1is -O- then X2is -C(O)-; (vii) when X1is -C(S)- then X2is - O-; (viii) when X1is -O- then X2is -C(S)-; (ix) when X1is -C(O)- then X2is -S-; or (x) when X1is -S- then X2is -C(O)-; wherein R1is hydrogen or C1-8 alkyl. In some embodiments of formula PCL-II: (i) when X1is -C(O)- then X2is -NR1-; (ii) when X1is -NR1- then X2is -C(O)-; (iii) when X1is -C(S)- then X2is -NR1-; (iv) when X1is -NR1- then X2is -C(S)-; (v) when X1is -C(O)- then X2is -O-; or (vi) when X1is -O- then X2is -C(O)-; wherein R1is hydrogen or C1-8 alkyl.

[0424] In some embodiments, a polymer-conjugated lipid comprises monomers of 2-(2-(2- aminoethoxy)ethoxy)acetic acid. In some embodiments, the polymer of the polymer- conjugated lipid is or comprises poly-2-(2-(2-aminoethoxy)ethoxy)acetic acid (pAEEA) or poly- 2-(2-(2-methylaminoethoxy)ethoxy)acetic acid (pMAEEA), or a derivative thereof, as defined herein. In some embodiments, a polymer conjugated lipid comprises monomers of unit PCL- ll-1 : ll-1

[0425] In some embodiments, a polymer conjugated lipid comprises, 5 to 50, 5 to 25 or 10 to 25 monomers of PCL-ll-1. In some embodiments, a polymer conjugated lipid comprises 14 to 17 monomers of PCL-ll-1. In some embodiments, a polymer conjugated lipid comprises 8 to 14 monomers of PCL-ll-1. In some embodiments, a polymer conjugated lipid is selected from the table below:

[0426] In some embodiments, an LNP comprises an polysarcosine-conjugated or a pAEEA / pMAEEA-conjugated lipid, as described herein. In some embodiments, nucleic acid particles (e.g., DNA or RNA particles) described herein comprise a polysarcosine-conjugated or a pAEEA / pMAEEA-conjugated lipid and are substantially free of a pegylated lipid (or do not contain a pegylated lipid).

[0427] In some embodiments, a lipid nanoparticle comprises about 0.5 to about 5.0 mol% of a polymer-conjugated lipid. In some embodiments, a lipid nanoparticle comprises about 1.0 to about 2.5 mol% of a polymer-conjugated lipid. In some embodiments, a lipid nanoparticle comprises about 1.5 to about 2.0 mol% of a polymer-conjugated lipid. In some embodiments, a lipid nanoparticle comprises about 1.5 to about 1.8 mol% of a polymer-conjugated lipid. In some embodiments, a lipid nanoparticle comprises about 1 .5 mol% to about 1 .8 mol% (relative to the total amount of lipids in a lipid nanoparticle) of a polymer-conjugated lipid selected from the group consisting of: DSPE-AEEA14-AC; VE-AEEA14-AC; ALC-0159 and PEG2000-DMG. In some embodiments, a lipid nanoparticle comprises about 1.5 mol% to about 1.8 mol% (relative to the total amount of lipids in a lipid nanoparticle) of a polymer-conjugated lipid selected from the group consisting of: DSPE-AEEA14-AC, VE-AEEA14-AC, and PEG2000- DMG. In some embodiments, a molar ratio of a cationic or cationically ionizable lipid to a polymer-conjugated lipid is from about 2:1 to about 8:1.

[0428] (i) Steroids

[0429] As described generally herein, lipid nanoparticles may further comprise a steroid. In some embodiments, a steroid is a sterol. In some embodiments, a sterol is p-sitosterol, stigmasterol, cholesterol, cholecalciferol, ergocalciferol, calcipotriol, botulin, lupeol, ursolic acid, oleanolic acid, cycloartenol, lanosterol, or a-tocopherol. In some embodiments, a sterol is cholesterol. In some embodiments, a lipid nanoparticle comprises about 39 to about 49 mol% of a steroid.

[0430] In some embodiments, a lipid nanoparticle comprises about 40 to about 46 mol% of a steroid.

[0431] In some embodiments, a lipid nanoparticle comprises about 40 to about 44 mol% of a steroid.

[0432] In some embodiments, a lipid nanoparticle comprises: about 40 to about 50 mol% of a cationically ionizable lipid; about 30 to about 45 mol% of a steroid (e.g., cholesterol); about 5 to about 15 mol% of a helper lipid (e.g., DSPC); and about 1 to about 2.5 mol% of a polymer conjugated lipid. In some embodiments, a lipid nanoparticle comprises: about 30 to about 60 mol% of a cationically ionizable lipid; about 18.5 to about 48.5 mol% of a steroid (e.g., cholesterol); about 0 to about 30 mol% of a helper lipid (e.g., DSPC); and about 0 to about 10 mol% of a polymer conjugated lipid. In some embodiments, a lipid nanoparticle comprises: about 35 to about 55 mol% of a cationically ionizable lipid; about 30 to about 40 mol% of a steroid (e.g., cholesterol); about 5 to about 25 mol% of a helper lipid (e.g., DSPC); and about 0 to about 10 mol% of a polymer conjugated lipid.

[0433] In some embodiments, a lipid nanoparticle comprises: 47.5 mol% di(41apidated41e-9-yl) 3,3’- ((2-(4-methylpiperazin-1-yl)ethyl)azanediyl)dipropionate (BHD-C2C2-PipZ); 10 mol% DSPC; 40.7 mol% cholesterol; and 1.8 mol% VE-AEEA14-AC. In some embodiments, a lipid nanoparticle comprises: 47.5 mol% di(41apidated41e-9-yl) 3,3’-((2-(4-methylpiperazin-1- yl)ethyl)azanediyl)dipropionate (BHD-C2C2-PipZ); 10 mol% DSPC; 40.7 mol% cholesterol; and 1.8 mol% PEG2000-DMG. In some embodiments, a lipid nanoparticle comprises: about 47.5 mol% of ALC-0315; about 10 mol% of DSPC; about 40.7 mol% of cholesterol; and about 1.8 mol% ofALC-159. In some embodiments, a lipid nanoparticle comprises: about 47.5 mol% of ALC-366; about 10 mol% of DSPC; about 40.7 mol% of cholesterol; and about 1.8 mol% of ALC-159. In some embodiments, a lipid nanoparticle comprises about 50 mol% of SM-102; about 1.5 mol% of PEG2000-DMG; about 10 mol% of DSPC; and about 38.5 mol% of cholesterol. In some embodiments, a lipid nanoparticle comprises: 47.5 mol% bis(2- octyldodecyl) 3,3’-((2-(1 -methylpyrrolidin-2-yl)ethyl)azanediyl)dipropionate (BODD-C2C2- 1 Me-Pyr); 10 mol% DSPC; 40.7 mol% cholesterol; and 1.8 mol% VE-AEEA14-AC. In some embodiments, a lipid nanoparticle comprises: 47.5 mol% bis(2-octyldodecyl) 3,3’-((2-(1- methylpyrrolidin-2-yl)ethyl)azanediyl)dipropionate (BODD-C2C2-1Me-Pyr); 10 mol% DSPC;

[0434] 40.7 mol% cholesterol; and 1.8 mol% PEG2000-DMG.

[0435] (it) Manufacturing

[0436] Lipids and lipid nanoparticles comprising nucleic acids and their method of preparation are known in the art, including, e.g., as described in U.S. Patent Publication Nos. 2016 / 0009637, 2015 / 0273068, 2014 / 0200257, 2013 / 0338210, 2013 / 0245107, 2013 / 0123338,

[0437] 2013 / 0017223, 2012 / 0183581 , 2012 / 0027803, 2011 / 0311583, 2011 / 0216622, 2011 / 0117125, 2007 / 0042031 , 2006 / 0083780, 2005 / 017054, 2004 / 0142025, 2007 / 0042031 , 1999 / 009076 and PCT Pub. Nos. WO 99 / 39741 , WO 2018 / 081480, WO 2017 / 004143, WO 2017 / 075531 , WO 2015 / 199952, WO 2013 / 086322, WO 2013 / 016058, WO 2013 / 086373, WO 2011 / 141705, WO 2022 / 016089, WO 2022 / 081752, the full disclosures of which are herein incorporated by reference in their entirety for the purposes described herein.

[0438] For example, in some embodiments, cationically ionizable lipids, helper lipids, and steroids are solubilized in an organic solvent such as ethanol, at a pre- determined weight or molar ratios / percentages (e.g., ones described herein). In some embodiments, lipid nanoparticles are prepared at a total lipid to nucleic acid (e.g., RNA) weight ratio of approximately 10:1 to 50:1. In some embodiments, such nucleic acid (e.g., RNA) can be diluted to 0.1 to 1.0 mg / mL (e.g., 0.4 mg / mL) in an acidic buffer, such as citrate or acetate having a pH of between about 4 to about 6.

[0439] In some embodiments, using an ethanol injection technique, a colloidal lipid dispersion comprising nucleic acids (e.g., RNAs) can be formed as follows: an ethanol solution comprising lipids, such as cationic lipids, helper lipids, steroids, and polymer-conjugated lipids, is combined with, e.g., injected into or continuously mixed with, an aqueous solution comprising nucleic acids.

[0440] In some embodiments, lipid and nucleic acid (e.g., RNA) solutions can be mixed at room temperature by pumping each solution (e.g., a lipid solution comprising a cationic lipid, a helper lipid, cholesterol, a conjugated lipid, and any other additives) at controlled flow rates into a mixing unit, for example, using piston pumps. In some embodiments, the flow rates of a lipid solution and a nucleic acid (e.g., RNA) solution into a mixing unit are maintained at a ratio of 1 :3. Upon mixing, nucleic acid-lipid particles are formed as the ethanolic lipid solution is diluted with aqueous nucleic acids (e.g., RNAs). The lipid solubility is decreased, while cationic lipids bearing a positive charge interact with the negatively charged nucleic acid (e.g., RNA).

[0441] In some embodiments, a solution comprising nucleic acid (e.g., RNA)-encapsulated lipid nanoparticles can be processed by one or more of concentration adjustment, buffer exchange, formulation, and / or filtration.

[0442] Particles

[0443] In some embodiments, an agent to be delivered to a subject, e.g., a nucleic acid, a polypeptide, a small molecule, and the like, is encapsulated in a particle. In some embodiments, the nucleic acid may be formulated in (e.g., encapsulated in) a particle, as further described herein. In some embodiments, a particle is a nucleic acid particle wherein the nucleic acid particle comprises a nucleic acid (e.g., DNA and / or RNA), and a cationic lipid, a cationically ionizable lipid, or a cationic polymer.

[0444] A “nucleic acid particle,” as used herein, refers to a particle that encompasses or contains a nucleic acid, and, is part of a composition (e.g., a pharmaceutical composition) comprising multiple nucleic acid particles, that is useful for (i) enhancing nucleic acid stability, e.g., during storage, (ii) improving biodistribution of the nucleic acid or delivering a nucleic acid to a target site of interest (e.g., cell, tissue, organ, and the like), and / or (iii) facilitating cell uptake of the nucleic acid. As described herein, a nucleic acid particle may be formed from i) at least one cationic or cationically ionizable lipid or lipid-like material; ii) at least one cationic polymer such as polyethyleneimine, protamine, or a mixture thereof (i.e., a mixture of i) and ii)), and iii) a nucleic acid. Nucleic acid particles described herein include lipid nanoparticles (LNP), lipoplexes (LPX), liposomes, and polyplexes (PLX).

[0445] Electrostatic interactions between positively charged molecules such as cationic polymers and cationic lipids and negatively charged nucleic acids are involved in particle formation. This results in complexation and spontaneous formation of nucleic acid particles. The characteristics of a particle (e.g., nanoparticle) are determined, at least in part, from the components used to form the particle and the process used to prepare the particle. A description of the different types of particles and their structures is provided in ACS Nano 2021 , 15, 11 , 16982-17015. In some embodiments, a nucleic acid particle described herein is a nanoparticle. As used in the present disclosure, “nanoparticle” refers to a particle having an average diameter suitable for parenteral administration and is less than 1000 nm in diameter. In some embodiments, a composition comprising nanoparticles can have an average nanoparticle size (e.g., mean diameter) of about 30 nm to about 150 nm, about 40 nm to about 120 nm, about 50 nm to about 100 nm, or about 60 nm to about 90 nm. In some embodiments, a composition comprising nanoparticles can have an average nanoparticle size (e.g., mean diameter) of about 40 nm to about 120 nm. The term “average diameter” or “mean diameter” refers to the mean hydrodynamic diameter of particles as measured by dynamic laser light scattering (DLS) with data analysis using an appropriate algorithm (e.g., the so-called cumulant algorithm for monodisperse samples), which provides as results the so-called Z-average with the dimension of a length, and the polydispersity index (PDI), which is dimensionless (Koppel, D., J. Chem. Phys. 57, 1972, pp 4814-4820, ISO 13321). Here “average diameter,” “mean diameter,” “diameter,” or “size” for particles is used synonymously with this value of the Z-average.

