PROCESSOS DE ENCAPSULAMENTO DE RNA MENSAGEIRO (MRNA) EM NANOPARTÍCULAS LIPÍDICAS (LNPS), COMPOSIÇÃO, E USO DE UM MRNA ENCAPSULADO EM NANOPARTÍCULAS LIPÍDICAS

Description

1 / 131 Processes for encapsulating messenger RNA (mRNA) in lipid nanoparticles (LNPs), composition, and use of an mRNA encapsulated in lipid nanoparticles. Separated from BR112023006699-9, filed on October 12, 2021. REFERENCE TO RELATED ORDERS

[001] This request claims priority over the US Provisional Request Serial No. 63 / 090.513, filed on October 12, 2020, the disclosure of which is incorporated herein by reference. BACKGROUND

[002] Messenger RNA therapy (MRT) is becoming an increasingly important approach for treating a variety of diseases. MRT involves administering messenger RNA (mRNA) to a patient in need of therapy to produce the protein encoded by the mRNA within the patient's body. Lipid nanoparticles are commonly used to encapsulate mRNA for efficient in vivo mRNA release. To improve the release of lipid nanoparticles, much effort has been focused on identifying new methods and compositions that can affect the intracellular release and / or expression of mRNA and can be adapted to a scalable and cost-effective manufacturing process. SUMMARY OF THE INVENTION

[003] The present invention provides, among other things, an improved, efficient and economical process for the preparation of a composition comprising mRNA-loaded lipid nanoparticles (mRNA-LNPs). The invention is based on the surprising finding that mixing an mRNA solution containing a low concentration of citrate (i.e., < 5 mM) and a lipid solution at room temperature (without preheating the mRNA solution and / or the lipid solution) resulted in high encapsulation efficiency, rate Petition 870260055239, dated 08 / 06 / 2026, page 15 / 305 2 / 131 mRNA recovery and more homogeneous and smaller particle sizes. Thus, in one aspect, the present invention provides an effective, reliable, energy-efficient, cost-effective and safe method of encapsulating mRNA in lipid nanoparticles, which can be used for therapeutic applications with a large-scale manufacturing process without the use of heat and high energy.

[004] In one aspect, the invention provides, among other things, a process for encapsulating messenger RNA (mRNA) in lipid nanoparticles (LNPs) comprising a mixing step of (a) an mRNA solution comprising one or more mRNAs with (b) a lipid solution comprising one or more cationic lipids, one or more non-cationic lipids and one or more PEG-modified lipids, to form mRNA encapsulated within LNPs (mRNALNPs) in an LNP-forming solution, wherein the mRNA solution comprises less than 5 mM citrate and wherein the mRNA-LNPs have an encapsulation efficiency greater than 60%.

[005] In some embodiments, an mRNA solution and a lipid solution are at room temperature before mixing. In some embodiments, an mRNA solution and a lipid solution are mixed at room temperature. In some embodiments, an mRNA solution and a lipid solution are at room temperature after mixing. In some embodiments, the process of encapsulating mRNA within the lipid nanoparticle is performed at room temperature, without heating.

[006] In some modes, the ambient temperature is less than about 35 °C. In some modes, the ambient temperature is less than about 32 °C. In some modes, the ambient temperature is less than about 30 °C. In some modes, the ambient temperature is less than about 28 °C. Petition 870260055239, dated 08 / 06 / 2026, p. 16 / 305 3 / 131 In some modes, the ambient temperature is less than about 26 °C. In some modes, the ambient temperature is less than about 25 °C. In some modes, the ambient temperature is less than about 24 °C. In some modes, the ambient temperature is less than about 23 °C. In some modes, the ambient temperature is less than about 22 °C. In some modes, the ambient temperature is less than about 21 °C. In some modes, the ambient temperature is less than about 20 °C. In some modes, the ambient temperature is less than about 19 °C. In some modes, the ambient temperature is less than about 18 °C. In some modes, the ambient temperature is less than about 16 °C.

[007] In some modes, the ambient temperature ranges from about 15 to 35 °C. In some modes, the ambient temperature ranges from about 16 to 32 °C.In some modalities, the ambient temperature varies from approximately 17 to 30°C. In some modalities, the ambient temperature varies from approximately 18 to 30°C. In some modalities, the ambient temperature varies from approximately 20 to 28°C. In some modalities, the ambient temperature varies from approximately 20 to 26°C. In some modalities, the ambient temperature varies from approximately 20 to 25°C. In some modalities, the ambient temperature varies from approximately 21 to 24°C. In some modalities, the ambient temperature varies from approximately 21 to 23°C.

[008] In some modes, the ambient temperature is around 16°C. In some modes, the ambient temperature is around 18°C. In some modes, the ambient temperature is around 20°C. In some modes, the ambient temperature is around 21°C. In some modes, the ambient temperature is around 22°C. In some modes, the ambient temperature is around 23°C. In some Petition 870260055239, dated 08 / 06 / 2026, p. 17 / 305 In some modalities, the ambient temperature is around 24°C. In some modalities, the ambient temperature is around 25°C. In some modalities, the ambient temperature is around 26°C. In some modalities, the ambient temperature is around 27°C. In some modalities, the ambient temperature is around 28°C. In some modalities, the ambient temperature is around 30°C. In some modalities, the ambient temperature is around 31°C. In some modalities, the ambient temperature is around 32°C.

[009] In some embodiments, the mRNA solution comprises less than about 10 mM of citrate buffer. In some embodiments, the mRNA solution comprises less than about 8.6 mM of citrate buffer. In some embodiments, the mRNA solution comprises less than about 6.0 mM of citrate buffer. In some embodiments, the mRNA solution comprises less than about 5.0 mM of citrate buffer. In some embodiments, the mRNA solution comprises less than about 4.0 mM of citrate buffer. In some embodiments, the mRNA solution comprises less than about 3.5 mM of citrate buffer. In some embodiments, the mRNA solution comprises less than about 3.0 mM of citrate buffer. In some embodiments, the mRNA solution comprises less than about 2.5 mM of citrate buffer. In some embodiments, the mRNA solution comprises less than about 2.0 mM of citrate buffer.In some embodiments, the mRNA solution comprises less than about 1.5 mM of citrate buffer. In some embodiments, the mRNA solution comprises less than about 1.25 mM of citrate buffer. In some embodiments, the mRNA solution comprises less than about 1.0 mM of citrate buffer. In some embodiments, the mRNA solution comprises less than about 0.9 mM of citrate buffer. Petition 870260055239, dated 08 / 06 / 2026, p. 18 / 305 5 / 131 In some embodiments, the mRNA solution comprises less than about 0.8 mM of citrate buffer. In some embodiments, the mRNA solution comprises less than about 0.7 mM of citrate buffer. In some embodiments, the mRNA solution comprises less than about 0.6 mM of citrate buffer. In some embodiments, the mRNA solution comprises less than about 0.5 mM of citrate buffer. In some embodiments, the mRNA solution comprises less than about 0.4 mM of citrate buffer. In some embodiments, the mRNA solution comprises less than about 0.3 mM of citrate buffer. In some embodiments, the mRNA solution comprises less than about 0.25 mM of citrate buffer. In some embodiments, the mRNA solution comprises less than about 0.2 mM of citrate buffer. In some embodiments, the mRNA solution comprises less than about 0.1 mM of citrate buffer. In some embodiments, the mRNA solution comprises less than about 0.05 mM of citrate buffer.In some embodiments, the mRNA solution comprises approximately 0 mM of citrate buffer.

[0010] In some embodiments, the mRNA solution comprises about 0 to 10 mM of citrate buffer. In some embodiments, the mRNA solution comprises about 1.5 to 7.5 mM of citrate buffer. In some embodiments, the mRNA solution comprises about 2.0 to 5.0 mM of citrate buffer. In some embodiments, the mRNA solution comprises about 2.5 to 3.5 mM of citrate buffer.

[0011] In some embodiments, the mRNA solution comprises approximately 0.1 mM citrate buffer. In some embodiments, the mRNA solution comprises approximately 0.2 mM citrate buffer. In some embodiments, the mRNA solution comprises approximately 0.25 mM citrate buffer. In some embodiments, the solution of Petition 870260055239, dated 08 / 06 / 2026, p. 19 / 305 6 / 131 mRNA comprises approximately 0.3 mM of citrate buffer. In some embodiments, the mRNA solution comprises approximately 0.4 mM of citrate buffer. In some embodiments, the mRNA solution comprises approximately 0.5 mM of citrate buffer. In some embodiments, the mRNA solution comprises approximately 0.6 mM of citrate buffer. In some embodiments, the mRNA solution comprises approximately 0.7 mM of citrate buffer. In some embodiments, the mRNA solution comprises approximately 0.8 mM of citrate buffer. In some embodiments, the mRNA solution comprises approximately 0.9 mM of citrate buffer. In some embodiments, the mRNA solution comprises approximately 1.0 mM of citrate buffer. In some embodiments, the mRNA solution comprises approximately 1.25 mM of citrate buffer. In some embodiments, the mRNA solution comprises approximately 1.5 mM of citrate buffer. In some embodiments, the mRNA solution comprises approximately 1.75 mM of citrate buffer.In some embodiments, the mRNA solution comprises approximately 2.0 mM of citrate buffer. In some embodiments, the mRNA solution comprises approximately 2.5 mM of citrate buffer. In some embodiments, the mRNA solution comprises approximately 3.0 mM of citrate buffer. In some embodiments, the mRNA solution comprises approximately 3.5 mM of citrate buffer. In some embodiments, the mRNA solution comprises approximately 4.0 mM of citrate buffer. In some embodiments, the mRNA solution comprises approximately 4.5 mM of citrate buffer. In some embodiments, the mRNA solution comprises approximately 5.0 mM of citrate buffer.

[0012] In some embodiments, the mRNA solution further comprises trehalose. In some embodiments, the mRNA solution comprises 20% trehalose. In some embodiments, the solution of Petition 870260055239, dated 08 / 06 / 2026, p. 20 / 305 7 / 131 mRNA comprises 15% trehalose. In some embodiments, the mRNA solution comprises 10% trehalose. In some embodiments, the mRNA solution comprises 5% trehalose.

[0013] In some embodiments, the process does not require a heating step of the mRNA solution and / or lipid solution.

[0014] In some embodiments, the mRNA solution comprises more than about 1 g of mRNA per 12 L of mRNA solution. In some embodiments, the mRNA solution comprises more than about 1 g of mRNA per 10 L of mRNA solution. In some embodiments, the mRNA solution comprises more than about 1 g of mRNA per 8 L of mRNA solution. In some embodiments, the mRNA solution comprises more than about 1 g of mRNA per 6 L of mRNA solution. In some embodiments, the mRNA solution comprises more than about 1 g of mRNA per 4 L of mRNA solution. In some embodiments, the mRNA solution comprises more than about 1 g of mRNA per 2 L of mRNA solution. In some embodiments, the mRNA solution comprises more than about 1 g of mRNA per 1 L of mRNA solution.

[0015] In some embodiments, the mRNA concentration in the mRNA solution is greater than about 0.1 mg / ml. In some embodiments, the mRNA concentration in the mRNA solution is greater than about 0.125 mg / ml. In some embodiments, the mRNA concentration in the mRNA solution is greater than about 0.25 mg / ml. In some embodiments, the mRNA concentration in the mRNA solution is greater than about 0.5 mg / ml. In some embodiments, the mRNA concentration in the mRNA solution is greater than about 1.0 mg / ml. In some embodiments, the mRNA concentration in the mRNA solution is greater than about 1.5 mg / ml. In some embodiments, the mRNA concentration in the mRNA solution is greater than about 2.0 mg / ml. Petition 870260055239, dated 08 / 06 / 2026, p. 21 / 305 8 / 131

[0016] In some embodiments, the mRNA solution and the lipid solution are mixed in a ratio (v / v) between 1:1 and 10:1. In some embodiments, the mRNA solution and the lipid solution are mixed in a ratio (v / v) between 2:1 and 6:1. In some embodiments, the mRNA solution and the lipid solution are mixed in a ratio (v / v) of about 2:1. In some embodiments, the mRNA solution and the lipid solution are mixed in a ratio (v / v) of about 3:1. In some embodiments, the mRNA solution and the lipid solution are mixed in a ratio (v / v) of about 4:1. In some embodiments, the mRNA solution and the lipid solution are mixed in a ratio (v / v) of about 5:1. In some embodiments, the mRNA solution and the lipid solution are mixed in a (v / v) ratio of approximately 6:1. In some embodiments, the mRNA solution and the lipid solution are mixed in a (v / v) ratio greater than approximately 2:1.In some embodiments, the mRNA solution and the lipid solution are mixed in a ratio (v / v) greater than about 3:1. In some embodiments, the mRNA solution and the lipid solution are mixed in a ratio (v / v) greater than about 4:1. In some embodiments, the mRNA solution and the lipid solution are mixed in a ratio (v / v) greater than about 5:1. In some embodiments, the mRNA solution and the lipid solution are mixed in a ratio (v / v) greater than about 6:1.

[0017] In some embodiments, the mRNA solution has a pH between 2.5 and 5.5. In some embodiments, the mRNA solution has a pH between 3.0 and 5.0. In some embodiments, the mRNA solution has a pH between 3.5 and 4.5. In some embodiments, the mRNA solution has a pH of about 3.0. In some embodiments, the mRNA solution has a pH of approximately 3.5. In some embodiments, the mRNA solution has a pH of approximately 4.0.In some forms, the mRNA solution has a pH of... Petition 870260055239, dated 08 / 06 / 2026, page 22 / 305 9 / 131 approximately 4.5. In some embodiments, the mRNA solution has a pH of approximately 5.0. In some embodiments, the mRNA solution has a pH of approximately 5.5.

[0018] In some embodiments, the mRNA solution comprises about 25 mM to 500 mM of NaCl. In some embodiments, the mRNA solution comprises about 37.5 mM to 350 mM of NaCl. In some embodiments, the mRNA solution comprises about 75 mM to 300 mM of NaCl. In some embodiments, the mRNA solution comprises about 100 mM to 300 mM of NaCl. In some embodiments, the mRNA solution comprises about 150 mM to 300 mM of NaCl. In some embodiments, the mRNA solution comprises about 37.5 mM of NaCl. In some embodiments, the mRNA solution comprises about 75 mM of NaCl. In some embodiments, the mRNA solution comprises about 100 mM of NaCl. In some embodiments, the mRNA solution comprises about 125 mM of NaCl. In some embodiments, the mRNA solution comprises about 150 mM of NaCl. In some embodiments, the mRNA solution comprises approximately 175 mM of NaCl.In some embodiments, the mRNA solution comprises approximately 200 mM NaCl. In some embodiments, the mRNA solution comprises approximately 225 mM NaCl. In some embodiments, the mRNA solution comprises approximately 250 mM NaCl. In some embodiments, the mRNA solution comprises approximately 300 mM NaCl. In some embodiments, the mRNA solution comprises approximately 350 mM NaCl.

[0019] In some embodiments, the mRNA solution comprises about 2.5 mM citrate, about 100 mM NaCl, and a pH of about 3.5. In some embodiments, the mRNA solution comprises about 3.0 mM citrate, about 100 mM NaCl, and a pH of about 3.5. In some embodiments, the mRNA solution comprises Petition 870260055239, dated 08 / 06 / 2026, p. 23 / 305 10 / 131 approximately 3.5 mM citrate, approximately 100 mM NaCl, and a pH of approximately 3.5. In some embodiments, the mRNA solution comprises approximately 2.5 mM citrate, approximately 150 mM NaCl, and a pH of approximately 3.5. In some embodiments, the mRNA solution comprises approximately 3.0 mM citrate, approximately 150 mM NaCl, and a pH of approximately 3.5. In some embodiments, the mRNA solution comprises approximately 3.5 mM citrate, approximately 150 mM NaCl, and a pH of approximately 3.5. In some embodiments, the mRNA solution comprises approximately 2.5 mM citrate, approximately 300 mM NaCl, and a pH of approximately 3.5. In some embodiments, the mRNA solution comprises about 3.0 mM citrate, about 300 mM NaCl, and a pH of about 3.5. In some embodiments, the mRNA solution comprises about 3.5 mM citrate, about 300 mM NaCl, and a pH of about 3.5.

[0020] In some embodiments, the mRNA solution comprises about 2.5 mM citrate, about 100 mM NaCl, and a pH of about 4.0. In some embodiments, the mRNA solution comprises about 3.0 mM citrate, about 100 mM NaCl, and a pH of about 4.0. In some embodiments, the mRNA solution comprises about 3.5 mM citrate, about 100 mM NaCl, and a pH of about 4.0. In some embodiments, the mRNA solution comprises about 2.5 mM citrate, about 150 mM NaCl, and a pH of about 4.0. In some embodiments, the mRNA solution comprises about 3.0 mM citrate, about 150 mM NaCl, and a pH of about 4.0. In some embodiments, the mRNA solution comprises about 3.5 mM citrate, about 150 mM NaCl, and a pH of about 4.0. In some embodiments, the mRNA solution comprises about 2.5 mM citrate, about 300 mM NaCl, and a pH of about 4.0.In some embodiments, the mRNA solution comprises approximately 3.0 mM citrate, approximately 300 mM NaCl, and a pH of approximately... Petition 870260055239, dated 08 / 06 / 2026, p. 24 / 305 11 / 131 of 4.0. In some embodiments, the mRNA solution comprises about 3.5 mM citrate, about 300 mM NaCl, and a pH of about 4.0.