[0446] A composition comprising nucleic acid particles can be characterized by its polydispersity index, that is, the relative uniformity of particles within a given composition. For example, compositions described herein may exhibit a polydispersity index (PDI) less than about 0.5, less than about 0.4, less than about 0.3, or about 0.2 or less of said nanoparticles. In some embodiments, a composition comprising nucleic acid particles, as described herein, may exhibit a PDI less than about 0.3. By way of example, a composition comprising nucleic acid particles described herein can exhibit a PDI in a range of about 0.1 to about 0.3, or about 0.2 to about 0.3. The polydispersity index of a given composition can be calculated based on dynamic light scattering measurements by the so-called cumulant analysis as mentioned in the definition of the “average diameter.” Under certain prerequisites, it can be taken as a measure of the size distribution of an ensemble of ribonucleic acid nanoparticles.

[0447] Nucleic acid particles described herein can be characterized by an “N / P ratio,” which is the molar ratio of cationic (nitrogen) groups (the “N” in N / P) in the cationic lipid or polymer to the anionic (phosphate) groups (the “P” in N / P) in RNA. It is understood that a cationic group is one that is either in permanently cationic form (e.g., N+), or one that is ionizable to become cationic (e.g., under certain pH conditions). Use of a single number in an N / P ratio (e.g., an N / P ratio of about 5) is intended to refer to that number over 1 , e.g., an N / P ratio of about 4 is intended to mean about 4:1. In some embodiments, a nucleic acid particle (e.g., an RNA LNP) described herein has an N / P ratio greater than or equal to 1 , greater than or equal to 2, or greater than or equal to 4. In some embodiments, a nucleic acid particle (e.g., an RNA LNP) described herein has an N / P ratio that is less than 24, less than 18, or less than 12. In some embodiments, a nucleic acid particle (e.g., an RNA LNP) described herein has an N / P ratio that is from about 2 to about 24, about 4 to about 18, about 4 to about 12, or about 4 to about 8. In some embodiments, a nucleic acid particle (e.g., a ribonucleic acid particle) described herein has an N / P ratio that is about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , or 12. In some embodiments, an N / P ratio for a nucleic acid particle (e.g., an RNA LNP) described herein is about 6.

[0448] Nucleic acid particles described herein can be prepared using a wide range of methods that may involve obtaining a colloid from at least one cationic or cationically ionizable lipid or lipid- like material and / or at least one cationic polymer and mixing the colloid with nucleic acid to obtain nucleic acid particles. As used herein, an “ionizable” lipid, e.g., a “cationically ionizable” lipid or “ionizable” polymer, e.g., a “cationically ionizable” polymer is a lipid or polymer that may be, in some embodiments, neutral at physiological pH, but is capable of becoming cationic (i.e. , becoming positively charged) at acidic pH.

[0449] The present disclosure describes particles comprising nucleic acid, at least one cationic or cationically ionizable lipid or lipid-like material, and / or at least one cationic polymer which associate with the nucleic acid to form nucleic acid particles (e.g., RNA nanoparticles) and compositions comprising such particles. The nucleic acid particles (e.g., RNA nanoparticles) may comprise nucleic acid which is complexed by different non-covalent interactions (e.g., electrostatic, hydrogen bonding, pi-stacking, van der Waals, etc.) to the particle. In some embodiments, the particles described herein are not viral particles, in particular, they are not infectious viral particles, i.e., they are not able to virally infect cells.

[0450] In a nucleic acid particle (e.g., RNA nanoparticle) composition, it is possible that each nucleic acid species is separately formulated as an individual nucleic acid particle formulation. In that case, each individual nucleic acid particle formulation will comprise one nucleic acid species. In some embodiments, a composition comprises more than one individual nucleic acid particle (e.g., RNA nanoparticle) formulation. Respective pharmaceutical compositions are referred to as “mixed particulate formulations.” Such mixed particulate formulations may be obtainable by forming, separately, individual nucleic acid particle formulations, and mixing these to produce a formulation comprising a mixed population of nucleic acid-containing particles. Alternatively, different nucleic acid species may be formulated together as a “combined particulate formulation.” Such formulations may be obtainable by mixing a combined formulation of different nucleic acid species with a particle-forming agent, to produce particles that comprise more than one nucleic acid species.

[0451] Lipid Nanoparticles (LNPs) In some embodiments, a particle described herein is a lipid nanoparticle (LNP). LNPs have emerged as particularly useful vehicles for delivery of nucleic acids, for example as described in Theranostics, 2022 Oct 24;12(17):7509-7531. It is understood that a LNP is structurally distinct from other nanoparticles previously used for nucleic acid delivery, such as a liposome, or a lipoplex. LNPs, as described herein, typically do not comprise a bilayer (uni-lamellar), or a concentric series of multiple bilayers (multi-lamellar) separated by aqueous compartments, enclosing a central aqueous compartment. Moreover, LNPs, as described herein, typically do not comprise a central aqueous core or compartment. LNPs as described herein typically comprise nucleic acids (e.g., DNA or RNA such as mRNA) and lipids forming a disordered, non-lamellar phase. LNPs as described herein may be considered as oil-in-water emulsions in which the LNP core materials are preferably in liquid state and hence have a melting point below body temperature. See, e.g., ACS Nano 2021 , 15, 11 , 16982-17015; Aldosari, et al., Pharmaceutics, 2021 , 13, 206.

[0452] LNPs described herein generally comprise four categories of lipids in addition to a nucleic acid agent (e.g., DNA or RNA such as mRNA): a cationic or cationically ionizable lipid (typically a cationically ionizable lipid), a polymer-conjugated lipid, a helper lipid, and a steroid. A person of skill in the art will understand that various combinations of these four categories of lipids can be used to prepare lipid nanoparticles for use in delivering nucleic acid agents.

[0453] (i) Cationic or cationically ionizable lipids

[0454] As described generally herein, a nucleic acid particle comprises a nucleic acid and a cationic or a cationically ionizable lipid. In some embodiments, a cationic or cationically ionizable lipid useful for incorporation into a nucleic acid particle are those lipids having a polar head group and an aliphatic tail. In some embodiments, a cationic lipid is one where the polar head group has a permanently positive charge (for example, comprising a quaternary ammonium group). In some embodiments, a cationically ionizable lipid is a lipid wherein, at a given pH and in the context of an LNP, the lipid becomes positively charged, such as at below physiological pH (e.g., below pH about 7.4) or neutral pH (e.g., a pH around 7 to 7.5), or in some embodiments, at a pH of less than 7 (e.g., less than 6). In some embodiments, a cationically ionizable lipid is one comprising polar head group that comprises one or more a tertiary amine groups (or secondary or primary amine group) that can become positively charged. LNPs typically comprise cationically ionizable lipids.

[0455] In some embodiments, a lipid nanoparticle comprises about 30 mol% to about 60 mol% of a cationic or cationically ionizable lipid. In some embodiments, a lipid nanoparticle comprises about 35 mol% to about 55 mol% of a cationic or cationically ionizable lipid. In some embodiments, a lipid nanoparticle comprises about 40 mol% to about 50 mol% of a cationic or cationically ionizable lipid. In some embodiments, a lipid nanoparticle comprises about 50 mol% of a cationic or cationically ionizable lipid. In some embodiments, a lipid nanoparticle comprises about 47.0, 47.1 , 47.2, 47.3, 47.4, 47.5, 47.6, 47.7, 47.8, 47.9, or 48.0 mol% of a cationic or cationically ionizable lipid. In some embodiments, a lipid nanoparticle comprises 47.5 mol% of a cationic or cationically ionizable lipid.

[0456] Suitable cationic or cationically ionizable lipids are readily identified by those of skill in the art. In some embodiments, a cationic lipid or cationically ionizable lipid is one provided in WO 2010 / 144740 or WO 2012 / 016184, which are incorporated herein by reference in their entirety. For example, in some embodiments, a cationic lipid is selected from N-(2,3-dioleyloxypropyl)- N,N,N-trimethylammonium chloride (DOTMA); N,N-distearyl-N,N-dimethylammonium bromide (DDAB), N-(2,3-dioleoyloxypropyl)-N,N,N-trimethylammonium chloride (DOTAP), N,N-dioleyl-N,N-dimethylammonium chloride (DODAC); 3-(N-(N',N'dimethylaminoethane)- carbamoyl)cholesterol (DC-Chol), and N-(1 ,2-dimyristyloxyprop-3-yl)-N,N-dimethyl-N- hydroxyethyl ammonium bromide (DMRIE). In some embodiments, a cationically ionizable lipid is selected from 1 ,2-dioleoyl-3-dimethylammonium propane (DODAP); N,N-dimethyl- (2,3-dioleoyloxypropyl)amine (DODMA); and 4-(dimethylamino)-butanoic acid, (10Z,13Z)-1- (9Z, 12Z)-9, 12-octadecadien-1 -yl-10, 13-nonadecadien-1 -yl ester (DLin-MC3-DMA).

[0457] In some embodiments, a cationically ionizable lipid is a lipid described in WO 2017 / 075531 or WO 2018 / 081480, each of which is incorporated by reference herein in its entirety. In some embodiments, a cationically ionizable lipid is a lipid represented by formula CL-I:

[0458] CL-I or a pharmaceutically acceptable salt thereof, wherein, as applied to formula CL-I: one of L1or L2is -O(C=O)-, -(C=O)O-, -C(=O)-, -O-, -S(O)X-, -S-S-, -C(=O)S-, -SC(=O)-, -NRaC(=O)-, -C(=O)NRa-, -NRaC(=O)NRa-, -OC(=O)NRa- or -NRaC(=O)O-, and the other of L1or L2is - O(C=O)-, -(C=O)O-, -C(=O)-, -O-, -S(O)X-, -S-S-, -C(=O)S-, -SC(=O)-, -NRaC(=O)-, - C(=O)NRa-, -NRaC(=O)NRa-, -OC(=O)NRa- or -NRaC(=O)O- or a direct bond; G1and G2are each independently unsubstituted C1-C12 alkylene or C1-C12 alkenylene; G3is C1-C24 alkylene, C1-C24 alkenylene, C3-C8 cycloalkylene, or C3-C8 cycloalkenylene; Rais H or C1-C12 alkyl; R1and R2are each independently C6-C24 alkyl or C6-C24 alkenyl; R3is H, OR5, CN, -C(=O)OR4, -OC(=O)R4or -NR5C(=O)R4; R4is C1-C12 alkyl; R5is H or Ci-C6alkyl; and x is 0, 1 or 2. In some embodiments, a cationically ionizable lipid is ((4-hydroxybutyl)azanediyl)bis(hexane- 6,1 -diyl) bis(2-hexyldecanoate) (ALC-0315) or ((3-hydroxypropyl)azanediyl)bis(nonane-9,1- diyl) bis(2-butyloctanoate) (ALC-366):

[0459] In some embodiments, a lipid nanoparticle comprises about 40 mol% to about 50 mol% of a cationically ionizable lipid. In some embodiments, a lipid nanoparticle comprises about 47.0, 47.1 , 47.2, 47.3, 47.4, 47.5, 47.6, 47.7, 47.8, 47.9, or 48.0 mol% of a cationically ionizable lipid. In some embodiments, a lipid nanoparticle comprises 47.5 mol% of a cationically ionizable lipid.