[0021] In some embodiments, the mRNA solution comprises about 2.5 mM citrate, about 100 mM NaCl, and a pH of about 4.5. In some embodiments, the mRNA solution comprises about 3.0 mM citrate, about 100 mM NaCl, and a pH of about 4.5. In some embodiments, the mRNA solution comprises about 3.5 mM citrate, about 100 mM NaCl, and a pH of about 4.5. In some embodiments, the mRNA solution comprises about 2.5 mM citrate, about 150 mM NaCl, and a pH of about 4.5. In some embodiments, the mRNA solution comprises about 3.0 mM citrate, about 150 mM NaCl, and a pH of about 4.5. In some embodiments, the mRNA solution comprises about 3.5 mM citrate, about 150 mM NaCl, and a pH of about 4.5. In some embodiments, the mRNA solution comprises about 2.5 mM citrate, about 300 mM NaCl, and a pH of about 4.5.In some embodiments, the mRNA solution comprises about 3.0 mM citrate, about 300 mM NaCl, and a pH of about 4.5. In some embodiments, the mRNA solution comprises about 3.5 mM citrate, about 300 mM NaCl, and a pH of about 4.5.

[0022] In some embodiments, the process also includes a post-mixing mRNA-LNP incubation step. In some embodiments, the mRNA-LNPs are incubated at a temperature between 21 °C and 65 °C. In some embodiments, the mRNA-LNPs are incubated at a temperature between 25 °C and 60 °C. In some embodiments, the mRNA-LNPs are incubated at a temperature between 30 °C and 55 °C. In some embodiments, the mRNA-LNPs are incubated at a temperature between 35 °C and Petition 870260055239, dated 08 / 06 / 2026, p. 25 / 305 12 / 131 50°C. In some embodiments, mRNA-LNPs are incubated at a temperature of approximately 26°C. In some embodiments, mRNA-LNPs are incubated at a temperature of approximately 30°C. In some embodiments, mRNA-LNPs are incubated at a temperature of approximately 31°C. In some embodiments, mRNA-LNPs are incubated at a temperature of approximately 32°C. In some embodiments, mRNA-LNPs are incubated at a temperature of approximately 35°C. In some embodiments, mRNA-LNPs are incubated at a temperature of approximately 38°C. In some embodiments, mRNA-LNPs are incubated at a temperature of approximately 40°C. In some embodiments, mRNA-LNPs are incubated at a temperature of approximately 42°C. In some modes, mRNA-LNPs are incubated at a temperature of approximately 45 °C. In some modes, mRNA-LNPs are incubated at a temperature of approximately 50 °C.In some embodiments, mRNA-LNPs are incubated at a temperature of approximately 55 °C. In some embodiments, mRNA-LNPs are incubated at a temperature of approximately 60 °C. In some embodiments, mRNA-LNPs are incubated at a temperature of approximately 65 °C.

[0023] In some embodiments, mRNA-LNPs are incubated for more than approximately 20 minutes. In some embodiments, mRNA-LNPs are incubated for more than approximately 30 minutes. In some embodiments, mRNA-LNPs are incubated for more than approximately 40 minutes. In some embodiments, mRNA-LNPs are incubated for more than approximately 50 minutes. In some embodiments, mRNA-LNPs are incubated for more than approximately 60 minutes. In some embodiments, mRNA-LNPs are incubated for more than approximately 70 minutes. In some methods, mRNA-LNPs are incubated for more than approximately... Petition 870260055239, dated 08 / 06 / 2026, p. 26 / 305 13 / 131 of 80 minutes. In some embodiments, mRNA-LNPs are incubated for more than about 90 minutes. In some embodiments, mRNA-LNPs are incubated for more than about 100 minutes. In some embodiments, mRNA-LNPs are incubated for more than about 120 minutes. In some embodiments, mRNA-LNPs are incubated for about 30 minutes. In some embodiments, mRNA-LNPs are incubated for about 40 minutes. In some embodiments, mRNA-LNPs are incubated for about 50 minutes. In some embodiments, mRNA-LNPs are incubated for about 60 minutes. In some embodiments, mRNA-LNPs are incubated for about 70 minutes. In some embodiments, mRNA-LNPs are incubated for about 80 minutes. In some modes, mRNA-LNPs are incubated for approximately 100 minutes. In some modes, mRNA-LNPs are incubated for approximately 120 minutes.In some modes, mRNA-LNPs are incubated for approximately 150 minutes. In some modes, mRNA-LNPs are incubated for approximately 180 minutes.

[0024] In some embodiments, the lipid solution comprises less than 50% non-aqueous solvent. In some embodiments, the lipid solution comprises less than 40% non-aqueous solvent. In some embodiments, the lipid solution comprises less than 30% non-aqueous solvent. In some embodiments, the lipid solution comprises less than 25% non-aqueous solvent. In some embodiments, the lipid solution comprises less than 20% non-aqueous solvent. In some embodiments, the lipid solution comprises less than 15% non-aqueous solvent. In some embodiments, the lipid solution comprises less than 10% non-aqueous solvent. In some embodiments, the lipid solution comprises Petition 870260055239, dated 08 / 06 / 2026, p. 27 / 305 14 / 131 less than 50% ethanol. In some embodiments, the lipid solution comprises less than 40% ethanol. In some embodiments, the lipid solution comprises less than 30% ethanol. In some embodiments, the lipid solution comprises less than 25% ethanol. In some embodiments, the lipid solution comprises less than 20% ethanol. In some embodiments, the lipid solution comprises less than 15% ethanol. In some embodiments, the lipid solution comprises less than 10% ethanol.

[0025] In some embodiments, the mRNA solution and the lipid solution are mixed within 40% ethanol, resulting in a suspension of lipid nanoparticles. In some embodiments, the mRNA solution and the lipid solution are mixed within 20% ethanol, resulting in a suspension of lipid nanoparticles. In some embodiments, the mRNA solution and the lipid solution are mixed within 15% ethanol, resulting in a suspension of lipid nanoparticles. In some embodiments, the mRNA solution and the lipid solution are mixed within 10% ethanol, resulting in a suspension of lipid nanoparticles.

[0026] In some embodiments, the lipid solution also comprises one or more cholesterol-based lipids.

[0027] In some embodiments, the lipid nanoparticles are further purified. In some embodiments, the lipid nanoparticles are purified by Tangential Flow Filtration.

[0028] In some embodiments, wherein the purified lipid nanoparticles have an average size of less than 200 nm. In some embodiments, wherein the purified lipid nanoparticles have an average size of less than 180 nm. In some embodiments, wherein the purified lipid nanoparticles have an average size of less than 150 nm. In some embodiments, wherein the purified lipid nanoparticles have Petition 870260055239, dated 08 / 06 / 2026, page 28 / 305 15 / 131 an average size of less than 100 nm. In some embodiments, the purified lipid nanoparticles have an average size of less than 90 nm. In some embodiments, the purified lipid nanoparticles have an average size of less than 80 nm. In some embodiments, the purified lipid nanoparticles have an average size of less than 70 nm. In some embodiments, the purified lipid nanoparticles have an average size of less than 60 nm. In some embodiments, the purified lipid nanoparticles have an average size of less than 50 nm. In some embodiments, the purified lipid nanoparticles have an average size of less than 40 nm.

[0029] In some embodiments, the purified lipid nanoparticles have an average size ranging from 40 to 150 nm. In some embodiments, the purified lipid nanoparticles have an average size ranging from 60 to 100 nm. In some embodiments, the purified lipid nanoparticles have an average size ranging from 40 to 70 nm.

[0030] In some embodiments, the lipid nanoparticles have a PDI of less than about 0.3. In some embodiments, the lipid nanoparticles have a PDI of less than about 0.2. In some embodiments, the lipid nanoparticles have a PDI of less than about 0.18. In some embodiments, the lipid nanoparticles have a PDI of less than about 0.15. In some embodiments, the lipid nanoparticles have a PDI of less than about 0.1.

[0031] In some embodiments, purified lipid nanoparticles have an encapsulation rate greater than about 60%. In some embodiments, purified lipid nanoparticles have an encapsulation rate greater than about Petition 870260055239, dated 08 / 06 / 2026, page 29 / 305 16 / 131 of 65%. In some embodiments, the purified lipid nanoparticles have an encapsulation rate greater than about 70%. In some embodiments, the purified lipid nanoparticles have an encapsulation rate greater than about 75%. In some embodiments, the purified lipid nanoparticles have an encapsulation rate greater than about 80%. In some embodiments, the purified lipid nanoparticles have an encapsulation rate greater than about 85%. In some embodiments, the purified lipid nanoparticles have an encapsulation rate greater than about 90%. In some embodiments, the purified lipid nanoparticles have an encapsulation rate greater than about 95%. In some embodiments, the purified lipid nanoparticles have an encapsulation rate greater than about 96%.In some embodiments, the purified lipid nanoparticles have an encapsulation rate greater than about 97%. In some embodiments, the purified lipid nanoparticles have an encapsulation rate greater than about 98%. In some embodiments, the purified lipid nanoparticles have an encapsulation rate greater than about 99%.

[0032] In some modalities, an N / P ratio is between 1 and 10. In some embodiments, the N / P ratio is between 2 and 6. In some embodiments, the N / P ratio is approximately 4. In some embodiments, the mRNA solution and the lipid solution are mixed at an N / P ratio between 1 and 10. In some embodiments, the mRNA solution and the lipid solution are mixed at an N / P ratio between 2 and 6. In some embodiments, the mRNA solution and the lipid solution are mixed at an N / P ratio of approximately 2. In some embodiments, the mRNA solution and the lipid solution are mixed Petition 870260055239, dated 08 / 06 / 2026, p. 30 / 305 17 / 131 in an N / P ratio of approximately 4. In some embodiments, the mRNA solution and the lipid solution are mixed in an N / P ratio of approximately 6.

[0033] In some embodiments, 5 g or more of mRNA are encapsulated in lipid nanoparticles in a single batch. In some embodiments, 10 g or more of mRNA are encapsulated in Lipid nanoparticles in a single batch. In some embodiments, 15 g or more of mRNA are encapsulated in lipid nanoparticles in a single batch. In some embodiments, 20 g or more of mRNA are encapsulated in lipid nanoparticles in a single batch. In some embodiments, 25 g or more of mRNA are encapsulated in lipid nanoparticles in a single batch. In some embodiments, 30 g or more of mRNA are encapsulated in lipid nanoparticles in a single batch. In some embodiments, 40 g or more of mRNA are encapsulated in lipid nanoparticles in a single batch. In some embodiments, 50 g or more of mRNA are encapsulated in lipid nanoparticles in a single batch. In some embodiments, 75 g or more of mRNA are encapsulated in lipid nanoparticles in a single batch. In some embodiments, 100 g or more of mRNA are encapsulated in lipid nanoparticles in a single batch.In some embodiments, 150 g or more of mRNA are encapsulated in lipid nanoparticles in a single batch. In some embodiments, 200 g or more of mRNA are encapsulated in lipid nanoparticles in a single batch. In some embodiments, 250 g or more of mRNA are encapsulated in lipid nanoparticles in a single batch. In some embodiments, 500 g or more of mRNA are encapsulated in... Petition 870260055239, dated 08 / 06 / 2026, page 31 / 305 18 / 131 Lipid nanoparticles in a single batch. In some embodiments, 750 g or more of mRNA are encapsulated in lipid nanoparticles in a single batch. In some embodiments, 1 kg or more of mRNA are encapsulated in lipid nanoparticles in a single batch. In some embodiments, 5 kg or more of mRNA are encapsulated in lipid nanoparticles in a single batch. In some embodiments, 10 kg or more of mRNA are encapsulated in Lipid nanoparticles in a single batch.

[0034] In some embodiments, the mRNA solution and the lipid solution are mixed in a flow pump with fewer pulses. In some embodiments, the pump is a gear pump. In some embodiments, the pump is a centrifugal pump. In some embodiments, the pump is a peristaltic pump.

[0035] In some embodiments, the buffer solution is mixed at a flow rate that varies between about 100 to 300 ml / minute, 300 to 600 ml / minute, 600 to 1200 ml / minute, 1200 to 2400 ml / minute, 2400 to 3600 ml / minute, 3600 to 4800 ml / minute or 4800 to 6000 ml / minute. In some embodiments, the buffer solution is mixed at a flow rate of about 220 ml / minute, about 600 ml / minute, about 1200 ml / minute, about 2400 ml / minute, about 3600 ml / minute, about 4800 ml / minute or about 6000 ml / minute.

[0036] In some embodiments, the citrate buffer is mixed at a flow rate ranging from about 100 to 300 ml / minute, 300 to 600 ml / minute, 600 to 1200 ml / minute, 1200 to 2400 ml / minute, 2400 to 3600 ml / minute, 3600 to 4800 ml / minute, or 4800 to 6000 ml / minute. In some embodiments, the citrate buffer is mixed at a flow rate of about 220 ml / minute, about 600 ml / minute, about 1200 ml / minute, about 2400 ml / minute, to Petition 870260055239, dated 08 / 06 / 2026, p. 32 / 305 19 / 131 about 3600 ml / minute, about 4800 ml / minute or about 6000 ml / minute.

[0037] In some embodiments, the mRNA solution is mixed at a flow rate ranging from about 150 to 250 ml / minute, 250 to 500 ml / minute, 500 to 1000 ml / minute, 1000 to 2000 ml / minute, 2000 to 3000 ml / minute, 3000 to 4000 ml / minute, or 4000 to 5000 ml / minute. In some embodiments, the mRNA solution is mixed at a flow rate of about 200 ml / minute, about 500 ml / minute, about 1000 ml / minute, about 2000 ml / minute, about 3000 ml / minute, about 4000 ml / minute, or about 5000 ml / minute.

[0038] In some embodiments, the mRNA solution is mixed at a flow rate of about 100 ml / minute. In some embodiments, the mRNA solution is mixed at a flow rate of about 200 ml / minute. In some embodiments, the mRNA solution is mixed at a flow rate of about 400 ml / minute. In some embodiments, the mRNA solution is mixed at a flow rate of about 500 ml / minute. In some embodiments, the mRNA solution is mixed at a flow rate of about 600 ml / minute. In some embodiments, the mRNA solution is mixed at a flow rate of about 800 ml / minute. In some embodiments, the mRNA solution is mixed at a flow rate of about 1000 ml / minute. In some embodiments, the mRNA solution is mixed at a flow rate of about 1200 ml / minute. In some embodiments, the mRNA solution is mixed at a flow rate of approximately 1400 ml / minute. In some embodiments, the mRNA solution is mixed at a flow rate of approximately 1600 ml / minute.In some embodiments, the mRNA solution is mixed at a flow rate of approximately 1800 ml / minute. In some embodiments, the mRNA solution is mixed at a flow rate of approximately 2000 ml / minute. In some embodiments, the mRNA solution... Petition 870260055239, dated 08 / 06 / 2026, p. 33 / 305 20 / 131 is mixed at a flow rate of approximately 2400 ml / minute. In some embodiments, the mRNA solution is mixed at a flow rate of approximately 3000 ml / minute. In some embodiments, the mRNA solution is mixed at a flow rate of approximately 4000 ml / minute.

[0039] In some embodiments, the lipid solution is mixed at a flow rate that varies from about 25 to 75 ml / minute, from about 75 to 200 ml / minute, from about 200 to 350 ml / minute, from about 350 to 500 ml / minute, from about 500 to 650 ml / minute, from about 650 to 850 ml / minute or from about 850 to 1000 ml / minute. In some embodiments, the lipid solution is mixed at a flow rate of approximately 50 ml / minute, approximately 100 ml / minute, approximately 150 ml / minute, approximately 200 ml / minute, approximately 250 ml / minute, approximately 300 ml / minute, approximately 350 ml / minute, approximately 400 ml / minute, approximately 450 ml / minute, approximately 500 ml / minute, approximately 550 ml / minute, approximately 600 ml / minute, approximately 650 ml / minute, approximately 700 ml / minute, approximately 750 ml / minute, approximately 800 ml / minute, approximately 850 ml / minute, approximately 900 ml / minute, approximately 950 ml / minute or at approximately 1000 ml / minute.

[0040] In some embodiments, the flow rate of the mRNA solution is the same as the flow rate of the lipid solution. In some embodiments, the flow rate of the mRNA solution is 2 times greater than the flow rate of the lipid solution. In some embodiments, the flow rate of the mRNA solution is 3 times greater than the flow rate of the lipid solution. In some embodiments, the flow rate of the mRNA solution is 4 times greater than the flow rate of the lipid solution. In some embodiments, the flow rate of the mRNA solution is 4.5 times greater than the flow rate of the lipid solution. In some embodiments, the flow rate of the mRNA solution is 5 times greater than the flow rate of the lipid solution. In some embodiments, the flow rate of the mRNA solution is 6 times greater than the flow rate of the lipid solution. In some embodiments, the flow rate of the mRNA solution is 8 Petition 870260055239, dated 08 / 06 / 2026, p. 34 / 305 21 / 131 times greater than the flow rate of the lipid solution. In some embodiments, the flow rate of the mRNA solution is 10 times greater than the flow rate of the lipid solution.

[0041] In some embodiments, the encapsulation efficiency of mRNA-LNP is at least 5% higher compared to an mRNA formed from the mRNA solution mixed with the lipid solution under the same conditions except that the mRNA solution has 10 mM citrate. In some embodiments, the encapsulation efficiency of mRNA-LNP is at least 10% higher compared to an mRNA formed from the mRNA solution mixed with the lipid solution under the same conditions except that the mRNA solution has 10 mM citrate. In some embodiments, the encapsulation efficiency of mRNA-LNP is at least 15% higher compared to an mRNA formed from the mRNA solution mixed with the lipid solution under the same conditions except that the mRNA solution has 10 mM citrate.In some embodiments, the encapsulation efficiency of mRNA-LNP is at least 20% higher compared to an mRNA-formed from mRNA solution mixed with lipid solution under the same conditions except with the mRNA solution containing 10 mM citrate.

[0042] In one aspect, the invention provides, among other things, a composition comprising mRNA encapsulated in lipid nanoparticles prepared by the process of the present invention.