[0460] In some embodiments, a cationic lipid is one described in WO 2017 / 049245, which is incorporated by reference in its entirety. In some embodiments, a cationic lipid is represented by formula CL-II or a pharmaceutically acceptable salt thereof, wherein, as applied to formula CL-II: Ri is selected from the group consisting of C5-30 alkyl, C5-20 alkenyl, -R*YR", -YR", and -R"M'R'; R2 and R3 are independently selected from the group consisting of H, C1-14 alkyl, C2-14 alkenyl, - R*YR", -YR", and -R*OR", or R2 and R3, together with the atom to which they are attached, form a heterocycle or carbocycle; R4 is selected from the group consisting of a C3-6 carbocycle, -(CH2)nQ, -(CH2)nCHQR, -CHQR, -CQR2, and unsubstituted C1-6 alkyl, where Q is selected from a carbocycle, heterocycle, -OR, -O(CH2)nNR2, -C(O)OR, OC(O)R, -CX3, -CX2H, -CXH2, - CN, -NR2, -C(O)NR2, -NRC(O)R, -NRS(O)2R, -NRC(O)NR2, -NRC(S)NR2, -NRR8, - O(CH2)nOR, -NRC(=NR9)NR2, -NRC(=CHR9)NR2, -OC(O)NR2, -NRC(O)OR, -N(OR)C(O)R, - N(OR)S(O)2R, -N(OR)C(O)OR, -N(OR)C(O)NR2, -N(OR)C(S)NR2, N(OR)C(=NR9)NR2, - N(OR)C(=CHR9)NR2, -C(=NR9)NR2, -C(=NR9)R, -C(O)NROR, and -CRNR2C(O)OR, and each n is independently selected from 1 , 2, 3, 4, and 5; each Rs is independently selected from the group consisting of C1-3 alkyl, C2-3 alkenyl, and H; each Rs is independently selected from the group consisting of C1-3 alkyl, C2-3 alkenyl, and H; M and M' are independently selected from -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-, -S-S-, an aryl group, and a heteroaryl group; R? is selected from the group consisting of C1-3 alkyl, C2-3 alkenyl, and H; Rs is selected from the group consisting of C3-6 carbocycle and heterocycle; R9 is selected from the group consisting of H, CN, NO2, C1-6 alkyl, -OR, -S(O)2R, -S(O)2NR2, C2-6 alkenyl, C3-6 carbocycle and heterocycle; each R is independently selected from the group consisting of C1-3 alkyl, C2-3 alkenyl, and H; each R' is independently selected from the group consisting of C1-18 alkyl, C2-18 alkenyl, - R*YR", -YR", and H; each R" is independently selected from the group consisting of C3-14 alkyl and C3-14 alkenyl; each R* is independently selected from the group consisting of C1-12 alkyl and C2-12 alkenyl; each Y is independently a C3-6 carbocycle; each X is independently selected from the group consisting of F, Cl, Br, and I; and m is selected from 5, 6, 7, 8, 9, 10, 11 , 12, and 13.

[0461] In some embodiments, a cationically ionizable lipid is heptadecan-9-yl 8-{(2-hydroxyethyl)[6- oxo-6-(undecyloxy)hexyl]amino}octanoate) (SM-102):

[0462] SM-102

[0463] In some embodiments, a cationically ionizable lipid is a lipid described in WO 2015 / 095340, which is incorporated by reference herein in its entirety. In some embodiments, a cationic lipid is represented by formula CL-III or a pharmaceutically acceptable salt thereof, wherein, as related to formula CL-III: n and p are each, independently, 0, 1 or 2; Li is -OC(O)-, -C(O)O- or a bond; R1is heterocyclyl, heterocyclyl-Ci-8-alkyl or heterocyclyl-Ci-8-alkoxyl, each of which may be optionally substituted with 1 , 2, 3, 4 or 5 groups, independently selected from halogen, formidamidine, Ci-s-alkyl, C3- 7-cycloalkyl, C3-7-cycloalkyl-Ci-8-alkyl, heterocyclyl, -[(Ci-C4)alkylene]v-N(R')R", -O-[(Ci- C4)alkylene]v-N(R')R" or -N(H)-[(Ci-C4)alkylene]v-N(R')R", where said (Ci-C4)alkylene is optionally substituted with one or more R groups; v is 0, 1 , 2, 3 or 4; R is hydrogen or -C1-8- alkyl or when v is 0 R is absent; R' and R", are each, independently, hydrogen, -Ci-8-alkyl; or R' and R" combine with the nitrogen to which they are bound, and optionally including another heteroatom selected from N, O and S, to form a 5-8 membered heterocycle or heteroaryl, optionally substituted with an -Ci-s-alkyl, hydroxy or cycloalkyl-Ci-8-;

[0464] R2and R3are each, independently, C7-22 alkyl, C12-22 alkenyl, C3-8 cycloalkyl optionally substituted with 1 , 2, or 3 C1-8 alkyl groups, ,

[0465] R4is selected from hydrogen, C1-14 alkyl,

[0466] In some embodiments, a cationically ionizable lipid is represented by

[0467] In some embodiments, a cationic lipid is one described in WO 2018 / 087753, which is incorporated herein by reference in its entirety.

[0468] In some embodiments, a cationic lipid is represented by formula CL-IV: or a pharmaceutically acceptable salt thereof, wherein, as applied for formula CL-IV: Y is O or NH; T is C or S; W is a bond, O, NH or S; R1is selected from the group consisting of: (a) NR4R5, wherein R4and R5are each independently a C1-C4 alkyl; or R4and R5together with the nitrogen to which they are attached form a 5 or 6 membered heterocyclic or heteroaromatic ring, optionally containing one or more additional heteroatoms selected from the group consisting of O, N and S; or NR4R5represent a guanidine group (-NHC(=NH)NH2); (b) the side chain of a natural or unnatural amino acid; and (c) a 5 or 6 membered heterocyclic or heteroaromatic ring containing one or more heteroatoms selected from the group consisting of O, N and S; R2and R3are selected from the group consisting of: (a) C10-C22 alkyl; (b) C10- C22 alkenyl; (c) C10-C22 alkynyl; (d) C4-C10 alkylene-Z-C4-C22 alkyl; and (e) C4-C10 alkylene-Z- C4-C22 alkenyl; Z is -O-C(=O)-, -C(=O)-O- or -O-; n is 0, 1 , 2, 3, 4, 5 or 6; m is 0 or 1 ; p is 0 or 1 ; and z is 0 or 2.

[0469] In some embodiments, a cationically ionizable lipid is selected from:

[0470] In some embodiments, a cationically ionizable lipid is one described in WO 2022 / 081750, which is incorporated herein by reference in its entirety.

[0471] In some embodiments, a cationically ionizable lipid is represented by formula CL-V-1 :

[0472] CL-V-1 or a pharmaceutically acceptable salt thereof, wherein, as applied to formula CL-V-1 : each R1and each R2is independently selected from the group consisting of H, an optionally substituted C1-C22 alkyl, optionally substituted C2-C22 alkenyl, optionally substituted C2- C22 alkynyl, optionally substituted C3-C6 cycloalkyl, optionally substituted C4- Ce heterocycloalkyl, optionally substituted C4-C6 alkylcycloalkyl, optionally substituted C4-C6 aryl, optionally substituted C3-C6 heteroaryl, optionally substituted C4-C8 aryloxy, optionally substituted C7-C10 arylalkyl, optionally substituted C5-C10 heteroaryl alkyl group, optionally substituted amine; or R1and R2can together form a 3-7 membered heterocycloalkyl or heteroaryl ring; each R3, R4, R13and R14is independently selected from the group consisting of an optionally substituted C1-C22 alkyl, optionally substituted C2-C22 alkenyl, optionally substituted C2-C22 alkynyl; each R5, R6, R7, R8, R9, R10, R15, and R16is independently selected from the group consisting of H, OH, halo, phenyl, benzyl, optionally substituted C1-C22 alkyl, optionally substituted C2-C22 alkenyl, optionally substituted C2-C22 alkynyl; each of w, x, y, and z is independently an integer from 0-10; each Q is independently an atom selected from O, NH, NR1, and S; each of m is an integer from 0 to 8, preferably 0, 1 , or 2; and each of L1and L2is independently selected from the group consisting of -C(=O)-; -OC(=O)-; -OC(=O)O-; - C(=O)O-; -C(=O)O(CR5R6R7)-; -NH-C(=O)-; -C(=O)NH-; -SO-; - SO2-; -SO3-; -NSO2-; -SO2N- ; -NH((Ci-C8)alkyl)-; -N((Ci-C8)alkyl)2-; -NH((C6)aryl)-; -N((C6)aryl)2-; -NHC(=O)NH-; - NHC(=O)O-; -OC(=O)NH-; -NHC(=O)NR1-; -NHC(=O)O-; -OC(=O)NR1-; -C(=O)R1-; -CO((Ci- C8)alkyl)-; -CO((C6)aryl)-; -CO2((Ci-C8)alkyl)-; - CO2((C6)aryl)-; -SO2((Ci-C8)alkyl)-; and - SO2((C6)aryl)-. In some embodiments, a cationically ionizable lipid is represented by formula CL-V-2: or a pharmaceutically acceptable salt thereof, wherein, as applied to CL-V-2: each R1’, R1, R2, R11, and R12is independently selected from the group consisting of H, an optionally substituted C1-C22 alkyl, optionally substituted C2-C22 alkenyl, optionally substituted C2-C22 alkynyl, optionally substituted C3-C6 cycloalkyl, optionally substituted C4- Ce heterocycloalkyl, optionally substituted C4-C6 alkylcycloalkyl, optionally substituted C4-C6 aryl, optionally substituted C3-C6 heteroaryl, optionally substituted C4-C8 aryloxy, optionally substituted C7-C10 arylalkyl, optionally substituted C5-C10 heteroarylalkyl group, optionally substituted amine; or R1and R2can together form cycloalkyl or heterocycloalkyl ring; if Q is S or O the R1attached to the S or O is an electron pair; each R3and R4is independently selected from the group consisting of an optionally substituted C1-C22 alkyl, optionally substituted C2- C22 alkenyl, optionally substituted C2-C22 alkynyl; each R5, R6, R7, R8, R9, and R10is independently selected from the group consisting of H, OH, halo, phenyl, benzyl, optionally substituted C1-C22 alkyl, optionally substituted C2-C22 alkenyl, optionally substituted C2- C22 alkynyl; each of x, y, and z is independently an integer from 0-10; G and Q are each independently an atom selected from CH, O, N, and S; each of m and n is an integer from 0- 8; and each of L1and L2is independently selected from the group consisting of -C(=O)-; - OC(=O)-; -OC(=O)O-; -C(=O)O-; -C(=O)O(CR1R2R3)-; -NH-C(=O)-; -C(=O)NH-; -SO-; -SO2-; - SO3-; -NSO2-; -SO2N-; -NH((Ci-C8)alkyl)-; -N((Ci-C8)alkyl)2-; -NH((C6)aryl)-; -N((C6)aryl)2-; - NHC(=O)NH-; -NHC(=O)O-; -OC(=O)NH-; -NHC(=O)NR1-; -NHC(=O)O-; -OC(=O)NR1-; - C(=O)R1-; -CO((Ci-C8)alkyl)-; -CO((C6)aryl)-; -CO2((Ci-C8)alkyl)-; -CO2((C6)aryl)-; -SO2((Ci- C8)alkyl)-; and -SO2((C8)aryl)-.

[0473] In some embodiments, a cationically ionizable lipid is selected from: di(heptadecan-9-yl) 3,3’- ((2-(4-methylpiperazin-1-yl)ethyl)azanediyl)dipropionate (BHD-C2C2-PipZ); bis(2- octyldodecyl) 3,3’-((2-(1 -methylpyrrolidin-2-yl)ethyl)azanediyl)dipropionate (BODD-C2C2- 1 Me-Pyr); bis(2-octyldodecyl) 3,3’-((2-(pyrrolidin-1-yl)ethyl)azanediyl)dipropionate (BODD- C2C2-Pyr); bis(2-octyldodecyl) 3,3’-(((1-methylpiperidin-3-yl)methyl)azanediyl)dipropionate (BODD-C2C2-1Me-3PipD); bis(2-octyldodecyl) 3,3’-((2-

[0474] (dimethylamino)ethyl)azanediyl)dipropionate (BODD-C2C2-DMA); bis(2-octyldodecyl) 3,3’- ((4-(4-methylpiperazin-1-yl)butyl)azanediyl)dipropionate (BODD-C2C4-PipZ); bis(2- octyldodecyl) 3,3’-((4-(pyrrolidin-1-yl)butyl)azanediyl)dipropionate (BODD-C2C4-Pyr); and bis(2-hexyldecyl) 3,3’-((4-(4-methylpiperazin-1-yl)butyl)azanediyl)dipropionate (BHD-C2C4- PipZ).

[0475] In some embodiments, a lipid nanoparticle (LNP) comprises a cationic or cationically ionizable lipid selected from the group consisting of: BHD-C2C2-PipZ, BODD-C2C2-1Me-Pyr, ALC- 0315, ALC-366, SM-102, HY-501 , EA-405, HY-405, DODMA, and Dlin-MC3-DMA. In some embodiments, a LNP comprises a cationic or cationically ionizable lipid selected from the group consisting of: BHD-C2C2-PipZ, BODD-C2C2-1 Me-Pyr, ALC-0315, SM-102, HY-501 , and DODMA. In some embodiments, a LNP comprises about 40 mol% to about 50 mol% (e.g., about 47.5 mol%) (relative to the total amount of lipids in a LNP) of a cationic or cationically ionizable lipid selected from the group consisting of: BHD-C2C2-PipZ; BODD-C2C2-1 Me-Pyr; ALC-0315; ALC-0366; SM-102; HY-501 ; EA-405; HY-405; DODMA; and Dlin-MC3-DMA. In some embodiments, a LNP comprises about 40 mol% to about 50 mol% (e.g., about 47.5 mol%) (relative to the total amount of lipids in a LNP) of a cationic or cationically ionizable lipid selected from the group consisting of: BHD-C2C2-PipZ; BODD-C2C2-1 Me-Pyr; ALC-315, SM- 102; HY-501 ; and DODMA.

[0476] (77) Helper lipids

[0477] As described herein, lipid nanoparticles of the present disclosure comprise a helper lipid. In some embodiments, a helper lipid is or comprises phosphatidylcholines, phosphatidylethanolamines, phosphatidylglycerols, phosphatidic acids, phosphatidylserines or sphingomyelin. In some embodiments, a helper lipid is a phospholipid. In some embodiments, a helper lipid is or comprises 1 ,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1 ,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1 ,2-dimyristoyl-sn-glycero-3- phosphocholine (DMPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPO), 1 ,2- dioleoyl-sn-glycero-3-phosphocholine (DOPC), phosphatidylethanolamines such as 1 ,2- dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), distearoyl-phosphatidylethanolamine (DSPE), sphingomyelins, N-palmitoyl-D-erythro-sphingosylphosphorylcholine (SM), 1 ,2-diacylglyceryl-3-O-4’-(N,N,N-trimethyl)-homoserine (DGTS), ceramides, and their derivatives. In some embodiments, a helper lipid is selected from the group consisting of DSPC, DOPC, DMPC, DPPC, POPC, DOPE, DSPE, and SM. In some embodiments, the helper lipid is selected from the group consisting of DSPC, DPPC, DMPC, DOPC, POPC, DOPE and SM. In some embodiments, the helper lipid is DSPC.

[0478] Helper lipids may be synthetic or naturally derived. Other helper lipids suitable for use in a lipid nanoparticle are described in WO 2021 / 026358, WO 2017 / 075531 , and WO 2018 / 081480, the entire contents of each of which are incorporated herein by reference. In some embodiments, a lipid nanoparticle comprises about 5 to about 15 mol% of a helper lipid. In some embodiments, a lipid nanoparticle comprises about 5 to about 15 mol% of a phospholipid. In some embodiments, a lipid nanoparticle comprises about 8 to about 12 mol% of a phospholipid. In some embodiments, a lipid nanoparticle comprises about 10 mol% of a phospholipid. In some embodiments, a lipid nanoparticle comprises about 5 to about 15 mol% of DSPC. In some embodiments, a lipid nanoparticle comprises about 8 to about 12 mol% of DSPC. In some embodiments, a lipid nanoparticle comprises about 10 mol% of DSPC.

[0479] (Hi) Polymer-conjugated lipids

[0480] As described herein, LNPs of the present disclosure comprise a polymer-conjugated lipid. In some embodiments, a polymer-conjugated lipid is a lipid conjugated to polyethylene glycol (a “PEG-lipid”). In some embodiments, a PEG-lipid is selected from pegylated diacylglycerol (PEG-DAG) such as 1 -(monomethoxy-polyethylene glycol)-2,3-dimyristoylglycerol (PEG- DMG) (e.g., 1 ,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (PEG2000- DMG)), a pegylated phosphatidylethanolamine (PEG-PE), a PEG succinate diacylglycerol (PEG-S-DAG) such as 4-O-(2’,3’-di(tetradecanoyloxy)propyl-1-O-(w- methoxy(polyethoxy)ethyl)butanedioate (PEG-S-DMG), 1 ,2-distearoyl-sn-glycero-3- phosphoethanolamine-N-[amino(polyethylene glycol)-2000] (DSPE-PEG2000 amine), a pegylated ceramide (PEG-cer), or a PEG dialkoxypropylcarbamate such as w- methoxy(polyethoxy)ethyl-N-(2,3-di(tetradecanoxy)propyl)carbamate, and 2,3- di(tetradecanoxy)propyl-N-(w-methoxy(polyethoxy)ethyl)carbamate. In some embodiments, a PEG group that is part of a PEG-lipid has, on average in a composition comprising one or more PEG-lipid molecules, a number average molecular weight (Mn) of about 2000 g / mol.

[0481] In some embodiments, a PEG-lipid is DMG-PEG. In some embodiments, a PEG-lipid is PEG2000-DMG:

[0482] In some embodiments, a PEG-lipid is provided in WO 2021 / 026358, WO 2017 / 075531 , or WO 2018 / 081480, each of which is incorporated by reference in its entirety.

[0483] In some embodiments, a PEG-lipid is a compound of Formula PCL-I: or a pharmaceutically acceptable salt thereof, wherein, as applied to formula PGL-I, R8and R9are each independently C10-C30 aliphatic, optionally interrupted by one or more ester bonds, and w is an integer from 30 to 60. In some embodiments, a compound of Formula PCL-I is 2- [(polyethylene glycol)-2000]-N,N-ditetradecylacetamide (ALC-0159). In some embodiments, a compound of Formula PCL-I is: or a pharmaceutically acceptable salt thereof, where n’ is an integer from about 45 to about 50.

[0484] In some embodiments, the PEG-lipid is represented by: wherein n has a mean value ranging from 30 to 60. In some embodiments, n is 50. In one embodiment, the PEG-conjugated lipid (pegylated lipid) is PEG2000-C-DMA which preferably refers to 3-N-[(w-methoxy poly(ethylene glycol)2000)carbamoyl]-1 ,2-dimyristyloxy- propylamine (MPEG-(2 kDa)-C-DMA) or methoxy-polyethylene glycol-2,3-bis(tetradecyloxy) propylcarbamate (2000).

[0485] In some embodiments, a PEG-lipid is selected from PEG-DAG, PEG-PE, PEG-S-DAG, PEG2000-DMG, ALC-159, PEG2000-C-DMA PEG-S-DMG, PEG-cer, and combinations thereof. In some embodiments, a PEG-lipid is ALC-0159 or PEG2000-DMG. In some embodiments, a PEG-lipid is ALC-0159. In some embodiments, a PEG-lipid is PEG2000- DMG.

[0486] In some embodiments, a polymer-conjugated lipid is a polysarcosine-conjugated lipid, also referred to herein as sarcosinylated lipid or pSar-lipid. The term “sarcosinylated lipid” refers to a molecule comprising both a lipid portion and a polysarcosine (poly(N-methylglycine)) portion. In some embodiments, a polymer-conjugated lipid is one described in WO 2024 / 028325, which is incorporated herein by reference in its entirety. In some embodiments, a polymer- conjugated lipid is represented by formula PCL-II: or a pharmaceutically acceptable salt thereof, wherein, as applied to formula PCL-II: X2and X1taken together are optionally substituted amide, optionally substituted thioamide, ester, or thioester; Y is -CH2-, -(CH2)2-, or-(CH2)3-; z is 2 to 24; and n is 1 to 100. In some embodiments of formula PCL-II: (i) when X1is -C(O)- then X2is -NR1-; (ii) when X1is -NR1- then X2is -C(O)- ; (iii) when X1is -C(S)- then X2is -NR1-; (iv) when X1is -NR1- then X2is -C(S)-; (v) when X1is -C(O)- then X2is -O-; (vi) when X1is -O- then X2is -C(O)-; (vii) when X1is -C(S)- then X2is - O-; (viii) when X1is -O- then X2is -C(S)-; (ix) when X1is -C(O)- then X2is -S-; or (x) when X1is -S- then X2is -C(O)-; wherein R1is hydrogen or C1-8 alkyl. In some embodiments of formula PCL-II: (i) when X1is -C(O)- then X2is -NR1-; (ii) when X1is -NR1- then X2is -C(O)-; (iii) when X1is -C(S)- then X2is -NR1-; (iv) when X1is -NR1- then X2is -C(S)-; (v) when X1is -C(O)- then X2is -O-; or (vi) when X1is -O- then X2is -C(O)-; wherein R1is hydrogen or C1-8 alkyl.

[0487] In some embodiments, a polymer-conjugated lipid comprises monomers of 2-(2-(2- aminoethoxy)ethoxy)acetic acid. In some embodiments, the polymer of the polymer- conjugated lipid is or comprises poly-2-(2-(2-aminoethoxy)ethoxy)acetic acid (pAEEA) or poly- 2-(2-(2-methylaminoethoxy)ethoxy)acetic acid (pMAEEA), or a derivative thereof. In some embodiments, a polymer-conjugated lipid comprises monomers of unit PCL-ll-1 : ll-1

[0488] In some embodiments, a polymer-conjugated lipid comprises, 5 to 50, 5 to 25 or 10 to 25 monomers of PCL-ll-1. In some embodiments, a polymer-conjugated lipid comprises 14 to 17 monomers of PCL-ll-1. In some embodiments, a polymer-conjugated lipid comprises 8 to 14 monomers of PCL-ll-1. In some embodiments, a polymer-conjugated lipid is selected from the table below:

[0489] In some embodiments, an LNP comprises an polysarcosine-conjugated or a pAEEA / pMAEEA-conjugated lipid, as described herein. In some embodiments, nucleic acid particles (e.g., DNA or RNA particles) described herein comprise a polysarcosine-conjugated or a pAEEA / pMAEEA-conjugated lipid and are substantially free of a pegylated lipid (or do not contain a pegylated lipid).

[0490] In some embodiments, a lipid nanoparticle comprises about 0.5 to about 5.0 mol% of a polymer-conjugated lipid. In some embodiments, a lipid nanoparticle comprises about 1.0 to about 2.5 mol% of a polymer-conjugated lipid. In some embodiments, a lipid nanoparticle comprises about 1.5 to about 2.0 mol% of a polymer-conjugated lipid. In some embodiments, a lipid nanoparticle comprises about 1.5 to about 1.8 mol% of a polymer-conjugated lipid. In some embodiments, a lipid nanoparticle comprises about 1 .5 mol% to about 1 .8 mol% (relative to the total amount of lipids in a lipid nanoparticle) of a polymer-conjugated lipid selected from the group consisting of: DSPE-AEEA14-AC; VE-AEEA14-AC; ALC-0159 and PEG2000-DMG. In some embodiments, a lipid nanoparticle comprises about 1.5 mol% to about 1.8 mol% (relative to the total amount of lipids in a lipid nanoparticle) of a polymer-conjugated lipid selected from the group consisting of: DSPE-AEEA14-AC, VE-AEEA14-AC, and PEG2000- DMG. In some embodiments, a molar ratio of a cationic or cationically ionizable lipid to a polymer-conjugated lipid is from about 2:1 to about 8:1.

[0491] (iv) Steroids

[0492] As described generally herein, lipid nanoparticles further comprise a steroid. In some embodiments, a steroid is a sterol. In some embodiments, a sterol is p-sitosterol, stigmasterol, cholesterol, cholecalciferol, ergocalciferol, calcipotriol, botulin, lupeol, ursolic acid, oleanolic acid, cycloartenol, lanosterol, or a-tocopherol. In some embodiments, a sterol is cholesterol. In some embodiments, a lipid nanoparticle comprises about 39 to about 49 mol% of a steroid.

[0493] In some embodiments, a lipid nanoparticle comprises about 40 to about 46 mol% of a steroid.

[0494] In some embodiments, a lipid nanoparticle comprises about 40 to about 44 mol% of a steroid.

[0495] In some embodiments, a lipid nanoparticle comprises: about 30 to about 60 mol% of a cationically ionizable lipid; about 18.5 to about 48.5 mol% of a steroid (e.g., cholesterol); about 0 to about 30 mol% of a helper lipid (e.g., DSPC); and about 0 to about 10 mol% of a polymer- conjugated lipid. In some embodiments, a lipid nanoparticle comprises: about 35 to about 55 mol% of a cationically ionizable lipid; about 30 to about 40 mol% of a steroid (e.g., cholesterol); about 5 to about 25 mol% of a helper lipid (e.g., DSPC); and about 0 to about 10 mol% of a polymer-conjugated lipid. In some embodiments, a lipid nanoparticle comprises: about 40 to about 50 mol% of a cationically ionizable lipid; about 30 to about 45 mol% of a steroid (e.g., cholesterol); about 5 to about 15 mol% of a helper lipid (e.g., DSPC); and about 1 to about 2.5 mol% of a polymer-conjugated lipid.