[0043] In some embodiments, the composition comprises 1 g or more of mRNA. In some embodiments, the composition comprises 5 g or more of mRNA. In some embodiments, the composition comprises 10 g or more of mRNA. In some embodiments, the composition comprises 15 g or more of mRNA. In some embodiments, the composition comprises 20 g or more of mRNA. In Petition 870260055239, dated 08 / 06 / 2026, page 35 / 305 22 / 131 In some embodiments, the composition comprises 25 g or more of mRNA. In some embodiments, the composition comprises 50 g or more of mRNA. In some embodiments, the composition comprises 75 g or more of mRNA. In some embodiments, the composition comprises 100 g or more of mRNA. In some embodiments, the composition comprises 125 g or more of mRNA. In some embodiments, the composition comprises 150 g or more of mRNA. In some embodiments, the composition comprises 250 g or more of mRNA. In some embodiments, the composition comprises 500 g or more of mRNA. In some embodiments, the composition comprises 1 kg or more of mRNA.

[0044] In some embodiments, mRNA comprises one or more modified nucleotides.

[0045] In some forms, the mRNA is unmodified.

[0046] In some forms, the mRNA is larger than about 0.5 kb. In some forms, the mRNA is larger than about 1 kb. In some forms, the mRNA is larger than about 2 kb. In some forms, the mRNA is larger than about 3 kb. In some forms, the mRNA is larger than about 4 kb. In larger than about 5 kb. In larger than about 6 kb. In larger than about 8 kb. In larger than about 10 kb. In larger than about 20 kb. In larger than about 20 kb. In larger than about 30 kb. In larger than about 40 kb. In some forms, the mRNA is larger than about 50 kb.

[0047] In one aspect, the process of encapsulating messenger RNA (mRNA) in lipid nanoparticles (LNPs) is provided comprising a mixing step of (a) an mRNA solution that Petition 870260055239, dated 08 / 06 / 2026, p. 36 / 305 23 / 131 comprises one or more mRNAs with (b) a lipid solution comprising one or more cationic lipids, one or more non-cationic lipids and one or more PEG-modified lipids, to form the mRNA encapsulated within LNPs (mRNA-LNPs) in an LNP-forming solution, wherein the mRNA solution comprises between 0.1 mM and 5 mM of citrate, and wherein the mRNA-LNPs have an encapsulation efficiency greater than 60%.

[0048] In some embodiments, the mRNA solution comprises between about 1 mM and 5 mM of citrate. In some embodiments, the mRNA solution comprises between about 1 mM and 4 mM of citrate. In some embodiments, the mRNA solution comprises between about 1 mM and 3 mM of citrate. In some embodiments, the mRNA solution comprises between about 1 mM and 2 mM of citrate. In some embodiments, the mRNA solution comprises between about 2 mM and 3 mM of citrate. In some embodiments, the mRNA solution comprises between about 3 mM and 4 mM of citrate. In some embodiments, the mRNA solution comprises between about 4 mM and 5 mM of citrate. In some embodiments, the mRNA solution comprises about 1 mM of citrate. In some embodiments, the mRNA solution comprises about 2 mM of citrate. In some embodiments, the mRNA solution comprises about 3 mM of citrate. In some embodiments, the mRNA solution comprises approximately 4 mM of citrate.In some embodiments, the mRNA solution comprises approximately 5 mM of citrate.

[0049] Other features, objects and advantages of the present invention are evident from the detailed description, drawings and claims that follow. It should be understood, however, that the detailed description, drawings and claims, while indicating embodiments of the present invention, are provided for illustrative purposes only, not as a limitation. Various alterations and modifications Petition 870260055239, dated 08 / 06 / 2026, p. 37 / 305 24 / 131 within the scope of the invention will become apparent to those skilled in the art. BRIEF DESCRIPTION OF THE DRAWINGS

[0050] FIG. 1 represents an exemplary graph showing encapsulation efficiencies of mRNA-LNPs prepared with various citrate concentrations in the mRNA solution. Encapsulation efficiencies were measured before (0 minutes) and after (90 minutes) incubation after mixing.

[0051] FIG. 2 represents an exemplary graph showing the encapsulation efficiency of mRNA-LNPs prepared with various concentrations of sodium chloride in the mRNA solution. Encapsulation efficiencies were measured before (0 minutes) and after (90 minutes) incubation after mixing.

[0052] FIG. 3 represents an exemplary graph showing the encapsulation efficiency of mRNA-LNPs prepared with various concentrations of citrate and sodium chloride in the mRNA solution. Encapsulation efficiencies were measured before (0 minutes) and after (90 minutes) incubation after mixing.

[0053] FIG. 4 represents an exemplary graph showing the encapsulation efficiency of mRNA-LNPs prepared with different (v / v) ratios of mRNA solution: lipid solution. Encapsulation efficiencies were measured before (0 minutes) and after (90 minutes) incubation after mixing.

[0054] FIG. 5 represents an exemplary graph showing the encapsulation efficiency of mRNA-LNPs prepared with various flow rates during the mixing process. Encapsulation efficiencies were measured before (0 minutes) and after (90 minutes) incubation following mixing. DEFINITIONS

[0055] In order for the present invention to be more easily Petition 870260055239, dated 08 / 06 / 2026, p. 38 / 305 25 / 131 understood, certain terms are first defined below. Additional definitions for the following terms and other terms are presented throughout the descriptive report. The publications and other reference materials referred to herein to describe the background of the invention and to provide further details regarding its practice are incorporated herein by reference.

[0056] Amino acid: As used herein, the term amino acid, in its broadest sense, refers to any compound and / or substance that can be incorporated into a polypeptide chain. In some embodiments, an amino acid has the general structure H2NC(H)(R)-COOH. In some embodiments, an amino acid is a naturally occurring amino acid. In some embodiments, an amino acid is a synthetic amino acid; in some embodiments, an amino acid is a d-amino acid; in some embodiments, an amino acid is an l-amino acid. Standard amino acid refers to any of the twenty standard l-amino acids commonly found in naturally occurring peptides. Non-standard amino acid refers to any amino acid except standard amino acids, regardless of whether it is synthetically prepared or obtained from a natural source.As used in this invention, synthetic amino acid encompasses chemically modified amino acids, including, but not limited to, salts, amino acid derivatives (such as amides) and / or substitutions. Amino acids, including carboxy- and / or amino-terminal amino acids in peptides, can be modified through methylation, amidation, acetylation, protecting groups and / or substitution by other chemical groups that can alter the circulating half-life of the peptide without adversely affecting its activity. Amino acids may participate in a disulfide bond. Amino acids may comprise one or more post-translational modifications, such as association with one or more chemical entities (e.g., groups). Petition 870260055239, dated 08 / 06 / 2026, page 39 / 305 26 / 131 methyl, acetate groups, acetyl groups, phosphate groups, formyl components, isoprenoid groups, sulfate groups, polyethylene glycol components, lipid components, carbohydrate components, biotin components, etc.). The term amino acid is used interchangeably with amino acid residue, and may refer to a free amino acid and / or an amino acid residue of a peptide. It will be evident from the context in which the term is used whether it refers to a free amino acid or a residue of a peptide.

[0057] Animal: As used herein, the term animal refers to any member of the animal kingdom. In some embodiments, animal refers to humans, at any stage of development. In some embodiments, animal refers to non-human animals, at any stage of development.In certain embodiments, the non-human animal is a mammal (e.g., a rodent, mouse, rat, rabbit, monkey, dog, cat, sheep, cattle, primate, and / or pig). In some embodiments, the animals include, but are not limited to, mammals, birds, reptiles, amphibians, fish, insects, and / or worms. In some embodiments, an animal may be a transgenic animal, a genetically modified animal, and / or a clone.

[0058] Approximately or about: As used in this invention, the term approximately or about, applied to one or more values ​​of interest, refers to a value similar to a stated reference value. In certain embodiments, the term approximately or about refers to a range of values ​​that fall within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or less in any direction (greater or less) from the mentioned reference value, unless otherwise stated or evident from the context (except when that number exceeds 100% of a possible value).

[0059] Combination: As used here, the term combine is Petition 870260055239, dated 08 / 06 / 2026, p. 40 / 305 27 / 131 used interchangeably with mixing or combination. Combination refers to joining discrete LNP particles with distinct properties in the same solution, for example, combining an mRNA-LNP and an empty LNP to obtain an mRNA-LNP composition. In some embodiments, the combination of the two LNPs is performed in a specific ratio of the components being combined. In some embodiments, the resulting composition obtained from the combination possesses a property distinct from either or both of its components.

[0060] Release: As used in this invention, the term release encompasses both local and systemic release. For example, mRNA release encompasses situations where an mRNA is released to a target tissue and the encoded protein is expressed and retained in the target tissue (also referred to as local distribution or local release), and situations where an mRNA is released to a target tissue and the encoded protein is expressed and secreted into the patient's circulatory system (e.g., serum) and systemically distributed and absorbed by other tissues (also referred to as systemic distribution or systemic release). In some embodiments, the release is pulmonary release, for example, comprising nebulization.

[0061] Efficacy: As used in this invention, the term efficacy or grammatical equivalents refers to an improvement in a biologically relevant outcome related to the release of mRNA encoding a relevant protein or peptide. In some embodiments, the biological outcome is protection against an ammonium chloride challenge at specific times after administration.

[0062] Encapsulation: As used herein, the term encapsulation or its grammatical equivalent refers to the process of confining a nucleic acid molecule within a Petition 870260055239, dated 08 / 06 / 2026, page 41 / 305 28 / 131 nanoparticle.

[0063] Expression: As used herein, expression of a nucleic acid sequence refers to the translation of an mRNA into a polypeptide, the assembly of multiple polypeptides (e.g., antibody heavy chain or light chain) into an intact protein (e.g., antibody), and / or post-translational modification of a fully assembled polypeptide or protein (e.g., antibody). In this application, the terms expression and production and their grammatical equivalents are used interchangeably.

[0064] Improve, increase or decrease: As used herein, the terms improve, increase or decrease or grammatical equivalents indicate values ​​relative to a baseline measurement, such as a measurement in the same individual before the start of the treatment described herein, or a measurement in a control individual (or several control individuals) in the absence of the treatment described herein. A control individual is an individual afflicted with the same form of disease as the individual being treated, who is approximately the same age as the individual being treated.

[0065] Impurities: As used in this invention, the term impurities refers to substances within a confined quantity of liquid, gas, or solid that differ from the chemical composition of the target material or compound. Impurities are also called contaminants.

[0066] In vitro: As used in this invention, the term in vitro refers to events that occur in an artificial environment, for example, in a test tube or reaction vessel, in cell culture, etc., rather than within a multicellular organism.

[0067] In vivo: As used in this invention, the term in vivo refers to events that occur within a multicellular organism, such as a human being and a non-human animal. In the context of Petition 870260055239, dated 08 / 06 / 2026, page 42 / 305 29 / 131 cell-based systems, the term can be used to refer to events that occur within a living cell (as opposed to, for example, in vitro systems).

[0068] Isolate: As used in this invention, the term isolate refers to a substance and / or entity that has been (1) separated from at least some of the components with which it was associated when initially produced (whether in nature and / or in an experimental setting) and / or (2) produced, prepared and / or manufactured by human hands. Isolated substances and / or entities may be separated from about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or more than about 99% of the other components with which they were initially associated.In some embodiments, the isolated agents are approximately 80%, approximately 85%, approximately 90%, approximately 91%, approximately 92%, approximately 93%, approximately 94%, approximately 95%, approximately 96%, approximately 97%, approximately 98%, approximately 99%, or more than approximately 99% pure. As used herein, a substance is pure if it is substantially free of other components. As used herein, the calculation of the percentage purity of isolated substances and / or entities should not include excipients (e.g., buffer, solvent, water, etc.).

[0069] Liposome: As used herein, the term liposome refers to any lamellar, multilamellar, or solid vesicle of nanoparticles. Typically, a liposome as used herein may be formed by mixing one or more lipids or by mixing one or more lipids and polymer(s). In some embodiments, a liposome suitable for the present invention contains cationic lipid(s) and optionally non-cationic lipid(s), optionally lipid(s) to Petition 870260055239, dated 08 / 06 / 2026, page 43 / 305 30 / 131 cholesterol base and / or optionally PEG-modified lipid(s).

[0070] Local distribution or release: As used herein, the terms local distribution, local release, or grammatical equivalents refer to tissue-specific release or distribution. Typically, local distribution or release requires that a peptide or protein (e.g., enzyme) encoded by mRNAs be translated and expressed intracellularly or with limited secretion that avoids entering the patient's circulatory system.

[0071] Messenger RNA (mRNA): As used herein, the term Messenger RNA (mRNA) refers to a polynucleotide that codes for at least one peptide, polypeptide, or protein. mRNA, as used in this invention, encompasses both modified and unmodified RNA. mRNA may contain one or more coding and non-coding regions. mRNA may be purified from natural sources, produced using recombinant expression systems and optionally purified, chemically synthesized, etc. When appropriate, for example, in the case of chemically synthesized molecules, mRNA may comprise nucleoside analogs such as analogs having chemically modified bases or sugars, backbone modifications, etc. An mRNA sequence is presented in the 5' to 3' direction unless otherwise indicated.In some embodiments, an mRNA is or comprises natural nucleosides (e.g., adenosine, guanosine, cytidine, uridine); nucleoside analogs (e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolopyrimidine, 3-methyladenosine, 5-methylcytidine, C-5 propynylcytidine, C-5 propynyluridine, 2-aminoadenosine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynyluridine, C5-propynylcytidine, C5-methylcytidine, 2-aminoadenosine, 7-deazadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, O(6)methylguanine, 2-thiocytidine, pseudouridine, and 5-methylcytidine); bases. Petition 870260055239, dated 08 / 06 / 2026, page 44 / 305 31 / 131 chemically modified; biologically modified bases (e.g., methylated bases); intercalated bases; modified sugars (e.g., 2-fluororibose, ribose, 2'-deoxyribose, arabinose, and hexose); and / or modified phosphate groups (e.g., phosphorothioates and 5'-N-phosphoramidite linkages).

[0072] N / P Ratio: As used herein, the term N / P ratio refers to a molar ratio of positively charged molecular units in the cationic lipids in a lipid nanoparticle relative to negatively charged molecular units in the mRNA encapsulated within that lipid nanoparticle. As such, the N / P ratio is typically calculated as the ratio of moles of amine groups in cationic lipids in a lipid nanoparticle relative to moles of phosphate groups in mRNA encapsulated within that lipid nanoparticle.

[0073] Nucleic acid: As used in this invention, the term nucleic acid, in its broadest sense, refers to any compound and / or substance that is or can be incorporated into a polynucleotide chain. In some embodiments, a nucleic acid is a compound and / or substance that is or can be incorporated into a polynucleotide chain via a phosphodiester bond. In some embodiments, nucleic acid refers to individual nucleic acid residues (e.g., nucleotides and / or nucleosides). In some embodiments, nucleic acid refers to a polynucleotide chain comprising individual nucleic acid residues. In some embodiments, nucleic acid encompasses RNA, as well as single-stranded and / or double-stranded DNA and / or cDNA. Furthermore, the terms nucleic acid, DNA, RNA, and / or similar terms include nucleic acid analogs, i.e., analogs having a different phosphodiester structure.For example, the so-called peptide nucleic acids, which are known in the art and have peptide bonds instead of phosphodiester bonds in the chain. Petition 870260055239, dated 08 / 06 / 2026, page 45 / 305 32 / 131 principal, are considered within the scope of the present invention. The term nucleotide sequence encoding an amino acid sequence includes all nucleotide sequences that are degenerate versions of each other and / or encode the same amino acid sequence. Nucleotide sequences encoding proteins and / or RNA may include introns. Nucleic acids may be purified from natural sources, produced using recombinant expression systems and optionally purified, chemically synthesized, etc. Where appropriate, for example, in the case of chemically synthesized molecules, nucleic acids may comprise nucleoside analogs such as analogs having chemically modified bases or sugars, backbone modifications, etc. A nucleic acid sequence is presented in the 5' to 3' direction unless otherwise indicated.In some embodiments, a nucleic acid is or comprises natural nucleosides (e.g., adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine, deoxythymidine, deoxyguanosine, and deoxycytidine); Nucleoside analogs (e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolopyrimidine, 3-methyladenosine, 5-methylcytidine, C-5 propynylcytidine, C-5 propynyluridine, 2-aminoadenosine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynyluridine, C5-propynylcytidine, C5-methylcytidine, 2-aminoadenosine, 7-deazadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, O(6)-methylguanine and 2-thiocytidine); chemically modified bases; biologically modified bases (e.g., methylated bases); intercalated bases; Modified sugars (e.g., 2'-fluororibose, ribose, 2'-deoxyribose, arabinose, and hexose); and / or modified phosphate groups (e.g., phosphorothioates and 5'-N-phosphoramidite linkages).In some embodiments, the present invention is specifically directed to unmodified nucleic acids, meaning nucleic acids (e.g., polynucleotides and residues, including nucleotides and / or). Petition 870260055239, dated 08 / 06 / 2026, page 46 / 305 33 / 131 nucleosides) that have not been chemically modified to facilitate or achieve release. In some embodiments, the T and U nucleotides are used interchangeably in sequence descriptions.

[0074] Patient: As used herein, the term patient or individual refers to any organism to which a supplied composition may be administered, for example, for experimental, diagnostic, prophylactic, cosmetic and / or therapeutic purposes. Typical patients include animals (e.g., mammals such as mice, rats, rabbits, non-human primates and / or humans). In some embodiments, a patient is a human being. A human being includes pre- and postnatal forms.

[0075] Pharmaceutically acceptable: The term pharmaceutically acceptable, as used herein, refers to substances which, within the scope of sound medical judgment, are suitable for use in contact with the tissues of humans and animals without excessive toxicity, irritation, allergic response or other problem or complication, consistent with a reasonable benefit / risk ratio.

[0076] Pharmaceutically acceptable salt: Pharmaceutically acceptable salts are well known in the art. For example, SM Berge et al., describes pharmaceutically acceptable salts in detail in J. Pharmaceutical Sciences (1977) 66:1-19. The pharmaceutically acceptable salts of the compounds of this invention include those derived from suitable inorganic and organic acids and bases. Examples of pharmaceutically acceptable non-toxic acid addition salts are salts of an amino group formed with inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid and perchloric acid or with organic acids such as acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid or malonic acid or through the use of other Petition 870260055239, dated 08 / 06 / 2026, page 47 / 305 34 / 131 methods used in the technique such as ion exchange. Other pharmaceutically acceptable salts include adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate, hydroiodide, 2-hydroxyethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, Stearate, succinate, sulfate, tartrate, thiocyanate, p-toluenesulfonate, undecanoate, valerate salts and the like. Salts derived from suitable bases include alkali metal, alkaline earth metal, ammonium and N+(C1-4 alkyl)4 salts.Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, and the like. Other pharmaceutically acceptable salts include, where appropriate, non-toxic ammonium, quaternary ammonium, and amine cations formed using counterions such as halide, hydroxide, carboxylate, sulfate, phosphate, nitrate, sulfonate, and aryl sulfonate. Other pharmaceutically acceptable salts include salts formed from the quaternization of an amine using an appropriate electrophile, for example, an alkyl halide, to form a quaternized alkylated amino salt.

[0077] Potency: As used in this invention, the term potency or grammatical equivalents refers to the level of expression of protein(s) or peptide(s) that the mRNA encodes and / or the resulting biological effect.

[0078] Salt: As used in this invention, the term salt refers to an ionic compound that results or may result from a neutralization reaction between an acid and a base. Petition 870260055239, dated 08 / 06 / 2026, page 48 / 305 35 / 131

[0079] Systemic distribution or release: As used herein, the terms systemic distribution, systemic release, or grammatical equivalent, refer to a mechanism or approach to release or distribution that affects the whole body or an entire organism. Typically, systemic distribution or release is carried out through the body's circulatory system, for example, the bloodstream. In comparison with the definition of local distribution or release.

[0080] Individual: As used herein, the term individual refers to a human being or any non-human animal (e.g., mouse, rat, rabbit, puppy, cat, cattle, pig, sheep, horse, or primate). A human being includes pre- and postnatal forms. In many modalities, an individual is a human being. An individual may be a patient, which refers to a human being presenting to a physician for diagnosis or treatment of a disease. The term individual is herein used interchangeably with person or patient. An individual may be afflicted with or susceptible to a disease or disorder, but may or may not exhibit symptoms of the disease or disorder.

[0081] Substantially: As used herein, the term substantially refers to the qualitative condition of exhibiting total or near-total extent or degree of a characteristic or property of interest. A person skilled in biological techniques will understand that biological and chemical phenomena rarely, if ever, go to completion and / or proceed to completeness or reach or avoid an absolute result. The term substantially is therefore used herein to capture the potential lack of completeness inherent in many biological and chemical phenomena.

[0082] Target tissues: As used in this invention, the term target tissues refers to any tissue affected by a disease to be treated. In some embodiments, target tissues include those Petition 870260055239, dated 08 / 06 / 2026, p. 49 / 305 36 / 131 tissues that exhibit pathology, symptoms, or characteristics associated with the disease.

[0083] Therapeutically Effective Amount: As used in this invention, the term therapeutically effective amount of a therapeutic agent means an amount that is sufficient, when administered to an individual suffering from or susceptible to a disease, disorder and / or condition, to treat, diagnose, prevent and / or delay the onset of the symptom(s) of the disease, disorder and / or condition. It will be observed by those skilled in the art that a therapeutically effective amount is typically administered through a dosing regimen comprising at least one unit dose.

[0084] Therapeutic Index: As used herein, Index The therapeutic index is the relationship between the concentration of a drug in the blood at which it becomes toxic and the concentration at which it is effective. The higher the therapeutic index, the safer the drug.

[0085] Treatment: As used in this invention, the term treat, treatment or treating refers to any method used to alleviate, improve, relieve, inhibit, prevent, delay the onset, reduce the severity and / or reduce the incidence partially or completely of one or more symptoms or characteristics of a given disease, disorder and / or condition. Treatment may be administered to an individual who does not show signs of a disease and / or shows only early signs of the disease for the purpose of reducing the risk of developing disease-related pathology.

[0086] Yield: As used herein, the term yield refers to the percentage of mRNA recovered after encapsulation compared to the total mRNA as starting material. In some embodiments, the term recovery is used interchangeably with the term yield. Petition 870260055239, dated 08 / 06 / 2026, p. 50 / 305 37 / 131 DETAILED DESCRIPTION

[0087] The present invention provides an improved process for manufacturing mRNA encapsulated in lipid nanoparticle (LNP) formulations to produce therapeutic mRNA compositions, such that the process does not require a heating step. The invention is based on the surprising discovery that mixing an mRNA solution in low-citrate buffer and a lipid solution at room temperature (without preheating the mRNA solution and / or the lipid solution) resulted in high encapsulation efficiency, mRNA recovery rate, and more homogeneous and smaller particle size. Thus, in one aspect, the present invention provides an effective, reliable, energy-efficient, cost-effective, and safer method of encapsulating mRNA in lipid nanoparticles, which can be used for large-scale manufacturing process therapeutic applications without the use of heating. Formation of liposomes encapsulating mRNA

[0088] The mRNA encapsulation method in lipid nanoparticles disclosed herein can be applied to various techniques that are currently known in the art. Several methods are described in U.S. Application No. 2011 / 0244026, U.S. Application No. 2016 / 0038432, U.S. Application No. 2018 / 0153822, U.S. Application No. 2018 / 0125989, and U.S. Provisional Application No. 62 / 877597, filed July 23, 2019, and can be used to practice the present invention, all of which are incorporated herein by reference. As used in this invention, Process A refers to a conventional method of encapsulating mRNA by mixing mRNA with a lipid mixture, without first pre-forming the lipids into lipid nanoparticles, as described in US 2016 / 0038432. As used herein, Process B refers to a process of encapsulating messenger RNA (mRNA) through Petition 870260055239, dated 08 / 06 / 2026, p. 51 / 305 38 / 131 of the mixture of pre-formed lipid nanoparticles with mRNA, as described in US 2018 / 0153822.

[0089] For nucleic acid release, achieving high encapsulation efficiencies is critical to protect the drug substance (e.g., mRNA) and reduce in vivo activity loss. Thus, the increased expression of a protein or peptide encoded by mRNA and its therapeutic effect is highly correlated with the mRNA encapsulation efficiency.

[0090] To achieve high encapsulation efficiency using the Process A described above typically includes a heating step of one or more of the solutions in 10 mM citrate buffer (i.e., applying heat from a heat source to the solution) to a temperature (or maintaining a temperature) higher than room temperature. As described in a published US Application No. 2016 / 0038432, heating one or more solutions increases mRNA encapsulation efficiency and recovery rate. Furthermore, Process A typically includes 10 to 100 mM citrate as a buffer in mRNA and / or lipid solutions. However, from a manufacturing standpoint, heating the mRNA and / or lipid solution requires significant energy and cost. Thus, in one aspect, the present invention provides a more economical and safer method of encapsulating mRNA in lipid nanoparticles, which can be used for large-scale manufacturing processes for therapeutic applications without the use of heat.The present invention discloses, for the first time, a process in which a high encapsulation rate can be achieved without heating the mRNA and / or lipid solutions prior to mixing, through the use of a low concentration of citrate (i.e., < 5 mM) in the mRNA solution. mRNA solution

[0091] Several methods can be used to prepare a Petition 870260055239, dated 08 / 06 / 2026, p. 52 / 305 39 / 131 suitable mRNA solution for the present invention. In some embodiments, the mRNA can be dissolved directly in a buffer solution described herein. In some embodiments, an mRNA solution can be generated by mixing an mRNA stock solution with a buffer solution before mixing with a lipid solution for encapsulation. In some embodiments, an mRNA solution can be generated by mixing an mRNA stock solution with a buffer solution immediately before mixing with a lipid solution for encapsulation. In some embodiments, a suitable mRNA stock solution may contain mRNA in water at a concentration equal to or greater than about 0.2 mg / ml, 0.4 mg / ml, 0.5 mg / ml, 0.6 mg / ml, 0.8 mg / ml, 1.0 mg / ml, 1.2 mg / ml, 1.4 mg / ml, 1.5 mg / ml, 1.6 mg / ml or 2.0 mg / ml. Consequently, in some embodiments, a suitable mRNA stock solution may contain mRNA in water at a concentration equal to or greater than about 0.2 mg / ml.In some embodiments, a suitable mRNA stock solution may contain mRNA in water at a concentration equal to or greater than about 0.4 mg / ml. In some embodiments, a suitable mRNA stock solution may contain mRNA in water at a concentration equal to or greater than about 0.5 mg / ml. In some embodiments, a suitable mRNA stock solution may contain mRNA in water at a concentration equal to or greater than about 0.6 mg / ml. In some embodiments, a suitable mRNA stock solution may contain mRNA in water at a concentration equal to or greater than about 0.8 mg / ml. In some embodiments, a suitable mRNA stock solution may contain mRNA in water at a concentration equal to or greater than about 1.0 mg / ml. In some embodiments, a suitable mRNA stock solution may contain mRNA in water at a concentration equal to or greater than about 1.2 mg / ml. In some embodiments, a stock solution of... Petition 870260055239, dated 08 / 06 / 2026, p. 53 / 305 40 / 131 A suitable mRNA stock solution may contain mRNA in water at a concentration equal to or greater than about 1.4 mg / ml. In some embodiments, a suitable mRNA stock solution may contain mRNA in water at a concentration equal to or greater than about 1.5 mg / ml. In some embodiments, a suitable mRNA stock solution may contain mRNA in water at a concentration equal to or greater than about 1.6 mg / ml. In some embodiments, a suitable mRNA stock solution may contain mRNA in water at a concentration equal to or greater than about 2.0 mg / ml. In some embodiments, a suitable mRNA stock solution may contain mRNA in water at a concentration equal to or greater than about 2.5 mg / ml, 3.0 mg / ml, 3.5 mg / ml, 4.0 mg / ml, 4.5 mg / ml, or 5.0 mg / ml. In some embodiments, a suitable mRNA stock solution contains mRNA at a concentration equal to or greater than about 1 mg / ml, about 10 mg / ml, about 50 mg / ml, or about 100 mg / ml.

[0092] Typically, a suitable mRNA solution may also contain a buffering agent and / or salt. Generally, buffering agents may include HEPES, ammonium sulfate, sodium bicarbonate, sodium citrate, sodium acetate, potassium phosphate, and sodium phosphate. In some embodiments, the appropriate concentration of the buffering agent may range from about 0.1 mM to 100 mM, 0.5 mM to 90 mM, 1.0 mM to 80 mM, 2 mM to 70 mM, 3 mM to 60 mM, 4 mM to 50 mM, 5 mM to 40 mM, 6 mM to 30 mM, 7 mM to 20 mM, 8 mM to 15 mM, or 9 to 12 mM. In some embodiments, the appropriate concentrations of the buffering agent may range from 2.0 mM to 4.0 mM.

[0093] In some embodiments, a buffer solution comprises less than about 5 mM citrate. In some embodiments, a buffer solution comprises less than about 3 mM citrate. In some embodiments, a buffer solution Petition 870260055239, dated 08 / 06 / 2026, page 54 / 305 41 / 131 comprises less than about 1 mM of citrate. In some embodiments, a buffer solution comprises less than about 0.5 mM of citrate. In some embodiments, a buffer solution comprises less than about 0.25 mM of citrate. In some embodiments, a buffer solution comprises less than about 0.1 mM of citrate. In some embodiments, a buffer solution does not comprise citrate.

[0094] Exemplary salts may include sodium chloride, magnesium chloride, and potassium chloride. In some embodiments, the appropriate concentration of salts in an mRNA solution may vary from about 1 mM to 500 mM, 5 mM to 400 mM, 10 mM to 350 mM, 15 mM to 300 mM, 20 mM to 250 mM, 30 mM to 200 mM, 40 mM to 190 mM, 50 mM to 180 mM, 50 mM to 170 mM, 50 mM to 160 mM, 50 mM to 150 mM, or 50 mM to 100 mM. The salt concentration in a suitable mRNA solution is equal to or greater than approximately 1 mM, 5 mM, 10 mM, 20 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, 80 mM, 90 mM, or 100 mM.

[0095] In some embodiments, a buffer solution comprises about 300 mM of NaCl. In some embodiments, a buffer solution comprises about 200 mM of NaCl. In some embodiments, a buffer solution comprises about 175 mM of NaCl. In some embodiments, a buffer solution comprises about 150 mM of NaCl. In some embodiments, a buffer solution comprises about 100 mM of NaCl. In some embodiments, a buffer solution comprises about 75 mM of NaCl. In some embodiments, a buffer solution comprises about 50 mM of NaCl. In some embodiments, a buffer solution comprises about 25 mM of NaCl.

[0096] In some embodiments, a suitable mRNA solution may have a pH ranging from approximately 3.5 to 6.5, 3.5 to 6.0, 3.5 to 5.5, 3.5 Petition 870260055239, dated 08 / 06 / 2026, p. 55 / 305 42 / 131 to 5.0, 3.5 to 4.5, 4.0 to 5.5, 4.0 to 5.0, 4.0 to 4.9, 4.0 to 4.8, 4.0 to 4.7, 4.0 to 4.6, or 4.0 to 4.5. In some embodiments, a suitable mRNA solution may have a pH equal to or not greater than approximately 3.5, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.2, 5.4, 5.6, 5.8, 6.0, 6.1, 6.3, and 6.5.

[0097] In some embodiments, a buffer solution has a pH of about 5.0. In some embodiments, a buffer solution has a pH of about 4.8. In some embodiments, a buffer solution has a pH of about 4.7. In some embodiments, a buffer solution has a pH of about 4.6. In some embodiments, a buffer solution has a pH of about 4.5. In some embodiments, a buffer solution has a pH of about 4.4. In some embodiments, a buffer solution has a pH of about 4.3. In some embodiments, a buffer solution has a pH of about 4.2. In some embodiments, a buffer solution has a pH of about 4.1. In some embodiments, a buffer solution has a pH of about 4.0. In some embodiments, a buffer solution has a pH of about 3.9. In some embodiments, a buffer solution has a pH of about 3.8. In some applications, a buffer solution has a pH of approximately 3.7.In some embodiments, a buffer solution has a pH of approximately 3.6. In some embodiments, a buffer solution has a pH of approximately 3.5. In some embodiments, a buffer solution has a pH of approximately 3.4.

[0098] In some embodiments, a stock solution of mRNA is mixed with a buffer solution using a pump. Exemplary pumps include, but are not limited to, pulseless flow pumps, gear pumps, peristaltic pumps, and centrifugal pumps.

[0099] Typically, the buffer solution is mixed at a rate Petition 870260055239, dated 08 / 06 / 2026, page 56 / 305 43 / 131 greater than that of the mRNA stock solution. For example, the buffer solution may be mixed at a rate at least 1x, 2x, 3x, 4x, 5x, 6x, 7x, 8x, 9x, 10x, 15x, or 20x greater than the rate of the mRNA stock solution. In some embodiments, a buffer solution is mixed at a flow rate ranging from about 100 to 6000 ml / minute (for example, about 100 to 300 ml / minute, 300 to 600 ml / minute, 600 to 1200 ml / minute, 1200 to 2400 ml / minute, 2400 to 3600 ml / minute, 3600 to 4800 ml / minute, 4800 to 6000 ml / minute, or 60 to 420 ml / minute). In some embodiments, a buffer solution is mixed at a flow rate of or greater than about 60 ml / minute, 100 ml / minute, 140 ml / minute, 180 ml / minute, 220 ml / minute, 260 ml / minute, 300 ml / minute, 340 ml / minute, 380 ml / minute, 420 ml / minute, 480 ml / minute, 540 ml / minute, 600 ml / minute, 1200 ml / minute, 2400 ml / minute, 3600 ml / minute, 4800 ml / minute, or 6000 ml / minute.

[00100] In some embodiments, an mRNA stock solution is mixed at a flow rate ranging from about 10 to 600 ml / minute (for example, about 5 to 50 ml / minute, about 10 to 30 ml / minute, about 30 to 60 ml / minute, about 60 to 120 ml / minute, about 120 to 240 ml / minute, about 240 to 360 ml / minute, about 360 to 480 ml / minute, or about 480 to 600 ml / minute). In some embodiments, a stock solution of mRNA is mixed at a flow rate of or greater than about 5 ml / minute, 10 ml / minute, 15 ml / minute, 20 ml / minute, 25 ml / minute, 30 ml / minute, 35 ml / minute, 40 ml / minute, 45 ml / minute, 50 ml / minute, 60 ml / minute, 80 ml / minute, 100 ml / minute, 200 ml / minute, 300 ml / minute, 400 ml / minute, 500 ml / minute, or 600 ml / minute.

[00101] In some embodiments, the mRNA stock solution is mixed at a flow rate ranging from about 10 to 30 ml / minute, about 30 to 60 ml / minute, about 60 to 120 ml / minute, about 120 to 240 ml / minute, about 240 to 360 ml / minute, about 360 to 480 ml / minute. Petition 870260055239, dated 08 / 06 / 2026, p. 57 / 305 44 / 131 ml / minute or about 480 to 600 ml / minute. In some embodiments, the mRNA stock solution is mixed at a flow rate of about 20 ml / minute, about 40 ml / minute, about 60 ml / minute, about 80 ml / minute, about 100 ml / minute, about 200 ml / minute, about 300 ml / minute, about 400 ml / minute, about 500 ml / minute, or about 600 ml / minute.