[0496] In some embodiments, a lipid nanoparticle comprises: 47.5 mol% di(heptadecan-9-yl) 3,3’-((2- (4-methylpiperazin-1-yl)ethyl)azanediyl)dipropionate (BHD-C2C2-PipZ); 10 mol% DSPC; 40.7 mol% cholesterol; and 1.8 mol% VE-AEEA14-AC. In some embodiments, a lipid nanoparticle comprises: 47.5 mol% di(heptadecan-9-yl) 3,3’-((2-(4-methylpiperazin-1- yl)ethyl)azanediyl)dipropionate (BHD-C2C2-PipZ); 10 mol% DSPC; 40.7 mol% cholesterol; and 1.8 mol% PEG2000-DMG. In some embodiments, a lipid nanoparticle comprises: about 47.5 mol% of ALC-0315; about 10 mol% of DSPC; about 40.7 mol% of cholesterol; and about 1.8 mol% ofALC-159. In some embodiments, a lipid nanoparticle comprises: about 47.5 mol% of ALC-366; about 10 mol% of DSPC; about 40.7 mol% of cholesterol; and about 1.8 mol% of ALC-159. In some embodiments, a lipid nanoparticle comprises about 50 mol% of SM-102; about 1.5 mol% of PEG2000-DMG; about 10 mol% of DSPC; and about 38.5 mol% of cholesterol. In some embodiments, a lipid nanoparticle comprises: 47.5 mol% bis(2- octyldodecyl) 3,3’-((2-(1 -methylpyrrolidin-2-yl)ethyl)azanediyl)dipropionate (BODD-C2C2- 1 Me-Pyr); 10 mol% DSPC; 40.7 mol% cholesterol; and 1.8 mol% VE-AEEA14-AC. In some embodiments, a lipid nanoparticle comprises: 47.5 mol% bis(2-octyldodecyl) 3,3’-((2-(1- methylpyrrolidin-2-yl)ethyl)azanediyl)dipropionate (BODD-C2C2-1Me-Pyr); 10 mol% DSPC; 40.7 mol% cholesterol; and 1.8 mol% PEG2000-DMG.

[0497] (v) Manufacturing

[0498] Lipids and lipid nanoparticles comprising nucleic acids and their method of preparation are known in the art, including, e.g., as described in U.S. Patent Publication Nos. 2016 / 0009637, 2015 / 0273068, 2014 / 0200257, 2013 / 0338210, 2013 / 0245107, 2013 / 0123338,

[0499] 2013 / 0017223, 2012 / 0183581 , 2012 / 0027803, 2011 / 0311583, 2011 / 0216622, 2011 / 0117125, 2007 / 0042031 , 2006 / 0083780, 2005 / 017054, 2004 / 0142025, 2007 / 0042031 , 1999 / 009076 and PCT Pub. Nos. WO 99 / 39741 , WO 2018 / 081480, WO 2017 / 004143, WO 2017 / 075531 , WO 2015 / 199952, WO 2013 / 086322, WO 2013 / 016058, WO 2013 / 086373, WO 2011 / 141705, WO 2022 / 016089, WO 2022 / 081752, the full disclosures of which are herein incorporated by reference in their entirety for the purposes described herein.

[0500] For example, in some embodiments, cationically ionizable lipids, helper lipids, and steroids are solubilized in an organic solvent such as ethanol, at a predetermined weight or molar ratios / percentages (e.g., ones described herein). In some embodiments, lipid nanoparticles are prepared at a total lipid to nucleic acid (e.g., RNA) weight ratio of approximately 10:1 to 50:1. In some embodiments, such nucleic acid (e.g., RNA) can be diluted to 0.1 to 1.0 mg / mL (e.g., 0.4 mg / mL) in an acidic buffer, such as citrate or acetate having a pH of between about 4 to about 6.

[0501] In some embodiments, using an ethanol injection technique, a colloidal lipid dispersion comprising nucleic acids (e.g., RNAs) can be formed as follows: an ethanol solution comprising lipids, such as cationic lipids, helper lipids, steroids, and polymer-conjugated lipids, is combined with, e.g., injected into or continuously mixed with, an aqueous solution comprising nucleic acids.

[0502] In some embodiments, lipid and nucleic acid (e.g., RNA) solutions can be mixed at room temperature by pumping each solution (e.g., a lipid solution comprising a cationic lipid, a helper lipid, cholesterol, a conjugated lipid, and any other additives) at controlled flow rates into a mixing unit, for example, using piston pumps. In some embodiments, the flow rates of a lipid solution and a nucleic acid (e.g., RNA) solution into a mixing unit are maintained at a ratio of 1 :3. Upon mixing, nucleic acid-lipid particles are formed as the ethanolic lipid solution is diluted with aqueous nucleic acids (e.g., RNAs). The lipid solubility is decreased, while cationic lipids bearing a positive charge interact with the negatively charged nucleic acid (e.g., RNA).

[0503] In some embodiments, a solution comprising nucleic acid (e.g., RNA)-encapsulated lipid nanoparticles can be processed by one or more of concentration adjustment, buffer exchange, formulation, and / or filtration.

[0504] Liposomes

[0505] In some embodiments, a nucleic acid particle is a liposome, wherein the liposome comprises a cationic lipid and a nucleic acid. Liposomes are lipid-based particles that comprise a bilayer (uni-lamellar) or a concentric series of multiple bilayers (multi-lamellar) separated by aqueous compartments, enclosing a central aqueous core that encapsulates the agent for delivery (e.g., a nucleic acid such as RNA). Different types of liposomes are described, including e.g., small and large unilamellar vesicles, multilamellar vesicles, multivesicular liposomes. Many suitable methods are known for manufacturing liposomes (see e.g., Shah S, et al., Adv Drug Deliv Rev. 2020;154-155:102-122), including e.g., solvent evaporation or lipid film hydration, solvent dispersion or reverse phase evaporation, optionally followed by processes to manipulate the size of the liposomes, such as e.g., sonication, homogenization and extrusion. Examples of liposomes that may be suitable for nucleic acid (e.g., RNA) delivery are described in PCT App. Pub. No. WO 2012 / 006378, WO 2013 / 006825, WO 2019 / 077053 and WO 2022 / 069632, each of which is incorporated herein by reference in its entirety.

[0506] In some embodiments, liposomes may be formed from one or more lipids selected from neutral lipids, phospholipids, cholesterol, and / or cationic lipids. In some embodiments, liposomes may comprise one or more phospholipids and optionally cholesterol. Suitable phospholipids for forming liposomes include DSPC, DPPC, DMPC, DOPC, DOPE, and DSPE. In some embodiments, a cationic lipid for use in a liposome is selected from 1 ,2-dioleoyl-3- dimethylammonium propane (DODAP), N,N-dimethyl-(2,3-dioleoyloxypropyl)amine (DODMA), N-(2,3-dioleyloxypropyl)-N,N,N-trimethylammonium chloride (DOTMA); N,N- distearyl-N,N-dimethylammonium bromide (DDAB), N-(2,3-dioleoyloxypropyl)-N,N,N- trimethylammonium chloride (DOTAP), 4-(dimethylamino)butanoic acid, and (10Z,13Z)-1- (9Z,12Z)-9,12-octadecadien-1-yl-10,13-nonadecadien-1-yl ester (Dlin-MC3-DMA). In some embodiments a cationic lipid for use in a liposome is selected from DOTMA and DOTAP. In some embodiments a cationic lipid is DOTMA. In some embodiments, a liposome may further comprise an additional lipid. In some embodiments, an additional lipid is a neutral lipid. As used herein, a “neutral lipid” refers to a lipid having a net charge of zero. Examples of suitable neutral lipids include, but are not limited to, 1 ,2-di-(9Z-octadecenoyl)-glycero-3-phosphoethanolamine (DOPE), 1 ,2-dioleoyl-glycero-3- phosphocholine (DOPC), diacylphosphatidylcholine, diacylphosphatidylethanolamine, ceramide, sphingomyelin, cephalin, cholesterol, and cerebroside. In specific embodiments, the second lipid is DOPE, cholesterol and / or DOPC.

[0507] Lipoplexes (LPX)

[0508] In some embodiments, a nucleic acid particle is a lipoplex, wherein the lipoplex comprises a cationic lipid and a nucleic acid. Lipoplex particles (LPX) may be prepared by mixing liposomes with nucleic acid (e.g., RNA, where lipoplex particles comprising RNA are referred to as “RNA lipoplex particles”). RNA LPX particles typically form spontaneously from electrostatic interactions between positively charged liposomes and negatively charged RNA, and typically have a multilamellar structure. LPX (e.g., RNA LPX) typically comprise one or more cationic lipids and optionally one or more additional lipids. Examples of lipoplexes that are suitable for nucleic acid (e.g., RNA) delivery, as well as methods of manufacture, are described in PCT App. Pub. No. WO 2019 / 077053 and WO 2022 / 069632, each of which is incorporated herein by reference in its entirety.

[0509] In some embodiments, a cationic lipid for use in a LPX is selected from DODAP, DODMA, DOTMA, DDAB, DOTAP, and Dlin-MC3-DMA. In some embodiments a cationic lipid for use in a LPX is selected from DOTMA and DOTAP. In some embodiments a cationic lipid for use in a LPX is DOTMA. In some embodiments, a LPX further comprises an additional lipid. In some embodiments, an additional lipid is a neutral lipid. As used herein, a “neutral lipid” refers to a lipid having a net charge of zero. Examples of suitable neutral lipids include, but are not limited to, DOPE, DOPC, diacylphosphatidyl choline, diacylphosphatidyl ethanol amine, ceramide, sphingomyelin, cephalin, cholesterol, and cerebroside. In specific embodiments, the second lipid is DOPE, cholesterol and / or DOPC.

[0510] In some embodiments, LPX may be manufactured by first preparing liposomes by injecting a solution of the lipids (e.g., DOTMA and DOPE) in ethanol into water or a suitable aqueous phase to form a liposome colloid. LPX may then be prepared by mixing the liposome colloid with a solution comprising nucleic acid (e.g., RNA). In one embodiment, RNA LPX particles comprise DOTMA and DOPE in a molar ratio of from about 10:0 to 1 :9, from about 4:1 to 1 :2, from about 3:1 to about 1 :1 , or about 2:1. In one embodiment, the ratio of positive charges (e.g., in DOTMA) to negative charges (e.g., in the RNA), in the RNA LPX particles, is from about 1 :2 to 1.9:2, or about 1.3:2.0. RNA LPX particles may have an average diameter that ranges from about 200 to about 800 nm, such as from about 300 nm to about 500 nm.

[0511] Polymer-based particles (Polyplexes) and other delivery systems

[0512] In some embodiments, a nucleic acid particle described herein is a polymer-based particle (i.e., a polyplex, PLX). In some embodiments, a nucleic acid particle is a polyplex particle, and comprises a cationic polymer and a nucleic acid. Examples of polyplex particles that are suitable for nucleic acid (e.g., RNA) delivery are described in PCT App. Pub. No. WO 2021 / 001417, which is incorporated herein by reference in its entirety. Nucleic acid polyplex particles typically form spontaneously from electrostatic interactions between positively charged cationic polymer (e.g., PEI) and negatively charged nucleic acid (e.g., RNA). In some embodiments a polyplex particle may further comprise one or more lipids, in which case it may be referred to as a lipopolyplex (LPLX). In some embodiments, a cationic polymer is a polycationic polymer, e.g., a polymer having one or more cationic or cationically ionizable groups. In some embodiments, one or more cationic or ionizable groups comprise a nitrogen atom. Cationic polymers useful for preparing complexes described herein can be homopolymers, heteropolymers, or block-co-polymers.

[0513] In some embodiments, a cationic polymer is poly(ethylenimine), poly(propylenimine), polybrene, polyallylamine, polyvinylamine, polyamidoamine, poly-L-lysine, poly-L-arginine, poly-L-histidine, poly(2-aminoethyl methacrylate), or a pharmaceutically acceptable salt thereof. In some embodiments, a cationic polymer is a homopolymer. It is understood that a cationic polymer described herein can be linear or branched. In some embodiments, a cationic polymer is linear. In some embodiments, a cationic polymer is poly(ethylenimine).

[0514] In some embodiments, a cationic polymer is a heteropolymer (e.g., a linear heteropolymer) comprising copolymers of one or more of poly(ethylenimine), poly(propylenimine), polybrene, polyallylamine, polyvinylamine, polyamidoamine, poly-L-lysine, poly-L-arginine, poly-L- histidine, and poly(2-aminoethyl methacrylate), or a pharmaceutically acceptable salt thereof. In some embodiments, a cationic polymer is a heteropolymer comprising poly(ethylenimine) and poly(propylenimine).

[0515] In some embodiments, a cationic polymer has between 250 and 2000 repeating monomer units (such as between 1500 and 2000 repeating monomer units). In some embodiments, a cationic polymer is a polymer described herein, having a number average molecular weight (Mn) of about 600 Daltons (Da) to about 400,000 Da (such as about 20,000 to about 120,000 Da).