[00102] In some embodiments, an mRNA solution is at room temperature. In some embodiments, an mRNA solution is at a temperature of about 20 to 25 °C. In some embodiments, an mRNA solution is at a temperature of about 21 to 23 °C. In some embodiments, an mRNA solution is not heated before mixing with a lipid solution. In some embodiments, an mRNA solution is kept at room temperature. Lipid Solution

[00103] According to the present invention, a lipid solution contains a mixture of lipids suitable for forming lipid nanoparticles for mRNA encapsulation. In some embodiments, a suitable lipid solution is derived from ethanol. For example, a suitable lipid solution may contain a mixture of desired lipids dissolved in pure ethanol (i.e., 100% ethanol). In another embodiment, a suitable lipid solution is derived from isopropyl alcohol. In another embodiment, a suitable lipid solution is derived from dimethyl sulfoxide. In another embodiment, a suitable lipid solution is a mixture of suitable solvents including, but not limited to, ethanol, isopropyl alcohol, and dimethyl sulfoxide.

[00104] A suitable lipid solution may contain a mixture of desired lipids at various concentrations. For example, a suitable lipid solution may contain a mixture of desired lipids at a total concentration of or greater than about 0.1 mg / ml, 0.5 Petition 870260055239, dated 08 / 06 / 2026, page 58 / 305 45 / 131 mg / ml, 1.0 mg / ml, 2.0 mg / ml, 3.0 mg / ml, 4.0 mg / ml, 5.0 mg / ml, 6.0 mg / ml, 7.0 mg / ml, 8.0 mg / ml, 9.0 mg / ml, 10 mg / ml, 15 mg / ml, 20 mg / ml, 30 mg / ml, 40 mg / ml, 50 mg / ml or 100 mg / ml. In some embodiments, a suitable lipid solution may contain a mixture of desired lipids in a total concentration ranging from about 0.1 to 100 mg / ml, 0.5 to 90 mg / ml, 1.0 to 80 mg / ml, 1.0 to 70 mg / ml, 1.0 to 60 mg / ml, 1.0 to 50 mg / ml, 1.0 to 40 mg / ml, 1.0 to 30 mg / ml, 1.0 to 20 mg / ml, 1.0 to 15 mg / ml, 1.0 to 10 mg / ml, 1.0 to 9 mg / ml, 1.0 to 8 mg / ml, 1.0 to 7 mg / ml, 1.0 to 6 mg / ml, or 1.0 to 5 mg / ml. In some embodiments, a suitable lipid solution may contain a mixture of desired lipids at a total concentration of up to about 100 mg / ml, 90 mg / ml, 80 mg / ml, 70 mg / ml, 60 mg / ml, 50 mg / ml, 40 mg / ml, 30 mg / ml, 20 mg / ml, or 10 mg / ml.

[00105] Any desired lipids can be mixed in any ratios suitable for mRNA encapsulation. In some embodiments, a suitable lipid solution contains a mixture of desired lipids including cationic lipids, auxiliary lipids (e.g., non-cationic lipids and / or cholesterol lipids), amphiphilic block copolymers (e.g., poloxamers), and / or PEGylated lipids. In some embodiments, a suitable lipid solution contains a mixture of desired lipids including one or more cationic lipids, one or more auxiliary lipids (e.g., non-cationic lipids and / or cholesterol lipids), and one or more PEGylated lipids.

[00106] In some embodiments, a lipid solution is at room temperature. In some embodiments, a lipid solution is at a temperature of about 20 to 25°C. In some embodiments, a lipid solution is at a temperature of about 21 to 23°C. In some embodiments, a lipid solution is not heated before mixing with a lipid solution. In some embodiments, Petition 870260055239, dated 08 / 06 / 2026, p. 59 / 305 46 / 131 a lipid solution is kept at room temperature.

[00107] In certain embodiments, the provided compositions comprise a liposome in which mRNA is associated on both surfaces of the liposome and encapsulated within the same liposome. For example, during the preparation of the compositions of the present invention, cationic liposomes can associate with mRNA through electrostatic interactions.

[00108] In some embodiments, the compositions and methods of the invention comprise mRNA encapsulated in a liposome. In some embodiments, one or more mRNA species may be encapsulated in the same liposome. In some embodiments, one or more mRNA species may be encapsulated in different liposomes. In some embodiments, the mRNAs are encapsulated in one or more liposomes, which differ in their lipid composition, molar ratio of lipid components, size, charge (zeta potential), targeting ligands and / or combinations thereof. In some embodiments, one or more liposomes may have a different composition of cationic lipids based on sterols, neutral lipid, PEG-modified lipid and / or combinations thereof. In some embodiments, one or more liposomes may have a different molar ratio of cationic lipid based on cholesterol, neutral lipid and PEG-modified lipid used to create the liposome. Encapsulation Process

[00109] As used herein, a process for the formation of mRNA-laden lipid nanoparticles (mRNA-LNPs) is used interchangeably with the term mRNA encapsulation or its grammatical variants. In some embodiments, mRNA-LNPs are formed by mixing an mRNA solution with a lipid solution, wherein the mRNA solution and / or the lipid solution are held at room temperature prior to mixing. Petition 870260055239, dated 08 / 06 / 2026, p. 60 / 305 47 / 131

[00110] In some embodiments, an mRNA solution and a lipid solution are mixed within the solution in such a way that the mRNA becomes encapsulated in the lipid nanoparticle. Such a solution is also referred to as an encapsulation formulation or solution.

[00111] A suitable encapsulation formulation or solution includes a solvent such as ethanol. For example, a suitable encapsulation formulation or solution includes about 10% ethanol, about 15% ethanol, about 20% ethanol, about 25% ethanol, about 30% ethanol, about 35% ethanol, or about 40% ethanol. In some embodiments, a suitable encapsulation formulation or solution includes a solvent such as isopropyl alcohol. For example, a suitable encapsulation formulation or solution includes about 10% isopropyl alcohol, about 15% isopropyl alcohol, about 20% isopropyl alcohol, about 25% isopropyl alcohol, about 30% isopropyl alcohol, about 35% isopropyl alcohol, or about 40% isopropyl alcohol.

[00112] In some embodiments, a suitable encapsulation formulation or solution includes a solvent such as dimethyl sulfoxide. For example, a suitable encapsulation formulation or solution includes about 10% dimethyl sulfoxide, about 15% dimethyl sulfoxide, about 20% dimethyl sulfoxide, about 25% dimethyl sulfoxide, about 30% dimethyl sulfoxide, about 35% dimethyl sulfoxide, or about 40% dimethyl sulfoxide.

[00113] In some embodiments, a suitable encapsulation formulation or solution may also contain a buffering agent or salt. Exemplary buffering agents may include HEPES, ammonium sulfate, sodium bicarbonate, sodium citrate, sodium acetate, potassium phosphate, and sodium phosphate. Exemplary salt Petition 870260055239, dated 08 / 06 / 2026, page 61 / 305 48 / 131 may include sodium chloride, magnesium chloride, and potassium chloride.

[00114] In some embodiments, ethanol, citrate buffer, and other destabilizing agents are absent during mRNA addition, and therefore the formulation requires no further downstream processing. In some embodiments, the formulation solution comprises trehalose. The lack of destabilizing agents and the stability of the trehalose solution increase the ease of scaling up the formulation and producing mRNA-encapsulated lipid nanoparticles.

[00115] In some embodiments, the lipid solution contains one or more cationic lipids, one or more non-cationic lipids, and one or more PEG lipids. In some embodiments, the lipids also contain one or more cholesterol lipids. In some embodiments, the lipids are present in an ethanolic stock solution.

[00116] In some embodiments, lipid and mRNA solutions are mixed using a pump system. In some embodiments, the pump system comprises a flow pump with fewer pulses. In some embodiments, the pump system is a gear pump. In some embodiments, a suitable pump is a peristaltic pump. In some embodiments, a suitable pump is a centrifugal pump. In some embodiments, the process using a pump system is carried out on a large scale. For example, in some embodiments, the process includes using pumps as described in this invention to mix a solution of at least about 1 mg, 5 mg, 10 mg, 50 mg, 100 mg, 500 mg, 1 g, 10 g, 50 g or 100 g or more of mRNA with a lipid solution, to produce mRNA encapsulated in lipid nanoparticles.In some embodiments, the process of mixing mRNA and lipid solutions provides a composition according to the present invention containing at least about 1 mg, 5 mg, 10 mg, 50 mg, 100 mg. Petition 870260055239, dated 08 / 06 / 2026, p. 62 / 305 49 / 131 500 mg, 1 g, 10 g, 50 g, or 100 g or more of encapsulated mRNA.

[00117] In some embodiments, a step combining lipid nanoparticles encapsulating mRNA with a lipid solution is performed using a pump system. This combination can be performed using a pump. In some embodiments, the mRNA and lipid solutions are mixed at a flow rate ranging from about 25 to 75 ml / minute, about 75 to 200 ml / minute, about 200 to 350 ml / minute, about 350 to 500 ml / minute, about 500 to 650 ml / minute, about 650 to 850 ml / minute, or about 850 to 1000 ml / minute.In some embodiments, an mRNA solution and a lipid solution are mixed at a flow rate of approximately 50 ml / minute, approximately 100 ml / minute, approximately 150 ml / minute, approximately 200 ml / minute, approximately 250 ml / minute, approximately 300 ml / minute, approximately 350 ml / minute, approximately 400 ml / minute, approximately 450 ml / minute, approximately 500 ml / minute, approximately 550 ml / minute, approximately 600 ml / minute, approximately 650 ml / minute, approximately 700 ml / minute, approximately 750 ml / minute, approximately 800 ml / minute, approximately 850 ml / minute, approximately 900 ml / minute, approximately 950 ml / minute ml / minute or approximately 1000 ml / minute.

[00118] In some embodiments, the mixing of an mRNA solution with a lipid solution is performed in the absence of any pump.

[00119] In some embodiments, the process according to the present invention includes maintaining at room temperature (i.e., not applying heat from a heating source to the solution) one or more of the solution comprising the lipids, the solution comprising the mRNA, and the mixed solution comprising the mRNA encapsulated with lipid nanoparticles. In some embodiments, the process includes the step of maintaining at room temperature one or both of the mRNA solution and the lipid solution before the mixing step. In Petition 870260055239, dated 08 / 06 / 2026, p. 63 / 305 50 / 131 In some embodiments, the process includes maintaining one or more of the solutions comprising the lipids and the solutions comprising the mRNA at room temperature during the mixing step. In some embodiments, the process includes the step of maintaining the lipid nanoparticle-encapsulated mRNA at room temperature after the mixing step. In some embodiments, the room temperature at which one or more of the solutions is maintained is at or below about 35 °C, 30 °C, 25 °C, 20 °C, or 16 °C. In some embodiments, the ambient temperature at which one or more of the solutions are maintained varies from about 15 to 35 °C, about 15 to 30 °C, about 15 to 25 °C, about 15 to 20 °C, about 20 to 35 °C, about 25 to 35 °C, about 30 to 35 °C, about 20 to 30 °C, about 25 to 30 °C, or about 20 to 25 °C. In some embodiments, the ambient temperature at which one or more of the solutions are maintained is 20 to 25 °C.

[00120] In some embodiments, the process according to the present invention includes performing the mRNA and lipid solution mixing step at room temperature to form lipid nanoparticles encapsulating mRNA.

[00121] In some embodiments, more than about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% of the purified nanoparticles have a size smaller than about 150 nm (e.g., smaller than about 145 nm, about 140 nm, about 135 nm, about 130 nm, about 125 nm, about 120 nm, about 115 nm, about 110 nm, about 105 nm, about 100 nm, about 95 nm, about 90 nm, about 85 nm, about nm, about 75 nm, about 70 nm, about 65 nm, about 60 nm, approximately 55 nm or approximately 50 nm). In some embodiments, substantially all purified nanoparticles have a size smaller than 150 nm (e.g., smaller than approximately 145 nm, approximately 140 nm, approximately 135 nm, approximately 130 nm, approximately Petition 870260055239, dated 08 / 06 / 2026, p. 64 / 305 51 / 131 125 nm, approximately 120 nm, approximately 115 nm, approximately 110 nm, approximately 105 nm, approximately 100 nm, approximately 95 nm, approximately 90 nm, approximately 85 nm, approximately 80 nm, approximately 75 nm, approximately 70 nm, approximately 65 nm, approximately 60 nm, approximately 55 nm, or approximately 50 nm). In some embodiments, more than approximately 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% of the purified nanoparticles have a size ranging from 50 to 150 nm. In some embodiments, substantially all purified nanoparticles have a size ranging from 50 to 150 nm. In some embodiments, more than approximately 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% of the purified nanoparticles have a size ranging from 80 to 150 nm. In some embodiments, substantially all purified nanoparticles have a size ranging from 80 to 150 nm.

[00122] In some embodiments, a process according to the present invention results in an encapsulation rate greater than about 90%, 95%, 96%, 97%, 98%, or 99%. In some embodiments, a process according to the present invention results in more than about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% mRNA recovery.

[00123] In some embodiments, a process according to the present invention comprises a post-mixing mRNA-LNP incubation step. A post-mixing mRNA-LNP incubation step is described in U.S. Provisional Application No. 62 / 847,837, filed May 14, 2019, and can be used to practice the present invention, which is incorporated herein by reference. Purification

[00124] In some embodiments, mRNA-LNPs are purified and / or concentrated. Several purification methods can be used. In some embodiments, mRNA-LNPs are purified by a Petition 870260055239, dated 08 / 06 / 2026, page 65 / 305 52 / 131 Tangential Flow Filtration (TFF) process. In some embodiments, mRNA-LNPs are purified by gravity-based normal flow filtration (NFF). In some embodiments, mRNA-LNPs are purified by any other suitable filtration process. In some embodiments, mRNA-LNPs are purified by centrifugation. In some embodiments, mRNA-LNPs are purified by chromatographic methods. Release Vehicles

[00125] According to the present invention, mRNA encoding a protein or peptide (e.g., a full length, fragment, or part of a protein or peptide) as described in this invention can be released as naked (unpackaged) RNA or via delivery vehicles. As used herein, the terms delivery vehicle, transfer vehicle, nanoparticle, or grammatical equivalent are used interchangeably.

[00126] Delivery vehicles may be formulated in combination with one or more additional nucleic acids, carriers, targeting ligands, or stabilizing reagents, or in pharmaceutical compositions where it is mixed with suitable excipients. For example, liposomes encapsulating mRNA may be formed as described above. Techniques for drug formulation and delivery may be found in Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa., latest edition. A particular delivery vehicle is selected based on its ability to facilitate the transfection of a nucleic acid into a target cell.

[00127] In some embodiments, mRNAs encoding at least one protein or peptide can be released via a single delivery vehicle. In some embodiments, mRNAs encoding at least one protein or peptide can be released Petition 870260055239, dated 08 / 06 / 2026, page 66 / 305 53 / 131 via one or more delivery vehicles, each of a different composition. In some embodiments, one or more mRNAs are encapsulated in the same lipid nanoparticles. In some embodiments, one or more mRNAs are encapsulated in separate lipid nanoparticles. In some embodiments, the lipid nanoparticles are empty.

[00128] According to various embodiments, suitable delivery vehicles include, but are not limited to, polymer-based carriers such as polyethyleneimine (PEI), lipid nanoparticles and liposomes, nanoliposomes, ceramide-containing nanoliposomes, proteoliposomes, both natural and synthetically derived exosomes, natural, synthetic and semi-synthetic lamellar bodies, nanoparticulates, calcium phosphorus silicate nanoparticulates, calcium phosphate nanoparticulates, silicon dioxide nanoparticulates, nanocrystalline particulates, semiconductor nanoparticulates, poly(D-arginine), sol-gels, nanodendrimers, starch-based delivery systems, micelles, emulsions, niosomes, multi-domain block polymers (vinyl polymers, polypropyl acrylic acid polymers, dynamic polyconjugates), dry powder formulations, plasmids, viruses, nucleotides of calcium phosphate, aptamers, peptides, and other vector markers.The use of bionanocapsules and other viral capsid protein assemblies as a suitable transfer vehicle is also contemplated. (Hum. Gene Ther. 2008 September; 19(9):887-95). Liposomal delivery vehicles

[00129] In some embodiments, a suitable delivery vehicle is a liposomal delivery vehicle, for example, a lipid nanoparticle. As used herein, liposomal delivery vehicles, for example, lipid nanoparticles, are generally characterized as microscopic vesicles having a water space. Petition 870260055239, dated 08 / 06 / 2026, page 67 / 305 54 / 131 internally sequestered from an external medium by a membrane of one or more double layers. Liposome double-layer membranes are typically formed by amphiphilic molecules, such as lipids of synthetic or natural origin comprising spatially separated hydrophilic and hydrophobic domains (Lasic, Trends Biotechnol., 16: 307-321, 1998). Liposome double-layer membranes can also be formed by amphiphilic polymers and surfactants (e.g., polymersomes, niosomes, etc.). In the context of the present invention, a liposomal delivery vehicle typically serves to transport a desired nucleic acid (e.g., mRNA) to a target cell or tissue. In some embodiments, a nanoparticle delivery vehicle is a liposome. In some embodiments, a liposome comprises one or more cationic lipids, one or more non-cationic lipids, one or more cholesterol-based lipids, or one or more PEG-modified lipids.In some embodiments, a liposome comprises no more than three distinct lipid components. In some embodiments, one distinct lipid component is a sterol-based cationic lipid. Cationic Lipids

[00130] As used herein, the term cationic lipids refers to any of the various lipid species that have a net positive charge at a selected pH, such as physiological pH.

[00131] Suitable cationic lipids for use in the compositions and methods of the invention include the cationic lipids as described in International Patent Publication WO 2010 / 144740, which is incorporated herein by reference. In certain embodiments, the compositions and methods of the present invention include a cationic lipid, (6Z,9Z,28Z,31Z)-heptatriaconta6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate, having a compound structure of: Petition 870260055239, dated 08 / 06 / 2026, page 68 / 305 55 / 131 and its pharmaceutically acceptable salts.

[00132] Other cationic lipids suitable for use in the compositions and methods of the present invention include ionizable cationic lipids as described in International Patent Publication WO 2013 / 149140, which is incorporated herein by reference. In some embodiments, the compositions and methods of the present invention include a cationic lipid of one of the following formulas: or a pharmaceutically acceptable salt thereof, wherein Ri and R2 are each independently selected from the group consisting of hydrogen, an optionally substituted C1-C20 alkyl, variably saturated or unsaturated, and an optionally substituted C6-C20 acyl, variably saturated or unsaturated; wherein Li and L2 are each independently selected from the group consisting of hydrogen, an optionally substituted C1-C30 alkyl, an optionally substituted C1-C30 alkenyl variably unsaturated, and an optionally substituted C1-C30 alkynyl; where m and o are each independently selected from the group consisting of zero and any positive integer (e.g., where m is three); and where n is zero or any positive integer (e.g., where n is one).In certain embodiments, the compositions and methods of the present invention include the cationic lipid (15Z,18Z)-N,N-dimethyl-6-(9Z,12Z)octadeca-9,12-dien-1-i)tetracosa-15,18-dien-1-amine (HGT5000), having a compound structure of: Petition 870260055239, dated 08 / 06 / 2026, page 69 / 305 56 / 131 (HGT-5000) and the pharmaceutically acceptable salt thereof. In certain embodiments, the compositions and methods of the present invention include the cationic lipid (15Z,18Z)-N,N-dimethyl-6-((9Z,12Z)-octadeca-9,12dien-1-yl)tetracosa-4,15,18-trien-1-amine (HGT5001), having a compound structure of: (HGT-5001) and the pharmaceutically acceptable salt thereof. In certain embodiments, the compositions and methods of the present invention include the cationic lipid and (15Z,18Z)-N,N-dimethyl-6-((9Z,12Z)-octadeca-9,12dien-1-yl)tetracosa-5,15,18-trien-1-amine (HGT5002), having a compound structure of: (HGT-5002) and the pharmaceutically acceptable salt thereof.

[00133] Other cationic lipids suitable for use in the compositions and methods of the invention include cationic lipids described as lipid amino alcohols in International Patent Publication WO 2010 / 053572, which is incorporated herein by reference. In certain embodiments, the compositions and methods of the present invention include a cationic lipid having a compound structure of: Petition 870260055239, dated 08 / 06 / 2026, page 70 / 305 57 / 131 and the pharmaceutically acceptable salt thereof.

[00134] Other cationic lipids suitable for use in the compositions and methods of the invention include the cationic lipids as described in International Patent Publication WO 2016 / 118725, which is incorporated herein by reference. In certain embodiments, the compositions and methods of the present invention include a cationic lipid having a compound structure of: and the pharmaceutically acceptable salt thereof.

[00135] Other cationic lipids suitable for use in the compositions and methods of the invention include the cationic lipids as described in International Patent Publication WO 2016 / 118724, which is incorporated herein by reference. In certain embodiments, the compositions and methods of the present invention include a cationic lipid having a compound structure of: and the pharmaceutically acceptable salt thereof.

[00136] Other cationic lipids suitable for use in the compositions and methods of the invention include a cationic lipid having Petition 870260055239, dated 08 / 06 / 2026, page 71 / 305 58 / 131 the formula of 14,25-ditridecyl 15,18,21,24-tetraza-octatriacontane, and the pharmaceutically acceptable salt thereof.

[00137] Other cationic lipids suitable for use in the compositions and methods of the invention include the cationic lipids as described in International Patent Publications WO 2013 / 063468 and WO 2016 / 205691, each of which is incorporated herein by reference. In some embodiments, the compositions and methods of the present invention include a cationic lipid of the following formula: or a pharmaceutically acceptable salt thereof, wherein each case RL is independently C6-C40 alkenyl optionally substituted. In certain embodiments, the compositions and methods of the present invention include a cationic lipid having a compound structure of: OH and the pharmaceutically acceptable salt thereof. In certain embodiments, the compositions and methods of the present invention include a cationic lipid having a compound structure of: Petition 870260055239, dated 08 / 06 / 2026, page 72 / 305 59 / 131 and the pharmaceutically acceptable salt thereof. In certain embodiments, the compositions and methods of the present invention include a cationic lipid having a compound structure of: and the pharmaceutically acceptable salt thereof. In certain embodiments, the compositions and methods of the present invention include a cationic lipid having a compound structure of: Petition 870260055239, dated 08 / 06 / 2026, page 73 / 305 60 / 131 and the pharmaceutically acceptable salt thereof.

[00138] Other cationic lipid compositions and methods of the invention as described in the Publication suitable for use in include the cationic lipids International Patent WO 2015 / 184256, which is incorporated herein by reference. In some embodiments, the compositions and methods of the present invention include a cationic lipid of the following formula: H3C-(CH2) OH H3C-(CH2)mx^NJ OH j (CRARg)nX x (CRARB)n| OH < ^^(CH2)m-CH3 HO^(CH2)m-CH3 or a pharmaceutically acceptable salt thereof, wherein each X independently is O or S; each Y independently is O or S; each m independently is 0 to 20; each n independently is 1 to 6; each Ra is independently hydrogen, optionally substituted C1-50 alkyl, optionally substituted C2-50 alkenyl, optionally substituted C2-50 alkynyl, optionally substituted C3-10 carbocyclyl, optionally substituted 3- to 14-membered heterocyclyl, optionally substituted C6-14 aryl, optionally substituted 5- to 14-membered heteroaryl or halogen; and each Rb is independently hydrogen, optionally substituted C1-50 alkyl, optionally substituted C2-50 alkenyl, optionally substituted C2-50 alkynyl, optionally substituted C3-10 carbocyclyl, optionally substituted 3- to 14-membered heterocyclyl, optionally substituted C6-14 aryl, optionally substituted 5- to 14-membered heteroaryl, or halogen.In certain modalities, the compositions and methods of the present. Petition 870260055239, dated 08 / 06 / 2026, page 74 / 305 61 / 131 inventions include a cationic lipid, Target 23, having a compound structure of: (Target 23) and the pharmaceutically acceptable salt thereof.

[00139] Other cationic lipids suitable for use in the compositions and methods of the invention include the cationic lipids as described in International Patent Publication WO 2016 / 004202, which is incorporated herein by reference. In some embodiments, the compositions and methods of the present invention include a cationic lipid having the structure of the compound: or a pharmaceutically acceptable salt thereof. In some embodiments, the compositions and methods of the present invention include a cationic lipid having the structure of the compound: Petition 870260055239, dated 08 / 06 / 2026, page 75 / 305 62 / 131 or a pharmaceutically acceptable salt thereof. In some embodiments, the compositions and methods of the present invention include a cationic lipid having the structure of the compound: or a pharmaceutically acceptable salt thereof.

[00140] Other cationic lipids suitable for use in the compositions and methods of the present invention include cationic lipids as described in U.S. Provisional Patent Application Serial Number 62 / 758,179, which is incorporated herein by reference. In some embodiments, the compositions and methods of the present invention include a cationic lipid of the following formula: or a pharmaceutically acceptable salt thereof, wherein each R1 and R2 is independently H or C1-C10 aliphatic; each m is independently an integer having a value from 1 to 4; each A is independently a covalent bond or arylene; each L1 is independently an ester, thioester, disulfide, or anhydrous group; each L2 is independently C2-C10 aliphatic; each X1 is independently H or OH; and each R3 is independently C6-C20 aliphatic. In some embodiments, the compositions and methods of the present invention include a cationic lipid of the following formula: Petition 870260055239, dated 08 / 06 / 2026, p. 76 / 305 63 / 131 (Compound 1) or a pharmaceutically acceptable salt thereof. In some embodiments, the compositions and methods of the present invention include a cationic lipid of the following formula: (Compound 2) or a pharmaceutically acceptable salt thereof. In some embodiments, the compositions and methods of the present invention include a cationic lipid of the following formula: (Compound 3) or a pharmaceutically acceptable salt thereof.

[00141] Other cationic lipids suitable for use in the compositions and methods of the present invention include the cationic lipids as described in J. McClellan, MC King, Cell 2010, 141, 210-217 and in Whitehead et al., Nature Communications (2014) 5:4277, which are incorporated herein by reference. In certain embodiments, the cationic lipids of the compositions and methods of the present invention include a cationic lipid having a structure of Petition 870260055239, dated 08 / 06 / 2026, page 77 / 305 64 / 131 composed of: and the pharmaceutically acceptable salt thereof.

[00142] Other cationic lipids suitable for use in the compositions and methods of the invention include the cationic lipids as described in International Patent Publication WO 2015 / 199952, which is incorporated herein by reference. In some embodiments, the compositions and methods of the present invention include a cationic lipid having the structure of the compound: and the pharmaceutically acceptable salt thereof. In some embodiments, the compositions and methods of the present invention include a cationic lipid having the structure of the compound: and the pharmaceutically acceptable salt thereof. In some Petition 870260055239, dated 08 / 06 / 2026, page 78 / 305 65 / 131 embodiments, the compositions and methods of the present invention include a cationic lipid having the structure of the compound: and the pharmaceutically acceptable salt thereof. In some embodiments, the compositions and methods of the present invention include a cationic lipid having the structure of the compound: and the pharmaceutically acceptable salt thereof. In some embodiments, the compositions and methods of the present invention include a cationic lipid having the structure of the compound: The pharmaceutically acceptable salt thereof. In some embodiments, the compositions and methods of the present invention include a cationic lipid having the structure of the compound: Petition 870260055239, dated 08 / 06 / 2026, page 79 / 305 66 / 131 and the pharmaceutically acceptable salt thereof. In some embodiments, the compositions and methods of the present invention include a cationic lipid having the structure of the compound: The pharmaceutically acceptable salt thereof. In some embodiments, the compositions and methods of the present invention include a cationic lipid having the structure of the compound: and the pharmaceutically acceptable salt thereof. In some embodiments, the compositions and methods of the present invention include a cationic lipid having the structure of the compound: and the pharmaceutically acceptable salt thereof. In some embodiments, the compositions and methods of the present invention include a cationic lipid having the structure of the compound: Petition 870260055239, dated 08 / 06 / 2026, page 80 / 305 67 / 131 and the pharmaceutically acceptable salt thereof. In some embodiments, the compositions and methods of the present invention include a cationic lipid having the structure of the compound: and the pharmaceutically acceptable salt thereof. In some embodiments, the compositions and methods of the present invention include a cationic lipid having the structure of the compound: and the pharmaceutically acceptable salt thereof. In some embodiments, the compositions and methods of the present invention include a cationic lipid having the structure of the compound: Petition 870260055239, dated 08 / 06 / 2026, page 81 / 305 68 / 131 and the pharmaceutically acceptable salt thereof.

[00143] Other cationic lipids suitable for use in the compositions and methods of the invention include the cationic lipids as described in International Patent Publication WO 2017 / 004143, which is incorporated herein by reference. In some embodiments, the compositions and methods of the present invention include a cationic lipid having the structure of the compound: and the pharmaceutically acceptable salt thereof. In some embodiments, the compositions and methods of the present invention include a cationic lipid having the structure of the compound: and the pharmaceutically acceptable salt thereof. In some embodiments, the compositions and methods of the present invention include a cationic lipid having the structure of the compound: and the pharmaceutically acceptable salt thereof. In some embodiments, the compositions and methods of the present invention include a cationic lipid having the structure of the compound: Petition 870260055239, dated 08 / 06 / 2026, page 82 / 305 69 / 131 and the pharmaceutically acceptable salt thereof. In some embodiments, the compositions and methods of the present invention include a cationic lipid having the structure of the compound: and the pharmaceutically acceptable salt thereof. In some embodiments, the compositions and methods of the present invention include a cationic lipid having the structure of the compound: and the pharmaceutically acceptable salt thereof. In some embodiments, the compositions and methods of the present invention include a cationic lipid having the structure of the compound: and the pharmaceutically acceptable salt thereof. In some embodiments, the compositions and methods of the present invention include a cationic lipid having the structure of the compound: Petition 870260055239, dated 08 / 06 / 2026, page 83 / 305 70 / 131 and the pharmaceutically acceptable salt thereof. In some embodiments, the compositions and methods of the present invention include a cationic lipid having the structure of the compound: and the pharmaceutically acceptable salt thereof. In some embodiments, the compositions and methods of the present invention include a cationic lipid having the structure of the compound: and the pharmaceutically acceptable salt thereof. In some embodiments, the compositions and methods of the present invention include a cationic lipid having the structure of the compound: and the pharmaceutically acceptable salt thereof. In some embodiments, the compositions and methods of the present invention include Petition 870260055239, dated 08 / 06 / 2026, page 84 / 305 71 / 131 a cationic lipid having the structure of a compound: and the pharmaceutically acceptable salt thereof. In some embodiments, the compositions and methods of the present invention include a cationic lipid having the structure of the compound: the and the pharmaceutically acceptable salt thereof. In some embodiments, the compositions and methods of the present invention include a cationic lipid having the structure of the compound: and the pharmaceutically acceptable salt thereof. In some embodiments, the compositions and methods of the present invention include a cationic lipid having the structure of the compound: and the pharmaceutically acceptable salt thereof. In some embodiments, the compositions and methods of the present invention include Petition 870260055239, dated 08 / 06 / 2026, page 85 / 305 72 / 131 a cationic lipid having the structure of a compound o. / and the pharmaceutically acceptable salt thereof. In some embodiments, the compositions and methods of the present invention include a cationic lipid having the structure of the compound: oeo pharmaceutically acceptable salt of the same.

[00144] Other cationic lipids suitable for use in the compositions and methods of the invention include the cationic lipids as described in International Patent Publication WO 2017 / 075531, which is incorporated herein by reference. In some embodiments, the compositions and methods of the present invention include a cationic lipid of the following formula: LÍ A2 R1G1G2R2 or a pharmaceutically acceptable salt thereof, wherein one of L1 or L2 is -O(C=O)-, -(C=O)O-, -C(=O)-, -O-, -S(O)X, -SS-, C(=O)S-, -SC(=O)-, -NRaC(=O)-, -C(=O)NRa-, NRaC(=O)NRa-, OC(=O)NRa-, or -NRaC(=O)O-; and the other of L1 or L2 is -O(C=O)-, (C=O)O-, -C(=O)-, -O-, -S(O)X, -SS-, -C(=O)S-, SC(=O)-, -NRaC(=O)-, -C(=O)NRa-, NRaC(=O)NRa-, -OC(=O)NRa- or -NRaC(=O)O- or a Petition 870260055239, dated 08 / 06 / 2026, page 86 / 305 73 / 131 direct linkage; G1 and G2 are each independently C1-C12 alkylene or unsubstituted C1-C12 alkenylene; G3 is C1-C24 alkylene, C1-C24 alkenylene, C3-C8 cycloalkylene, C3-C8 cycloalkenylene; Ra is H or C1-C12 alkyl; R1 and R2 are each independently C6-C24 alkyl or C6-C24 alkenyl; R3 is H, OR5, CN, -C(=O)OR4, -OC(=O)R4 or -NR5C(=O)R4; R4 is C1-C12 alkyl; R5 is H or C1-C12 alkyl; ex is 0, 1 or 2.

[00145] Other cationic lipids suitable for use in the compositions and methods of the invention include the cationic lipids as described in International Patent Publication WO 2017 / 117528, which is incorporated herein by reference. In some embodiments, the compositions and methods of the present invention include a cationic lipid having the structure of the compound: and the pharmaceutically acceptable salt thereof. In some embodiments, the compositions and methods of the present invention include a cationic lipid having the structure of the compound: and the pharmaceutically acceptable salt thereof. In some embodiments, the compositions and methods of the present invention include a cationic lipid having the structure of the compound: Petition 870260055239, dated 08 / 06 / 2026, page 87 / 305 74 / 131 and the pharmaceutically acceptable salt thereof.

[00146] Other cationic lipids suitable for use in the compositions and methods of the invention include the cationic lipids as described in International Patent Publication WO 2017 / 049245, which is incorporated herein by reference. In some embodiments, the cationic lipids of the compositions and methods of the present invention include a compound of one of the following formulas: and the pharmaceutically acceptable salt thereof. For Petition 870260055239, dated 08 / 06 / 2026, page 88 / 305 75 / 131 any of these four formulas, R4 is independently selected from -(CH2)nQ and -(CH2)nCHQR; Q is selected from the group consisting of -OR, -OH, -O(CH2)nN(R)2, -OC(O)R, -CX3, -CN, N(R)C(O)R, -N(H)C(O)R, -N(R)S(O)2R, -N(H)S(O)2R, -N(R)C(O)N(R)2, N(H)C(O)N(R)2, -N(H)C(O)N(H)(R), -N(R)C(S)N(R)2i-N(H)C(S)N(R)2, N(H)C(S)N(H)(R), and a heterocycle; en is 1, 2, or 3. In certain embodiments, the compositions and methods of the present invention include a cationic lipid having a compound structure of: and the pharmaceutically acceptable salt thereof. In certain embodiments, the compositions and methods of the present invention include a cationic lipid having a compound structure of: and the pharmaceutically acceptable salt thereof. In certain embodiments, the compositions and methods of the present invention include a cationic lipid having a compound structure of: and the pharmaceutically acceptable salt thereof. In certain embodiments, the compositions and methods of the present invention include a cationic lipid having a compound structure of: Petition 870260055239, dated 08 / 06 / 2026, page 89 / 305 76 / 131 and the pharmaceutically acceptable salt thereof.