[0516] In some embodiments, a complex comprises a cationic polymer and a nucleic acid, wherein the cationic polymer is or comprises a polyamine derivative (e.g., a carboxylated polyamine derivative). Suitable polyamine derivatives for delivery of nucleic acids, such as RNA, are described in WO 2014 / 053245 and WO 2014 / 056590, both of which are incorporated herein by reference in their entirety.

[0517] In some embodiments, a polyamine derivative comprises: a polyamine moiety comprising a plurality of amino groups; a plurality of carboxylated substituents comprising a carboxyl group bonded via a hydrophobic linker to amino groups of said polyamine moiety, wherein each of said carboxylated substituents comprises from 6 to 40 carbon atoms, preferably from 6 to 20 carbon atoms, and more preferably from 8 to 16 carbon atoms, and each of said hydrophobic linker may comprise from 1 to 3 heteroatoms selected from O, N, and S; and a plurality of hydrophobic substituents bonded to amino groups of said polyamine moiety, wherein each of said hydrophobic substituents comprises at least 2 carbon atoms, preferably from 6 to 40 carbon atoms, and may comprise from 1 to 3 heteroatoms selected from O, N, and S provided said hydrophobic substituent has at least 6 carbon atoms.

[0518] In some embodiments, a polyamine derivative which is useful herein as delivery vehicle for polyanions is a polyalkylenimine (e.g., polyethylenimine) derivative having one or more carboxyalkyl substituents comprising from 6 to 40 carbon atoms, and one or more hydrophobic substituents selected from hydrocarbon substituents having at least 2 carbon atoms, preferably from 6 to 40 carbon atoms, wherein each of said hydrophobic substituents may be or may comprise an alkyl group and / or each of said hydrophobic substituents may be or may comprise an aryl group.

[0519] In some embodiments, the polyamine derivative has (i) a linear polyethylenimine moiety of from 2 to 500 kDa (in terms of number average molecular weight), or (ii) a branched polyethylenimine moiety of from 0.5 to 200 kDa (in terms of number average molecular weight); and the carboxylated substituents have from 10 to 16 carbon atoms and are n- alkylcarboxylic acids and the hydrophobic substituents have from 1 to 12 carbon atoms and are alkyls, preferably n-alkyls, and / or alkylarylalkyls. Other suitable polymers include, for example, Viromer® and jetPEI® (Polyplus). Other suitable polymers or lipidoids useful for delivery of nucleic acids, such as RNA, are described in WO 2014 / 207231 , WO 2016 / 097377 and WO 2024 / 042236, all of which are incorporated herein by reference in their entirety. Other delivery systems suitable for nucleic acid (e.g., RNA) delivery, which are based on oligosaccharide compounds, are described in WO 2023 / 067121 , WO 2023 / 067123, WO 2023 / 067124, WO 2023 / 067125, and WO 2023 / 067126, all of which are incorporated herein by reference in their entirety.

[0520] In order to improve the targeting of LNPs and the delivery of payloads to specific target cells, ligands (e.g. antibodies or nanobodies) for specific target molecules (e.g. cell-specific receptors) can be provided on the surface of the LNPs. As used herein, such LNPs may be termed as “functionalized particles”, “functionalized lipid particles”, or “functionalized LNPs”.

[0521] Such functionalized LNPs may comprise, bind to or interact with, a compound comprising a targeting moiety that binds a target on target cells. Thus, a “functionalized LNP” may be understood as a particle that exhibits preferential interaction with target cells expressing or exhibiting a particular primary target as defined herein (such as a marker or antigen, preferably on the cell surface) which is preferentially recognized by the targeting moiety of the particle.

[0522] Usually, a receptor-specific ligand is chosen based on its affinity towards a specific cell type, e.g., selecting CD3 receptors that are found on the surface of T cells for modulating the immune system. Thus, immobilizing ligands on the surface of a LNP may improve cell-specific targeting and internalization through receptor-mediated endocytosis followed by endosomal escape and delivery of the cargo into the cytosol.

[0523] In some embodiments, the LNPs are not functionalised LNPs (i.e. non functionalised LNPs).

[0524] In some embodiments, the LNPs are functionalised LNPs comprising one or more moieties.

[0525] In some embodiments, the one or more moieties are present on the surface of the LNPs.

[0526] It will be understood that “present on the surface of the LNPs” may mean that the one or more moieties are (i) attached to the surface of the LNPs; or (ii) otherwise connected to the surface of the LNPs, e.g. through an intermediate molecule.

[0527] In some embodiments, the one or more moieties each comprise a targeting domain.

[0528] In some embodiments, the targeting domain comprises one or more protein-based molecules. In some embodiments, the protein-based molecules are selected from the list consisting of: an antibody or fragment thereof, a peptide, a receptor, and a cell surface molecule.

[0529] In some embodiments, the targeting domain comprises an antibody or fragment thereof.

[0530] As used herein, “antibody” may refer to a protein or polypeptide having an antigen binding site or antigen-binding domain which comprises at least one complementarity determining region (CDR).

[0531] In some embodiments, the antibody or fragment thereof may comprise a heavy chain variable (VH) domain. In some embodiments, the antibody or fragment thereof may comprise a light chain variable (VL) domain. In some embodiments, the antibody or fragment thereof may comprise a VH domain and a VL domain.

[0532] In some embodiments, the VH domain may comprise one or more complementarity determining regions (CDRs). In some embodiments, the VH domain may comprise one, two or three CDRs. In some embodiments, the VH domain may comprise three CDRs. It will be understood that CDRs of the VH domain may be termed HCDRs. It will also be understood that each of the three CDRs of the VH domain may be termed HCDR1 , HCDR2 and HCDR3 respectively.

[0533] In some embodiments, the VL domain may comprise one or more CDRs. In some embodiments, the VL domain may comprise one, two or three CDRs. In some embodiments, the VL domain may comprise three CDRs. It will be understood that CDRs of the VL domain may be termed LCDRs. It will also be understood that each of the three CDRs of the VL domain may be termed LCDR1 , LCDR2 and LCDR3 respectively.

[0534] In some embodiments, the antibody may comprise 6 complementarity determining regions (CDRs). It will be understood that such an antibody may be a classical antibody molecule. In some embodiments, the antibody may comprise 3 CDRs and have an antigen binding site which is equivalent to that of a single domain antibody (sdAb). A sdAb (i.e. a nanobody) may be defined as an antibody fragment comprising of a single monomeric variable antibody domain. The sdAb may be a single chain variable domain which may be a heavy chain variable (VH) domain or light chain variable (VL) domain, having 3 CDRs.

[0535] “Heavy chain variable region” or “VH” refers to the fragment of the heavy chain of an antigenbinding domain or antibody that contains three CDRs interposed between flanking stretches known as framework regions, which are more highly conserved than the CDRs and form a scaffold to support the CDRs. “Light chain variable region” or “VL” refers to the fragment of the light chain of an antigen-binding domain or antibody that contains three CDRs interposed between framework regions.

[0536] “Complementarity determining region” or “CDR” with regard to an antigen-binding domain or antibody or antigen-binding fragment thereof refers to a highly variable loop in the variable region of the heavy chain of the light chain of an antibody. CDRs can interact with the antigen conformation and largely determine binding to the antigen (although some framework regions are known to be involved in binding). The heavy chain variable region and the light chain variable region each contain 3 CDRs (heavy chain CDRs 1 , 2 and 3 and light chain CDRs 1 , 2 and 3, numbered from the amino to the carboxy terminus).

[0537] A number of definitions of the CDRs are commonly in use. The Kabat definition is based on sequence variability and is the most commonly used (see http: / / www.bioinf.org.uk / abs / ). The ImMunoGeneTics information system (IMGT) (see http: / / www.imgt.org) can also be used. According to this system, a complementarity determining region (CDR-IMGT) is a loop region of a variable domain, delimited according to the IMGT unique numbering for V domain. There are three CDR-IMGT in a variable domain: CDR1-IMGT (loop BC), CDR2-IMGT (loop C'C"), and CDR3-IMGT (loop FG). Other definitions of the CDRs have also been developed, such as the Chothia, the AbM and the contact definitions (see http: / / www.imgt.org). The CDRs of the sdAbs according to the present invention may be defined using any suitable system, such as any suitable system known in the art.

[0538] In some embodiments, the antibody or fragment thereof may be selected from the list consisting of: a single-chain variable fragment (scFv), a Fab, a F(ab)’2, a Fv, a single domain antibody, a nanobody, a VHH antibody, a monoclonal antibody or fragment thereof, a humanized antibody or fragment thereof, a chimeric antibody or fragment thereof, a bifunctional antibody, and a bispecific antibody.

[0539] In some embodiments, the antibody or fragment thereof comprises a nanobody.

[0540] In some embodiments, the antibody or fragment thereof comprises a scFv.

[0541] In some embodiments, the antibody or fragment thereof comprises a camelid heavy chain- only antibody (VHH). In some embodiments, the targeting domain is capable of binding to T cells, B cells, NK cells, monocytes, macrophages, mast cells, basophils, eosinophils or dendritic cells.

[0542] In some embodiments, the antibody or fragment thereof is capable of binding to T cells, B cells, NK cells, monocytes, macrophages, mast cells, basophils, eosinophils or dendritic cells.

[0543] In some embodiments, the targeting domain is a T cell targeting domain, a NK cell targeting domain or a B cell targeting domain.

[0544] In some embodiments, the antibody or fragment thereof is a T cell targeting antibody or fragment thereof, a NK cell targeting antibody or fragment thereof or a B cell targeting antibody or fragment thereof.

[0545] In some embodiments, the targeting domain is a T cell targeting domain. In other words, the targeting domain is capable of specifically binding to a cell surface marker expressed on T cells.

[0546] In some embodiments, the antibody or fragment thereof is a T cell targeting antibody or fragment thereof. In other words, the antibody or fragment thereof is capable of specifically binding to a cell surface marker expressed on T cells.

[0547] In some embodiments, the targeting domain is a B cell targeting domain. In other words, the targeting domain is capable of specifically binding to a cell surface marker expressed on B cells.

[0548] In some embodiments, the antibody or fragment thereof is a B cell targeting antibody or fragment thereof. In other words, the antibody or fragment thereof is capable of specifically binding to a cell surface marker expressed on B cells.

[0549] In some embodiments, the targeting domain is a NK cell targeting domain. In other words, the targeting domain is capable of specifically binding to a cell surface marker expressed on NK cells.

[0550] In some embodiments, the antibody or fragment thereof is a NK cell targeting antibody or fragment thereof. In other words, the antibody or fragment thereof is capable of specifically binding to a cell surface marker expressed on NK cells. In some embodiments, the T cell targeting domain targets CD3.

[0551] In some embodiments, the antibody or fragment thereof binds to CD3.

[0552] In some embodiments, the T cell targeting domain targeting CD3 is a nanobody, a scFv, or a VHH.

[0553] In some embodiments, the antibody or fragment thereof binding to CD3 is a nanobody, a scFv, or a VHH.

[0554] In some embodiments, each moiety is directly linked to an LNP.

[0555] In some embodiments, each moiety is indirectly linked to an LNP.

[0556] In some embodiments, each moiety is linked to an LNP by an intermediate linker molecule.

[0557] In some embodiments, each moiety may further comprise a binding domain that is capable of binding to an LNP.

[0558] In some embodiments, each moiety may further comprise a binding domain that is capable of binding to an LNP, wherein the binding domain is capable of binding to an intermediate linker molecule present on the surface of the LNP.

[0559] In other words, each moiety may comprise:

[0560] (i) a targeting domain (such as the antibody or fragment thereof described above); and

[0561] (ii) a binding domain capable of binding to an intermediate linker molecule present on the surface of the LNP.

[0562] In some embodiments, the targeting domain and the binding domain are linked by a spacer.

[0563] In some embodiments, the binding domain that is capable of binding to the intermediate linker molecule present on the surface of the LNP may also comprise an antibody or fragment thereof.

[0564] It will be understood that when the binding domain comprises an antibody or fragment thereof capable of binding to the intermediate linker molecule present on the surface of the LNP, this antibody or fragment thereof binds to a different target than the antibody or fragment thereof of the targeting domain as described above.

[0565] In some embodiments, the binding domain comprises an antibody or fragment thereof comprising a nanobody, a scFv, or a VHH.

[0566] In some embodiments, the intermediate linker molecule comprises a portion capable of inserting / integrating into the LNP.

[0567] In some embodiments, the intermediate linker molecule comprises a lipid component and a peptide component. In some embodiments, the lipid component and peptide component are linked by a spacer.

[0568] It will be understood that the lipid component of the intermediate linker molecule can insert / integrate into the LNP. It will be understood that the peptide component of the intermediate linker molecule can bind to the binding domain described above.