[00147] Other cationic lipids suitable for use in the compositions and methods of the invention include the cationic lipids as described in International Patent Publications WO 2017 / 173054 and WO 2015 / 095340, each of which is incorporated herein by reference. In certain embodiments, the compositions and methods of the present invention include a cationic lipid having a compound structure of: and the pharmaceutically acceptable salt thereof. In certain embodiments, the compositions and methods of the present invention include a cationic lipid having a compound structure of: the and the pharmaceutically acceptable salt thereof. In certain embodiments, the compositions and methods of the present invention include a cationic lipid having a compound structure of: Petition 870260055239, dated 08 / 06 / 2026, page 90 / 305 77 / 131 and the pharmaceutically acceptable salt thereof. In certain embodiments, the compositions and methods of the present invention include a cationic lipid having a compound structure of: and the pharmaceutically acceptable salt thereof.

[00148] Other cationic lipids suitable for use in the compositions and methods of the present invention include cleavable cationic lipids as described in International Patent Publication WO 2012 / 170889, which is incorporated herein by reference. In some embodiments, the compositions and methods of the present invention include a cationic lipid of the following formula: wherein Ri is selected from the group consisting of imidazole, guanidinium, amino, imine, enamine, an optionally substituted alkylamino (for example, an alkylamino such as dimethylamino) and pyridyl; where R2 is selected from the group consisting of one of the following two formulas: and wherein R3 and R4 are each independently selected from the group consisting of an optionally substituted, variably saturated or unsaturated C6-C20 alkyl and a Petition 870260055239, dated 08 / 06 / 2026, p. 91 / 305 78 / 131 C6-C20 acyl group optionally substituted, variably saturated or unsaturated; and wherein n is zero or any positive integer (e.g., one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty or more). In certain embodiments, the compositions and methods of the present invention include a cationic lipid, HGT4001, having a compound structure of: (HGT4001) and the pharmaceutically acceptable salt thereof. In certain embodiments, the compositions and methods of the present invention include a cationic lipid, HGT4002, having a compound structure of: (HGT4002) and the pharmaceutically acceptable salt thereof. In certain embodiments, the compositions and methods of the present invention include a cationic lipid, HGT4003, having a compound structure of: (HGT4003) and the pharmaceutically acceptable salt thereof. In certain embodiments, the compositions and methods of the present invention include a cationic lipid, HGT4004, having a compound structure of: Petition 870260055239, dated 08 / 06 / 2026, page 92 / 305 79 / 131 (HGT4004) and the pharmaceutically acceptable salt thereof. In certain embodiments, the compositions and methods of the present invention include a cationic lipid HGT4005, having a compound structure of: (HGT4005) and the pharmaceutically acceptable salt thereof.

[00149] Other cationic lipids suitable for use in the compositions and methods of the present invention include cleavable cationic lipids as described in International Application No. PCT / US2019 / 032522, which is incorporated herein by reference. In certain embodiments, the compositions and methods of the present invention include a cationic lipid that is any of the general formulas or any of the structures (1a)-(21a) and (1b)-(21b) and (22)-(237) described in International Application No. PCT / US2019 / 032522. In certain embodiments, the compositions and methods of the present invention include a cationic lipid having a structure according to Formula (I'), Rxé independently -H, -L1-R1, or -L5A-L5B-B'; Each of L1, L2, and L3 is independently a covalent bond, -C(O)-, -C(O)O-, -C(O)S-, or -C(O)NRL-; each L4Ae L5Aé independently -C(O)-, -C(O)O- or C(O)NRL-; Petition 870260055239, dated 08 / 06 / 2026, p. 93 / 305 80 / 131 each L4Be L5Bis independently C1-C20 alkylene; C2-C20 alkenylene; or C2-C20 alkynylene; each B and B' is NR4R5 or a 5 to 10 membered nitrogen-containing heteroaryl; each R1, R2, and R3 is independently C6-C30 alkyl, CeC30 alkenyl or C6-C30 alkynyl; each R4 and R5 is independently hydrogen, C1-C10 alkyl; C2-C10 alkenyl; or C2-C10 alkynyl; and each RL is independently hydrogen, C1-C20 alkyl, C2C20 alkenyl or C2-C20 alkynyl.

[00150] In certain embodiments, the compositions and methods of the present invention include a cationic lipid that is Compound (139) of International Application No. PCT / US2019 / 032522, having a compound structure of: (18:1 carbon-ribose lipid tail).

[00151] In some embodiments, the compositions and methods of the present invention include the cationic lipid, N-[1-(2,3-dioleyyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA). (Feigner et al. (Proc. Nat'l Acad. Sci. 84, 7413 (1987); US Pat. No. 4,897,355, which is incorporated herein by reference). Other suitable cationic lipids for the compositions and methods of the present invention include, for example, 5-carboxyspermylglycinedioctadecylamide (DOGS); 2,3-dioleyyloxy-N-[2(spermine-carboxamido)ethyl]-N,N-dimethyl-1-propanaminomethylamide (DOSPA) (Behr et al. Proc. Nat.'l Acad. Sci. 86, 6982 Petition 870260055239, dated 08 / 06 / 2026, page 94 / 305 81 / 131 (1989), Pat. US No. 5,171,678; Pat. US No. 5,334,761); 1,2-Dioleoyl3-Dimethylammonium-Propane (DODAP); 1,2-Dioleoyl-3-TrimethylammoniumPropanp (DOTAP).

[00152] Additional exemplary cationic lipids suitable for the compositions and methods of the present invention also include: 1,2-distearyloxy-N,N-dimethyl-3-aminopropane (DSDMA); 1,2-dioleyyloxy-N,N-dimethyl-3-aminopropane (DODMA); 1,2-dilinoleyyloxy-N,N-dimethyl-3-aminopropane (DLinDMA); 1,2-dilinolenyloxy-N,N-dimethyl-3-aminopropane (DLenDMA); N-dioleyl-N,N-dimethylammonium chloride (DODAC); N,N-distearyl-N,N-dimethylammonium bromide (DDAB); N-(1,2-dimyristyloxypropyl-3-yl)-N,N-dimethyl-N-hydroxyethyl ammonium bromide (DMRIE); 3-dimethylamino-2-(cholest-5-en-3-beta-oxybutan-4-oxy)-1-(cis,cis-9,12-octadecadienoxy)propane (CLinDMA); 2-[5'-(cholest-5en-3-beta-oxy)-3'-oxapentoxy)-3-dimethyl-1-(cis,cis-9',1-2'octadecadienoxy)propane dioleyloxybenzylamine (CpLinDMA); N,N-dimethyl-3,4dimethylaminopropane dimethylpropylamine dimethylaminopropane dimethylaminopropane (DMOBA); 1,2-N,N'-dioleylcarbamyl-3(DOcarbDAP); 2,3-Dilinoleoylóxi-N,N(DLinDAP); 1,2-N,N'-Dilinoleylcarbamyl-3(DLincarbDAP); 1,2-Dilinoleoylcarbamyl-3(DLinCDAP); 2,2-dilinoleyl-4-dimethylaminomethyl[l,3]-dioxolane (DLin-K-DMA); 2-((8-[(3P)-cholest-5-en-3-ylóxi]octyl)óxi)N,N-dimethyl-3-[(9Z, 12Z)-octadeca-9,12-dien-1 -ylóxi]propane-1 -amine (Octyl-CLinDMA); (2R)-2-((8-[(3beta)-cholest-5-en-3-ylóxi]octyl)óxi)-N,Ndimethyl-3-[(9Z, 12Z)-octadeca-9,12-dien-1 -ylóxi]propan-1 -amine (OctylCLinDMA (2R)); (2S)-2-((8-[(3P)-cholest-5-en-3-ylóxi]octyl)óxi)-N,fsldimethyl-3-[(9Z, 12Z)-octadeca-9,12-dien-1 -ylóxi]propan-1 -amine (OctylCLinDMA (2S)); 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLinK-XTC2-DMA); and 2-(2,2-di((9Z,12Z)-octadeca-9,1 2-dien-1-yl)-1,3dioxolan-4-yl)-N,N-dimethylyanamine (DLin-KC2-DMA) (ver, WO 2010 / 042877, which is here incorporated into the reference title; Semple et Petition 870260055239, dated 08 / 06 / 2026, p. 95 / 305 82 / 131 al., Nature Biotech. 28: 172-176 (2010)). (Heyes, J., et al., J Controlled Release 107: 276-287 (2005); Morrissey, DV., et al., Nat. Biotechnol. 23(8): 1003-1007 (2005); International Patent Publication WO 2005 / 121348). In some embodiments, one or more of the cationic lipids comprise at least one imidazole, dialkylamino, or guanidinium component.

[00153] In some embodiments, one or more cationic lipids suitable for the compositions and methods of the present invention include 2,2-Dilinoleyl-1-4-dimethylaminoethyl-1-[1,3]-dioxolane (XTC); (3aR,5s,6aS)-N,N-dimethyl-2,2-di((9Z,12Z)-octadeca-9,12dienyl)tetrahydro-3aH-cyclopenta[d] [1,3]dioxol-5-amine (ALNY-100) and / or 4,7,13-tris(3-oxo-3-(undecylamino)propyl)-N1,N16-diundecyl-4,7,10,13tetrazaexadecane-1,16-diamide (NC98-5).

[00154] In some embodiments, the compositions of the present invention include one or more cationic lipids that constitute at least about 5%, 10%, 20%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65% or 70%, measured by weight, of the total lipid content in the composition, for example, a lipid nanoparticle. In some embodiments, the compositions of the present invention include one or more cationic lipids that constitute at least about 5%, 10%, 20%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65% or 70%, measured as a molar %, of the total lipid content in the composition, for example, a lipid nanoparticle.In some embodiments, the compositions of the present invention include one or more cationic lipids that constitute about 30 to 70% (for example, about 30 to 65%, about 30 to 60%, about 30 to 55%, about 30 to 50%, about 30 to 45%, about 30 to 40%, about 35 to 50%, about 35 to 45% or about 35-40%), measured by weight, of the total lipid content in the composition, for example, a lipid nanoparticle. In some embodiments, the compositions of the present invention include one or... Petition 870260055239, dated 08 / 06 / 2026, p. 96 / 305 83 / 131 more cationic lipids that constitute about 30 to 70% (for example, about 30 to 65%, about 30 to 60%, about 30 to 55%, about 30 to 50%, about 30 to 45%, about 30 to 40%, about 35 to 50%, about 35 to 45% or about 35 to 40%), measured as molar %, of the total lipid content in the composition, for example, a lipid nanoparticle. Non-Cationic / Auxiliary Lipids

[00155] In some embodiments, liposomes contain one or more non-cationic (auxiliary) lipids. As used herein, the term non-cationic lipid refers to any neutral, zwitterionic, or anionic lipid. As used herein, the term anionic lipid refers to any of several lipid species that carry a net negative charge at a selected pH, such as physiological pH.Non-cationic lipids include, but are not limited to, distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoylphosphatidylethanolamine (DOPE), palmitoylleoylphosphatidylcholine (POPC), palmitoylleoylphosphatidylethanolamine (POPE), dioleoyl-phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoyl phosphatidyl ethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoyl-phosphatidyl-ethanolamine (DSPE), phosphatidylserine, sphingolipids, cerebrosides, gangliosides, 16-O-monomethyl PE, 16-O-dimethyl PE, 181-trans PE, 1-stearoyl-2-oleoyl-phosphatidyethanolamine (SOPE), or a mixture thereof.

[00156] In some embodiments, a non-cationic lipid is a neutral lipid, that is, a lipid that does not carry a net charge under the conditions in which the composition is formulated and / or administered.

[00157] In some embodiments, such non-cationic lipids may be used alone, but are preferably used in combination with other compounds. Petition 870260055239, dated 08 / 06 / 2026, page 97 / 305 84 / 131 combination with other lipids, for example, cationic lipids.

[00158] In some embodiments, a non-cationic lipid may be present in a molar ratio (% molar) of about 5% to about 90%, about 5% to about 70%, about 5% to about 50%, about 5% to about 40%, about 5% to about 30%, about 10% to about 70%, about 10% to about 50%, or about 10% to about 40% of the total lipids present in a composition. In some embodiments, total non-cationic lipids may be present in a molar ratio (% molar) of about 5% to about 90%, about 5% to about 70%, about 5% to about 50%, about 5% to about 40%, about 5% to about 30%, about 10% to about 70%, about 10% to about 50%, or about 10% to about 40% of the total lipids present in a composition.In some embodiments, the percentage of non-cationic lipid in a liposome may be greater than about 5 molar percent, greater than about 10 molar percent, greater than about 20 molar percent, greater than about 30 molar percent, or greater than about 40 molar percent. In some embodiments, the percentage of total non-cationic lipids in a liposome may be greater than about 5 molar percent, greater than about 10 molar percent, greater than about 20 molar percent, greater than about 30 molar percent, or greater than about 40 molar percent. In some embodiments, the percentage of non-cationic lipid in a liposome is no more than about 5 molar percent, no more than about 10 molar percent, no more than about 20 molar percent, no more than about 30 molar percent, or no more than about 40 molar percent.In some embodiments, the percentage of total non-cationic lipids in a liposome may be no more than about 5 molar percent, no more than about 10 molar percent, no more than about 20 molar percent, no more than about 30 molar percent, or no more than about 40 molar percent.

[00159] In some embodiments, a non-cationic lipid may be Petition 870260055239, dated 08 / 06 / 2026, page 98 / 305 85 / 131 present in a weight ratio (% by weight) of about 5% to about 90%, about 5% to about 70%, about 5% to about 50%, about 5% to about 40%, about 5% to about 30%, about 10% to about 70%, about 10% to about 50% or about 10% to about 40% of the total lipids present in a composition. In some embodiments, total non-cationic lipids may be present in a weight ratio (% by weight) of about 5% to about 90%, about 5% to about 70%, about 5% to about 50%, about 5% to about 40%, about 5% to about 30%, about 10% to about 70%, about 10% to about 50%, or about 10% to about 40% of the total lipids present in a composition. In some embodiments, the percentage of non-cationic lipid in a liposome may be greater than about 5% by weight, greater than about 10% by weight, greater than about 20% by weight, greater than about 30% by weight, or greater than about 40% by weight.In some embodiments, the percentage of total non-cationic lipids in a liposome may be greater than about 5% by weight, greater than about 10% by weight, greater than about 20% by weight, greater than about 30% by weight, or greater than about 40% by weight. In some embodiments, the percentage of non-cationic lipid in a liposome is no more than about 5% by weight, no more than about 10% by weight, no more than about 20% by weight, no more than about 30% by weight, or no more than about 40% by weight. In some embodiments, the percentage of total non-cationic lipids in a liposome may be no more than about 5% by weight, no more than about 10% by weight, no more than about 20% by weight, no more than about 30% by weight, or no more than about 40% by weight. Non-Cationic / Auxiliary Lipids

[00160] In some embodiments, liposomes comprise one or more cholesterol-based lipids. For example, cationic lipids to Petition 870260055239, dated 08 / 06 / 2026, page 99 / 305 Suitable cholesterol bases include, for example, DC-Choi (N,N-dimethyl-N-ethylcarboxamidocholesterol), 1,4-bis(3-N-oleylaminopropyl)piperazine (Gao, et al. Biochem. Biophys. Res. Comm. 179, 280 (1991); Wolf et al. BioTechniques 23, 139 (1997); Pat. US No. 5,744,335), or imidazole cholesterol ester (ICE), which has the following structure, N (ICE).

[00161] In embodiments, a cholesterol-based lipid is cholesterol.

[00162] In some embodiments, the cholesterol-based lipid may comprise a molar ratio (molar %) of about 1% to about 30% or about 5% to about 20% of the total lipids present in a liposome. In some embodiments, the percentage of cholesterol-based lipid in the lipid nanoparticle may be greater than about 5% molar, greater than about 10% molar, greater than about 20% molar, greater than about 30% molar, or greater than about 40% molar. In some embodiments, the percentage of cholesterol-based lipid in the lipid nanoparticle may be no more than about 5 molar percent, no more than about 10 molar percent, no more than about 20 molar percent, no more than about 30 molar percent, or no more than about 40 molar percent.

[00163] In some embodiments, a cholesterol-based lipid may be present in a weight ratio (% by weight) of about 1% to about 30% or about 5% to about 20% of the total lipids present in a liposome. In some embodiments, the percentage of cholesterol-based lipid in the lipid nanoparticle may be greater than about 5% by weight, greater than about 10% by weight, Petition 870260055239, dated 08 / 06 / 2026, p. 100 / 305 87 / 131 greater than about 20% by weight, greater than about 30% by weight, or greater than about 40% by weight. In some embodiments, the percentage of cholesterol-based lipid in the lipid nanoparticle may be no more than about 5% by weight, no more than about 10% by weight, no more than about 20% by weight, no more than about 30% by weight, or no more than about 40% by weight. PEG-Modified Lipids

[00164] In some embodiments, the liposome comprises one or more PEGylated lipids.

[00165] For example, the use of polyethylene glycol (PEG) modified phospholipids and derivatized lipids such as derivatized ceramides (PEG-CER), including N-Octanoyl-Sphingosine-1[Succinyl(Methoxy Polyethylene Glycol)-2000] (C8 PEG-2000 ceramide) are also contemplated by the present invention, alone or preferably in combination with other lipid formulations comprising the transfer vehicle (e.g., a lipid nanoparticle).

[00166] The PEG-modified lipids contemplated include, but are not limited to, a polyethylene glycol chain up to 5 kDa in length covalently linked to a lipid with C6-C20 length alkyl chain(s). In some embodiments, a PEG-modified or PEGylated lipid is PEGylated cholesterol or PEG-2K. The addition of such components may prevent complex aggregation and may also provide a means to increase the lifetime of circulation and enhance the release of the lipid-nucleic acid composition to target tissues (Klibanov et al. (1990) FEBS Letters, 268 (1): 235-237), or they may be selected for rapid in vivo formulation exchange (see US Pat. No. 5,885,613). Particularly useful exchangeable lipids are PEG-ceramides having longer acyl chains. Petition 870260055239, dated 08 / 06 / 2026, p. 101 / 305 88 / 131 short (e.g., C14 or C18).