[0569] In some embodiments, the peptide component comprises a peptide tag, an affinity tag and / or an epitope tag.

[0570] In some embodiments, the epitope tag may be any epitope tag as defined in W02020053239.

[0571] A peptide tag may comprise from about 5 to about 50 amino acids. In some embodiments, the peptide tag comprises from about 8 to about 30 amino acids. In some embodiments, the peptide tag comprises from about 10 to about 20 amino acids.

[0572] In some embodiments, the peptide component comprises an ALFA peptide tag.

[0573] In some embodiments, an ALFA peptide tag comprises the amino acid sequence: -AA0-AA1 -AA2-AA3-AA4-AA5-AA6-AA7-AA8-AA9-AA10-AA11 -AA12-AA13-AA14-, wherein the amino acids of AA0, AA1 , AA2, AA3, AA4, AA5, AA6, AA7, AA8, AA9, AA10, AA11 , AA12, AA13 and AA14 are:

[0574] AA0 is Pro or deleted;

[0575] AA1 is Ser, Gly, Thr, or Pro;

[0576] AA2 is Arg, Gly, Ala, Glu, or Pro;

[0577] AA3 is Leu, lie, or Vai; AA4 is Glu or Gin;

[0578] AA5 is Glu or Gin;

[0579] AA6 is Glu or Gin;

[0580] AA7 is Leu, lie, or Vai;

[0581] AA8 is Arg, Ala, Gin, or Glu;

[0582] AA9 is Arg, Ala, Gin, or Glu;

[0583] AA10 is Arg;

[0584] AA11 is Leu;

[0585] AA12 is Thr, Ser, Asp, Glu, Pro, Ala, or deleted;

[0586] AA13 is Glu, Lys, Pro, Ser, Ala, Asp, or deleted; and

[0587] AA14 is Pro or deleted.

[0588] In some embodiments, the lipid component comprises a polyethylene glycol (PEG) lipid.

[0589] In some embodiments, the peptide component comprises an ALFA peptide tag and the lipid component comprises a polyethylene glycol (PEG) lipid.

[0590] In some embodiments, the binding domain comprises an anti-ALFA binding domain.

[0591] In some embodiments, the binding domain comprises an antibody or fragment thereof comprising an anti-ALFA binding domain.

[0592] In some embodiments, the anti-ALFA binding domain is a nanobody, a scFv, or a VHH.

[0593] In some embodiments, the anti-ALFA binding domain is a nanobody (NbALFA).

[0594] In some embodiments, the targeting domain comprises a VHH, and the binding domain comprises NbALFA.

[0595] In some embodiments, the targeting domain comprises a VHH binding to CD3, and the binding domain comprises NbALFA.

[0596] OTHER EMBODIMENTS

[0597] The present invention also provides a method of purifying nucleic acid from a composition of LNPs comprising a cargo of nucleic acids, comprising the steps of: (i) incubating a sample of the composition with a reagent that releases the cargo from the LNPs;

[0598] (ii) purifying the nucleic acids in the sample;

[0599] (iii) incubating the purified nucleic acids with an enzyme, wherein the enzyme is a DNase or an RNase;;

[0600] (iv) incubating the result of step (iii) with proteinase K; and

[0601] (v) purifying the resulting product obtained from step (iv).

[0602] It will be understood that the embodiments and definitions of the LNPs, cargo, nucleic acids, and steps (i) to (v) described above with regards to the method of measuring nucleic acid integrity may also apply to the method of purifying nucleic acid from a composition of LNPs as described herein.

[0603] In some embodiments where the nucleic acid to be purified is RNA, the method is performed in the presence of an RNase inhibitor.

[0604] In some embodiments where the nucleic acid to be purified is RNA, the method is performed in RNase-free conditions.

[0605] In some embodiments where the nucleic acid to be purified is DNA, the method is performed in the presence of a DNase inhibitor.

[0606] In some embodiments where the nucleic acid to be purified is DNA, the method is performed in DNase-free conditions.

[0607] GENERAL TERMS AND DEFINITIONS

[0608] This disclosure is not limited by the exemplary methods and materials disclosed herein, and any methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of this disclosure. Numeric ranges are inclusive of the numbers defining the range. Unless otherwise indicated, any nucleic acid sequences are written left to right in 5' to 3' orientation; amino acid sequences are written left to right in amino to carboxy orientation, respectively.

[0609] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limits of that range is also specifically disclosed. Each smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in that stated range is encompassed within this disclosure. The upper and lower limits of these smaller ranges may independently be included or excluded in the range, and each range where either, neither or both limits are included in the smaller ranges is also encompassed within this disclosure, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in this disclosure.

[0610] It must be noted that as used herein and in the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise.

[0611] The terms "comprising", "comprises" and "comprised of' as used herein are synonymous with "including", "includes" or "containing", "contains", and are inclusive or open-ended and do not exclude additional, non-recited members, elements or method steps. The terms "comprising", "comprises" and "comprised of' also include the term "consisting of.

[0612] The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that such publications constitute prior art to the claims appended hereto.

[0613] NUMBERED PARAGRAPHS

[0614] The present invention may be described by way of the following numbered paragraphs:

[0615] 1 . A method of measuring nucleic acid integrity in a composition of lipid nanoparticles (LNPs) comprising a cargo of nucleic acids, wherein the method comprises the steps of:

[0616] (i) incubating a sample of the composition with a reagent that releases the cargo from the LNPs;

[0617] (ii) purifying the nucleic acids in the sample;

[0618] (iii) incubating the purified nucleic acids with an enzyme, wherein the enzyme is a DNase or an RNase;

[0619] (iv) incubating the resulting product obtained from step (iii) with proteinase K;

[0620] (v) purifying the resulting product obtained from step (iv); and

[0621] (vi) measuring the nucleic acid integrity in the sample.

[0622] 2. The method according to paragraph 1 , wherein the reagent in step (i) comprises a detergent.

[0623] 3. The method according to paragraph 2, wherein the detergent is selected from the list consisting of: 2-[4-(2,4,4-trimethylpentan-2-yl)phenoxy]ethanol, N-tetradecyl-N,N-dimethyl-3- ammonio-1 -propanesulfonate, and sodium dodecyl sulfate (SDS).

[0624] 4. The method according to paragraph 2 or paragraph 3, wherein the detergent is Triton; optionally wherein the Triton is Triton X-100.

[0625] 5. The method according to paragraph 4, wherein the concentration of the Triton is 10- 40% m / v, optionally wherein the concentration of the Triton is 15-25% m / v.

[0626] 6. The method according to paragraph 5, wherein the concentration of the T riton is 20% m / v.

[0627] 7. The method according to any one of paragraphs 1-6, wherein the reagent in step (i) further comprises an alcohol.

[0628] 8. The method according to paragraph 7, wherein the alcohol is ethanol.

[0629] 9. The method according to paragraph 8, wherein the concentration of the ethanol is 10-40% v / v, optionally wherein the concentration of the ethanol is 25-35% v / v. 10. The method according to paragraph 9, wherein the concentration of the ethanol is 30% v / v.

[0630] 11. The method according to any one of paragraphs 1-10, wherein the nucleic acid integrity is RNA integrity and the enzyme in step (iii) is a DNase.

[0631] 12. The method according to any one of paragraphs 1-10, wherein the nucleic acid integrity is DNA integrity and the enzyme in step (iii) is an RNase.

[0632] 13. The method according to any one of paragraphs 1-11, wherein the DNase in step (iii) is selected from the list consisting of: DNase-H, DNase I, DNase1L1, DNase 1L2, DNase1 L3, DNase II a and DNase II [3.

[0633] 14. The method according to paragraph 13, wherein the DNase is DNase-l.

[0634] 15. The method according to any one of paragraphs 1-11, 13 or 14, wherein the concentration of the DNase in the sample in step (iii) is 0.01-1 U / pL; optionally wherein the concentration of the DNase in the sample in step (iii) is 0.01-0.4 U / pL.

[0635] 16. The method according to paragraph 15, wherein the concentration of the DNase in the sample in step (iii) is about 0.03 ll / pl.

[0636] 17. The method according to any one of paragraphs 1-11 or 13-15, wherein all of the DNA in step (iii) is digested by the DNase.

[0637] 18. The method according to any one of paragraphs 1-10, or 12, wherein the RNase is selected from the list consisting of RNase I, RNase II, RNase III, RNase H, RNase L, RNase P, RNase, PhyM, RNase T1 , RNase T2, RNase U2, Rnase V, RNase E, RNase G, and Monarch® RNase.

[0638] 19. The method according to paragraph 18, wherein the RNase is Monarch® RNase.

[0639] 20. The method according to any one of paragraphs 1-10, 12, 18 or 19, wherein the concentration of the RNase in the sample in step (iii) is 0.1-2 mg / ml; optionally wherein the concentration of the RNase in the sample in step (iii) is 0.1-1 mg / ml 21. The method according to paragraphs 20, wherein the concentration of the RNase in the sample in step (iii) is about 0.65 mg / ml.

[0640] 22. The method according to any one of paragraphs 1-10, 12 or 18-21, wherein all of the RNA in step (iii) is digested by the RNase.

[0641] 23. The method according to any one of paragraphs 1-22, wherein all of the enzyme from step (iii) is digested by the proteinase K in step (iv).

[0642] 24. The method according to any one of paragraphs 1-23, wherein the concentration of the proteinase K in the sample in step (iv) is 0.1-2 U / rnL.

[0643] 25. The method according to any one of paragraphs 1-24, wherein the concentration of the proteinase K in the sample in step (iv) is about 1 U / rnL.

[0644] 26. The method according to any one of paragraphs 1-25, wherein the nucleic acid integrity measured in step (vi) is determined by analysing nucleic acid size and / or nucleic acid size distribution.

[0645] 27. The method according to any one of paragraphs 1-26, wherein the nucleic acid integrity is measured using capillary gel electrophoresis or classical gel electrophoresis.

[0646] 28. The method according to paragraph 27, wherein the nucleic acid integrity is measured using capillary gel electrophoresis

[0647] 29. The method according to paragraph 28, wherein the nucleic acid integrity is measured using a Fragment Analyser.

[0648] 30. The method according to any one of paragraphs 1-29, wherein the LNPs are functionalised LNPs comprising one or more moieties.

[0649] 31. The method according to paragraph 30, wherein the one or more moieties are present on the surface of the LNPs; optionally wherein the one or more moieties comprise a targeting domain.

[0650] 32. The method according to any one of paragraphs 1-31 , wherein the cargo of nucleic acids comprises (i) DNA, (ii) RNA; or (iii) DNA and RNA. 33. The method according to paragraph 32, wherein the cargo of nucleic acids comprises DNA and RNA.

[0651] 34. The method according to any one of paragraphs 1-33, wherein the nucleic acids purified in step (ii) are DNA or RNA.

[0652] 35. The method according to any one of paragraphs 1-33, wherein the nucleic acids purified in step (ii) are DNA and RNA.

[0653] 36. The method according to any one of paragraphs 32-35, wherein the DNA is selected from the list consisting of: genomic DNA and cDNA.

[0654] 37. The method according to any one of paragraphs 32-36, wherein the RNA is selected from the list consisting of: mRNA, miRNA, siRNA and shRNA.

[0655] 38. A method of purifying nucleic acid from a composition of LNPs comprising a cargo of nucleic acids, comprising the steps of:

[0656] (i) incubating a sample of the composition with a reagent that releases the cargo from the LNPs;

[0657] (ii) purifying the nucleic acids in the sample;

[0658] (iii) incubating the purified nucleic acids with an enzyme, wherein the enzyme is a DNase or an RNase;;

[0659] (iv) incubating the result of step (iii) with proteinase K; and

[0660] (v) purifying the resulting product obtained from step (iv).

[0661] The invention will now be further described by way of Examples, which are meant to serve to assist one of ordinary skill in the art in carrying out the invention and are not intended in any way to limit the scope of the invention. EXAMPLES

[0662] Example 1

[0663] In order to study the impact the LNP disruption buffer, also referred to herein as RNA releasing buffer, samples of RNA (100 pg / mL) in nuclease free water (Fig.1 (-) RNA releasing buffer) or in LNP disruption buffer (aqueous solution containing 20% Triton X-100 (m / v) and 30% EtOH (v / v), Fig.1 (+) RNA releasing buffer) were treated with different concentrations of DNase-l in the presence of 10x TRIS MgCI2 buffer pH 7.5.. Final concentrations of DNase-l in the samples ranged from 0.5 U / pL to 4 LI / pL (cf. Fig.1 ) that were prepared out of a 50 ll / pl DNase- I stock (EN0523, Thermo Fisher Scientific) .

[0664] After 20 minutes incubation at 37 °C, RNA was isolated by using the QIAGEN RNeasy purification kit according to manufacturer's protocol. The RNA integrity was measured using capillary electrophoresis (Agilent Fragment Analyzer, Standard Sensitive RNA integrity kit, Agilent; Prerun 8.0 kV, 30 sec., Sample Injection 5.0 kV, 4 sec., Separation 8.0 kV, 40.0 min.). Samples treated with 0 LI / pL DNase but in the presence of 10x TRIS MgCI2 buffer pH 7.5 and naked RNA in nuclease free water without any DNase-l treatment and without the presence of 10x TRIS MgCI2 buffer pH 7.5 served as control groups.

[0665] Although, and among other DNase enzymes known in the art, DNase-l is described as highly specific for DNA, a decreasing RNA integrity was measured at increasing DNase-l concentrations. Surprisingly, this effect was massively increased in the presence of LNP disruption buffer, i.e. it was not possible to reliable measure the RNA integrity of the RNA of an LNP encapsulated DNA / RNA cargo mix after DNA digest and / or LNP disruption.

[0666] Example 2

[0667] LNPs (in LNP buffer: 20 mM Hepes buffer pH 6.0), optionally comprising a ligand, containing a 1 :1 DNA-RNA cargo mix (50 pg / ml DNA and 50 pg / ml RNA, i.e. total 100 pg / mL ) were treated with an aqueous solution containing 20% Triton X-100 (m / v) and 30% EtOH (v / v) for LNP disruption [(1) Cargo release].

[0668] The RNA-DNA cargo mix was purified from the other ingredients (e.g. LNP disruption buffer, disrupted LNPs, ligands) by using the QIAGEN RNeasy kit according to manufacturer's protocol [(2) First purification of free cargo]. The RNA-DNA cargo mix was mixed with DNase- I (stock solution (1 U / pL), RNase-frei / #Cat. 10849700 / Fisher Scientific GmbH) and 10x TRIS MgCI2 buffer pH 7.5. DNase-1 concentration in the sample was about 0.03 LI / pL [(3) Digestion of DNA],

[0669] Afterwards, DNase-1 and optionally LNP ligands were digested by adding Proteinase-K with an incubation for 10 min at 37 °C [(4) Digestion of DNase and optionally the LNP ligands], RNA was purified by using the QIAGEN RNeasy kit according to manufacturer's protocol [(5) Second purification)] and analyzed by capillary electrophoresis (Agilent Fragment Analyzer; Modified Standard Sensitive Protocol; Prerun 8.0 kV, 30 sec., Sample Injection 7.0 kV, 8 sec., Separation 8.0 kV, 40.0 min.). This workflow is visualized in Fig. 2.

[0670] Example 3

[0671] Functionalized LNPs , i.e. LNPs comprising a ligand, containing a 1 :1 DNA-RNA cargo mix (50 pg / ml DNA and 50 pg / ml RNA, i.e. total 100 pg / mL were treated as follows:

[0672] • Sample #1 / #2 = RNA-DNA cargo mix encapsulated in LNPs (LNP buffer: 20 mM Hepes buffer pH 6.0) treated as described in Example 2;

[0673] • Sample #3 / #4 = Non-LNP encapsulated RNA-DNA cargo mix in 20 mM Hepes buffer pH 6.0 and treated such as #1 I #2;

[0674] • Sample #5 / #6 = RNA-DNA cargo mix encapsulated in LNPs treated as described in example 2 without the first purification step after LNP disruption;

[0675] • Sample #7 / #8 = Non-LNP encapsulated RNA-DNA cargo mix in 20 mM Hepes buffer pH 6.0 and treated such as #5 1 #&,

[0676] • Sample #9 = RNA as was used for DNA / RNA cargo samples stored in 10 mM HPESES / EDTA pH 7.1 was used as RNA control sample;

[0677] • Sample #10 = DNA as was used for DNA / RNA cargo samples stored nuclease free water as DNA control sample.

[0678] Technical duplicates measured as triplicates in Fragment Analyzer.

[0679] To summarize the results, it shows that not only the LNP disruption buffer but also the residual disrupted LNPs and / or ligands affect the RNA integrity measurement. The assay according to the invention surprisingly showed that it effectively prevents DNA from interfering with RNA in integrity measurement by either overlapping with the RNA peak profile or forming RNA-DNA aggregates (see also Figure 4 A). By using this assay, reliable RNA integrity measurement results can be achieved since also DNase, LNP disruption buffer, disrupted LNPs and / or ligands are removed from the sample (see also Figure 4 B). Example 4

[0680] In order to compare the impact of DNase digestion and purification steps on RNA integrity measurement, functionalized LNPs containing a 1 :1 DNA-RNA cargo mix (50 pg / ml DNA and 50 pg / ml RNA, i.e. total 100 pg / mL ) were used.

[0681] A first sample (Fig. 4A) was treated with an aqueous solution containing 20% Triton X-100 (m / v) and 30% EtOH (v / v) for LNP disruption without a further digestion step with DNase-l or purification steps and RNA integrity was analyzed by capillary electrophoresis (Agilent Fragment Analyzer; Modified Standard Sensitive Protocol) A second sample was treated as described in Example 2 (Fig. 4B).

[0682] While for the second sample highly pure RNA was measured and RNA integrity was measurable, it was not possible to measure RNA integrity for the first sample due to contamination of DNA and / or protein / ligand and / or disrupted LNPs

[0683] Example 5

[0684] The protocol as described in example 2 was modified to allow for the quality measurement of the DNA of a LNP encapsulating a DNA-RNA cargo mix and optionally comprising a ligand on its surface.

[0685] LNPs, optionally comprising a ligand, containing a 1 :1 DNA-RNA cargo mix (total content of 100 pg / mL ) were treated with an aqueous solution containing 20% Triton X-100 (m / v) and 30% EtOH (v / v) for LNP disruption [(1) Cargo release]. The RNA-DNA cargo mix was purified from the other ingredients (LNP disruption buffer, disrupted LNPs, ligands) by using the QIAGEN RNeasy kit according to manufacturer's protocol [(2) First purification of free cargo].

[0686] The RNA-DNA cargo mix was treated with Monarch® RNase A (T3018L, New England Biolabs) with and 10x TRIS MgCI2 buffer pH 7.5. Monarch® RNase A concentration in the sample was about 0.65 mg / mL [(3) Digestion of RNA], Afterwards, Monarch® RNase Al was digested by adding Proteinase-K with an incubation for 10 min at 37 °C [(4) Digestion of Monarch® RNase A and optionally the residual LNP ligands],

[0687] DNA was purified by using the QIAGEN RNeasy kit according to manufacturer's protocol [(5) Second purification)] and analyzed by capillary electrophoresis (Agilent Fragment Analyzer; Intern Plasmid DNA Protocol; Prerun 10.0 kV, 30 sec., Sample Injection 5.0 kV, 15 sec., Separation 10.0 kV, 30.0 min.). This workflow is visualized in Fig. 5.

[0688] Example 6

[0689] To compare the impact on the enzymatic sample preparation for DNA or RNA integrity measurement in samples comprising a RNA-DNA cargo mix in LNPs, DNA integrity or RNA integrity was measured in samples as follows (data shown in Figure 6):

[0690] A) DNA integrity was measured by capillary electrophoresis (Agilent Fragment Analyzer; Intern Plasmid DNA Protocol; Prerun 10.0 kV, 30 sec., Sample Injection 5.0 kV, 15 sec., Separation 10.0 kV, 30.0 min.) without any RNase sample treatment;

[0691] B) RNA integrity was measured by capillary electrophoresis (Agilent Fragment Analyzer; Standard Sensitive RNA integrity kit, Agilent; Prerun 8.0 kV, 30 sec., Sample Injection 5.0 kV, 4 sec., Separation 8.0 kV, 40.0 min.) without any DNase treatment;

[0692] C) DNA integrity was measured by capillary electrophoresis (Agilent Fragment Analyzer; Intern Plasmid DNA Protocol; Prerun 10.0 kV, 30 sec., Sample Injection 5.0 kV, 15 sec., Separation 10.0 kV, 30.0 min.) after sample treatment as described in example 5 (see Figure 5);

[0693] D) RNA integrity was measured by capillary electrophoresis (Agilent Fragment Analyzer; Standard Sensitive RNA integrity kit, Agilent; Prerun 8.0 kV, 30 sec., Sample Injection 5.0 kV, 4 sec., Separation 8.0 kV, 40.0 min.) after sample treatment as described in example 2 (see figure 2).

[0694] All publications mentioned in the above specification are herein incorporated by reference. Various modifications and variations of the described methods and system of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention which are obvious to those skilled in molecular biology or related fields are intended to be within the scope of the following claims.

Claims

CLAIMS1. A method of measuring nucleic acid integrity in a composition of lipid nanoparticles (LNPs) comprising a cargo of nucleic acids, wherein the method comprises the steps of:(i) incubating a sample of the composition with a reagent that releases the cargo from the LNPs;(ii) purifying the nucleic acids in the sample;(iii) incubating the purified nucleic acids with an enzyme, wherein the enzyme is a DNase or an RNase;(iv) incubating the resulting product obtained from step (iii) with proteinase K;(v) purifying the resulting product obtained from step (iv); and(vi) measuring the nucleic acid integrity in the sample.

2. The method according to claim 1, wherein the reagent in step (i) comprises a detergent; optionally wherein the detergent is selected from the list consisting of: Triton, Zwittergent, and sodium dodecyl sulfate (SDS); further optionally wherein the detergent is Triton; further optionally wherein the detergent is Triton X-100.

3. The method according to claim 2, wherein the concentration of the Triton is 10-40% m / v, optionally wherein the concentration of the Triton is 15-25% m / v, further optionally wherein the concentration of the Triton is 20% m / v.

4. The method according to any one of claims 1-3, wherein the reagent in step (i) further comprises an alcohol; optionally wherein the alcohol is ethanol, further optionally wherein the concentration of the ethanol is 10-40% v / v, optionally wherein the concentration of the ethanol is 25-35% v / v, further optionally wherein the concentration of the ethanol is 30% v / v.

5. The method according to any one of claims 1-4, wherein:(i) the nucleic acid integrity is RNA integrity and the enzyme in step (iii) is a DNase; or(ii) the nucleic acid integrity is DNA integrity and the enzyme in step (iii) is an RNase.

6. The method according to any one of claims 1-5, wherein the concentration of the DNase in the sample in step (iii) is 0.01-1 U / pL; optionally where the concentration of the DNase in the sample in step (iii) is 0.01-0.4 U / pL; further optionally wherein the concentration of the DNase in the sample in step (iii) is about 0.03 ll / pl; further optionally wherein all of the DNA in step (iii) is digested by the DNase.

7. The method according to any one of claims 1-5, wherein the concentration of the concentration of the RNase in the sample in step (iii) is 0.1-2 mg / ml; optionally where the concentration of the RNase in the sample in step (iii) is 0.1-1 mg / ml ; further optionally wherein concentration of the RNase in the sample in step (iii) is about 0.65 mg / ml; further optionally wherein all of the RNA in step (iii) is digested by the RNase.

8. The method according to any one of claims 1-7, wherein the concentration of the proteinase K in the sample in step (iv) is 0.1-2 U / rnL; optionally wherein the concentration of the proteinase K in the sample in step (iv) is about 1 U / rnL; further optionally wherein all of the enzyme from step (iii) is digested by the proteinase K in step (iv).

9. The method according to any one of claims 1-8, wherein the nucleic acid integrity measured in step (vi) is determined by analysing nucleic acid size and / or nucleic acid size distribution.

10. The method according to any one of claims 1-9, wherein the nucleic acid integrity is measured using capillary gel electrophoresis or classical gel electrophoresis.

11. The method according to claim 10, wherein the nucleic acid integrity is measured using capillary gel electrophoresis; optionally wherein the nucleic acid integrity is measured using a Fragment Analyser.

12. The method according to any one of claims 1-11, wherein the LNPs are functionalised LNPs comprising one or more moieties; optionally wherein the one or more moieties are present on the surface of the LNPs; further optionally wherein the one or more moieties comprise a targeting domain.

13. The method according to any one of claims 1-12, wherein the cargo of nucleic acids comprises (i) DNA, (ii) RNA; or (iii) DNA and RNA.

14. The method according to any one of claims 1-33, wherein the nucleic acids purified in step (ii) are DNA and / or RNA.

15. The method according to claim 13 or claim 14, wherein:(i) the DNA is selected from the list consisting of: genomic DNA and cDNA; and / or(ii) the RNA is selected from the list consisting of: mRNA, miRNA, siRNA and shRNA.

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