[00167] The PEG-modified phospholipid and derivatized lipids of the present invention may comprise a molar ratio of about 0% to about 20%, about 0.5% to about 20%, about 1% to about 15%, about 4% to about 10%, or about 2% of the total lipid present in the liposome transfer vehicle. In some embodiments, one or more PEG-modified lipids constitute about 4% of the total lipids by molar ratio. In some embodiments, one or more PEG-modified lipids constitute about 5% of the total lipids by molar ratio. In some embodiments, one or more PEG-modified lipids constitute about 6% of the total lipids by molar ratio. Amphiphilic block copolymers

[00168] In some embodiments, a suitable delivery vehicle contains amphiphilic block copolymers (e.g., poloxamers).

[00169] Various amphiphilic block copolymers can be used in the practice of the present invention. In some embodiments, an amphiphilic block copolymer is also referred to as a surfactant or a nonionic surfactant.

[00170] In some embodiments, an amphiphilic polymer suitable for the invention is selected from poloxamers (Pluronic®), poloxamines (Tetronic®), polyoxyethylene glycol sorbitan alkyl esters (polysorbates) and polyvinylpyrrolidones (PVPs). Poloxamers

[00171] In some embodiments, a suitable amphiphilic polymer is a poloxamer. For example, a suitable poloxamer has the following structure: Petition 870260055239, dated 08 / 06 / 2026, page 102 / 305 89 / 131 where a is an integer between 10 and 150 and b is an integer between 20 and 60. For example, a is around 12 and b is around 20, or a is around 80 and b is around 27, or a is around 64 and b is around 37, or a is around 141 and b is around 44, or a is around 101 and b is around 56.

[00172] In some embodiments, a poloxamer suitable for the invention has ethylene oxide units from about 10 to about 150. In some embodiments, a poloxamer has ethylene oxide units from about 10 to about 100.

[00173] In some embodiments, a suitable poloxamer is poloxamer 84. In some embodiments, a suitable poloxamer is poloxamer 101. In some embodiments, a suitable poloxamer is poloxamer 105. In some embodiments, a suitable poloxamer is poloxamer 108. In some embodiments, a suitable poloxamer is poloxamer 122. In some embodiments, a suitable poloxamer is poloxamer 123. In some embodiments, a suitable poloxamer is poloxamer 124. In some embodiments, a suitable poloxamer is poloxamer 181. In some embodiments, a suitable poloxamer is poloxamer 182. In some embodiments, a suitable poloxamer is poloxamer 183. In some embodiments, a suitable poloxamer is poloxamer 184. In some embodiments, a A suitable poloxamer is poloxamer 185. In some embodiments, a suitable poloxamer is poloxamer 188. In some embodiments, a suitable poloxamer is poloxamer 212. In some embodiments, a suitable poloxamer is poloxamer 215.In some applications, a suitable poloxamer is poloxamer 217. Petition 870260055239, dated 08 / 06 / 2026, page 103 / 305 90 / 131 In some embodiments, a suitable poloxamer is poloxamer 231. In some embodiments, a suitable poloxamer is poloxamer 234. In some embodiments, a suitable poloxamer is poloxamer 235. In some embodiments, a suitable poloxamer is poloxamer 237. In some embodiments, a suitable poloxamer is poloxamer 238. In some embodiments, a suitable poloxamer is poloxamer 282. In some embodiments, a suitable poloxamer is poloxamer 284. In some embodiments, a suitable poloxamer is poloxamer 288. In some embodiments, a suitable poloxamer is poloxamer 304. In some embodiments, a suitable poloxamer is poloxamer 331. In some embodiments, a suitable poloxamer is poloxamer 333. In some embodiments, a suitable poloxamer is poloxamer 334. In some embodiments, a suitable poloxamer is poloxamer 335. In some embodiments, a suitable poloxamer is poloxamer 338. In some embodiments, a suitable poloxamer is poloxamer 401.In some embodiments, a suitable poloxamer is poloxamer 402. In some embodiments, a suitable poloxamer is poloxamer 403. In some embodiments, a suitable poloxamer is poloxamer 407. In some embodiments, a suitable poloxamer is a combination thereof.

[00174] In some embodiments, a suitable poloxamer has an average molecular weight of about 4000 g / mol to about 20,000 g / mol. In some embodiments, a suitable poloxamer has an average molecular weight of about 1000 g / mol to about 50,000 g / mol. In some embodiments, a suitable poloxamer has an average molecular weight of about 1000 g / mol. In some embodiments, a suitable poloxamer has an average molecular weight of about 2000 g / mol. In some embodiments, a suitable poloxamer has a Petition 870260055239, dated 08 / 06 / 2026, page 104 / 305 91 / 131 average molecular weight of about 3000 g / mol. In some embodiments, a suitable poloxamer has an average molecular weight of about 4000 g / mol. In some embodiments, a suitable poloxamer has an average molecular weight of about 5000 g / mol. In some embodiments, a suitable poloxamer has an average molecular weight of about 6000 g / mol. In some embodiments, a suitable poloxamer has an average molecular weight of about 7000 g / mol. In some embodiments, a suitable poloxamer has an average molecular weight of about 8000 g / mol. In some embodiments, a suitable poloxamer has an average molecular weight of about 9000 g / mol. In some embodiments, a suitable poloxamer has an average molecular weight of about 10000 g / mol. In some embodiments, a suitable poloxamer has an average molecular weight of about 20,000 g / mol. In some embodiments, a suitable poloxamer has an average molecular weight of about 25,000 g / mol.In some embodiments, a suitable poloxamer has an average molecular weight of about 30,000 g / mol. In some embodiments, a suitable poloxamer has an average molecular weight of about 40,000 g / mol. In some embodiments, a suitable poloxamer has an average molecular weight of about 50,000 g / mol. Other amphiphilic polymers

[00175] In some embodiments, an amphiphilic polymer is a poloxamine, for example, tetronic 304 or tetronic 904.

[00176] In some embodiments, an amphiphilic polymer is a polyvinylpyrrolidone (PVP), such as PVP with a molecular weight of 3 kDa, 10 kDa or 29 kDa.

[00177] In some embodiments, an amphiphilic polymer is a polyethylene glycol ether (Brij), polysorbate, sorbitan, and derivatives thereof. In some embodiments, an amphiphilic polymer is a Petition 870260055239, dated 08 / 06 / 2026, page 105 / 305 92 / 131 polysorbate, as is PS 20.

[00178] In some embodiments, an amphiphilic polymer is polyethylene glycol ether (Brij), poloxamer, polysorbate, sorbitan, or derivatives thereof.

[00179] In some embodiments, an amphiphilic polymer is a polyethylene glycol ether. In some embodiments, a suitable polyethylene glycol ether is a compound of Formula (S-1): or a salt or isomer thereof, wherein: t is an integer between 1 and 100; R1BRIJ is independently C10-40 alkyl, C10-40 alkenyl or C10-40 alkynyl; and optionally one or more methylene groups of R5PEG are independently substituted with C3-10 carbocyclylene, 4- to 10-membered heterocyclylene, C6-warylene, 4- to 10-membered heteroarylene, -N(RN)-, -O-, -S-, -C(O)-, -C(O)N(RN)-, -NRNC(O)-, -NR C(O)N(R)-, -C(O)O- -OC(O)-, -OC(O)O- - OC(O)N(RN)-, -NRNC(O)O- C(O)S- -SC(O)-, -C(=NRN)-,— C(=NR)N(R)—, - NRNC(=NRn)- NRNC(=NRN)N(RN)-, -C(S)-, -C(S)N(RN)-, -NRNC(S)-, -NRNC(S)N(RN)-, S(O)-, -OS(O)-, -S(O)O- -OS(O)O- -OS(O)2- -S(O)2O- -OS(O)2O- N(RN)S(O)-, - S(O)N(RN)- -N(RN)S(O)N(RN)- -OS(O)N(RN)- N(RN)S(O)0- -S(O)2- -N(RN)S(O)2- - S(O)2N(RN)-, -N(RN)S(O)2N(RN)- OS(O)2N(Rn)- or -N(Rn)S(O)2O-; and in each case RN is independently hydrogen, C1-6 alkyl or a nitrogen protecting group.

[00180] In a certain embodiment, R1BRIJé C is alkyl. For example, polyethylene glycol ether is a compound with Formula (S-1a): or a salt or isomer thereof, wherein s is an integer between 1 and 100. Petition 870260055239, dated 08 / 06 / 2026, page 106 / 305 93 / 131

[00181] In some embodiments, R1BRIJé C alkenyl. For example, a suitable polyethylene glycol ether is a compound of Formula (Slb): (S-lb), or a salt or isomer thereof, wherein s is an integer between 1 and 100.

[00182] Typically, an amphiphilic polymer (e.g., a poloxamer) is present in a formulation in an amount lower than its critical micelle concentration (CMC). In some embodiments, an amphiphilic polymer (e.g., a poloxamer) is present in the mixture in an amount about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, or about 50% lower than its CMC. In some embodiments, an amphiphilic polymer (e.g., a poloxamer) is present in the mixture in an amount approximately 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1% lower than its CMC.In some embodiments, an amphiphilic polymer (e.g., a poloxamer) is present in the mixture in an amount approximately 55%, 60%, 65%, 70%, 75%, 80%, 90%, or 95% lower than its CMC.

[00183] In some embodiments, less than about 0.1%, 0.09%, 0.08%, 0.07%, 0.06%, 0.05%, 0.04%, 0.03%, 0.02%, or 0.01% of the original amount of amphiphilic polymer (e.g., poloxamer) present in the formulation remains after removal. In some embodiments, a residual amount of amphiphilic polymer (e.g., poloxamer) remains in a formulation after removal. As used herein, a residual amount means a Petition 870260055239, dated 08 / 06 / 2026, page 107 / 305 94 / 131 remaining quantity after substantially all of the substance (an amphiphilic polymer described herein as a poloxamer) in a composition has been removed. A residual quantity may be detected using a known qualitative or quantitative technique. A residual quantity may not be detected using a known technique.

[00184] In some embodiments, a suitable release vehicle comprises less than 5% amphiphilic block copolymers (e.g., poloxamers). In some embodiments, a suitable release vehicle comprises less than 3% amphiphilic block copolymers (e.g., poloxamers). In some embodiments, a suitable release vehicle comprises less than 2.5% amphiphilic block copolymers (e.g., poloxamers). In some embodiments, a suitable release vehicle comprises less than 2% amphiphilic block copolymers (e.g., poloxamers). In some embodiments, a suitable release vehicle comprises less than 1.5% amphiphilic block copolymers (e.g., poloxamers). In some embodiments, a suitable release vehicle comprises less than 1% amphiphilic block copolymers (e.g., poloxamers).In some embodiments, a suitable release vehicle comprises less than 0.5% (e.g., less than 0.4%, 0.3%, 0.2%, 0.1%) of amphiphilic block copolymers (e.g., poloxamers). In some embodiments, a suitable release vehicle comprises less than 0.09%, 0.08%, 0.07%, 0.06%, 0.05%, 0.04%, 0.03%, 0.02%, or 0.01% of amphiphilic block copolymers (e.g., poloxamers). In some embodiments, a suitable release vehicle comprises less than 0.01% of amphiphilic block copolymers (e.g., poloxamers). In some embodiments, a suitable release vehicle contains a residual amount of amphiphilic polymers (e.g.,...). Petition 870260055239, dated 08 / 06 / 2026, page 108 / 305 95 / 131 poloxamers). As used herein, a residual quantity means an amount remaining after substantially all of the substance (an amphiphilic polymer described herein as a poloxamer) in a composition has been removed. A residual quantity can be detected using a known qualitative or quantitative technique. A residual quantity cannot be detected using a known technique. Polymers

[00185] In some embodiments, a suitable delivery vehicle is formulated using a polymer as a carrier, alone or in combination with other carriers, including various lipids described in this invention. Thus, in some embodiments, the liposomal delivery vehicles, as used herein, also encompass nanoparticles comprising polymers. Suitable polymers may include, for example, polyacrylates, polyalkylcyanoacrylates, polylactide, polylactidepolyglycolide copolymers, polycaprolactones, dextran, albumin, gelatin, alginate, collagen, chitosan, cyclodextrins, protamine, PEGylated protamine, PLL, PEGylated PLL, and polyethyleneimine (PEI). When PEI is present, it may be branched PEI with a molecular weight ranging from 10 to 40 kDa, for example, 25 kDa branched PEI (Sigma #408727).

[00186] According to various embodiments, the selection of cationic lipids, non-cationic lipids, PEG-modified lipids, cholesterol-based lipids and / or amphiphilic block copolymers comprising the lipid nanoparticle, as well as the relative molar ratio of such components (lipids) to each other, is based on the characteristics of the selected lipid(s), the nature of the intended target cells, and the characteristics of the nucleic acid to be released. Additional considerations include, for example, alkyl chain saturation, as well as size, charge, pH, pKa, fusogenicity and. Petition 870260055239, dated 08 / 06 / 2026, page 109 / 305 96 / 131 tolerability of the selected lipid(s). Thus, the molar ratios can be adjusted accordingly. Relationship of Distinct Lipid Components

[00187] A liposome suitable for the present invention may include one or more of the cationic lipids, non-cationic lipids, cholesterol lipids, PEG-modified lipids, amphiphilic block copolymers and / or polymers described herein in various relationships. In some embodiments, a lipid nanoparticle comprises five and no more than five distinct nanoparticle components. In some embodiments, a lipid nanoparticle comprises four and no more than four distinct nanoparticle components. In some embodiments, a lipid nanoparticle comprises three and no more than three distinct nanoparticle components. As non-limiting examples, a suitable liposome formulation may include a selected combination of cKK-E12 (also known as ML2), DOPE, cholesterol and DMG-PEG2K; C12-200, DOPE, cholesterol and DMG-PEG2K; HGT4003, DOPE, cholesterol and DMG-PEG2K; ICE, DOPE, cholesterol and DMG-PEG2K; or ICE, DOPE and DMG-PEG2K.

[00188] In several embodiments, cationic lipids (e.g., cKK-E12, C12-200, ICE and / or HGT4003) constitute about 30 to 60% (e.g., about 30 to 55%, about 30 to 50%, about 30 to 45%, about 30 to 40%, about 35 to 50%, about 35 to 45% or about 35 to 40%) of the liposome in terms of molar ratio. In some embodiments, the percentage of cationic lipids (e.g., cKK-E12, C12-200, ICE, and / or HGT4003) is equal to or greater than about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, or about 60% of the liposome in terms of molar ratio.

[00189] In some embodiments, the ratio of cationic lipids to non-cationic lipids to cholesterol-based lipids to Petition 870260055239, dated 08 / 06 / 2026, p. 110 / 305 97 / 131 PEG-modified lipids may be between approximately 30:60:25, 35:20, and 30:1:15, respectively. In some embodiments, the ratio of cationic lipids to non-cationic lipids to cholesterol-based lipids to PEG-modified lipids is approximately 40:30:20:10, respectively. In some embodiments, the ratio of cationic lipids to non-cationic lipids to cholesterol-based lipids to PEG-modified lipids is approximately 40:30:25:5, respectively. In some embodiments, the ratio of cationic lipids to non-cationic lipids to cholesterol-based lipids to PEG-modified lipids is approximately 40:32:25:3, respectively. In some embodiments, the ratio of cationic lipids to non-cationic lipids to cholesterol-based lipids to PEG-modified lipids is approximately 50:25:20:5.

[00190] In embodiments where a lipid nanoparticle comprises three and no more than three distinct lipid components, the total lipid content ratio (i.e., the ratio of lipid component (1):lipid component (2):lipid component (3)) can be represented as x:y:z, where (y + z) = 100 - x.

[00191] In some embodiments, each of x, y, ez represents molar percentages of the three distinct lipid components, and the relationship is a molar relationship.

[00192] In some embodiments, each of x, y, ez represents weight percentages of the three distinct lipid components, and the ratio is a weight ratio.

[00193] In some embodiments, the lipid component (1), represented by the variable x, is a cationic sterol-based lipid.

[00194] In some embodiments, the lipid component (2), represented by the variable y, is an auxiliary lipid. Petition 870260055239, dated 08 / 06 / 2026, page 111 / 305 98 / 131

[00195] In some embodiments, the lipid component (3), represented by the variable z, is a PEG lipid.

[00196] In some embodiments, the variable x, representing the molar percent of the lipid component (1) (for example, a sterol-based cationic lipid), is at least about 10%, about 20%, about 30%, about 40%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90% or about 95%.

[00197] In some embodiments, the variable x, representing the molar percentage of the lipid component (1) (for example, a sterol-based cationic lipid), is no more than about 95%, about 90%, about 85%, about 80%, about 75%, about 70%, about 65%, about 60%, about 55%, about 50%, about 40%, about 30%, about 20%, or about 10%. In embodiments, the variable x is nothing more than about 65%, about 60%, about 55%, about 50%, about 40%.

[00198] In some embodiments, the variable x, representing the molar percent of the lipid component (1) (for example, a sterol-based cationic lipid), is: at least about 50%, but less than about 95%; at least about 50%, but less than about 90%; at least about 50%, but less than about 85%; at least about 50%, but less than about 80%; at least about 50%, but less than about 75%; at least about 50%, but less than about 70%; at least about 50%, but less than about 65%; or at least about 50%, but less than about 60%. In the embodiments, the variable x is at least about 50%, but less than about 70%; at least about 50%, but less than about 65%; or at least about 50%, but less than about 60%.

[00199] In some modalities, the variable x, representing the Petition 870260055239, dated 08 / 06 / 2026, p. 112 / 305 99 / 131 weight percentage of the lipid component (1) (for example, a sterol-based cationic lipid), is at least about 10%, about 20%, about 30%, about 40%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90% or about 95%.

[00200] In some embodiments, the variable x, representing the weight percentage of the lipid component (1) (for example, a sterol-based cationic lipid), is no more than about 95%, about 90%, about 85%, about 80%, about 75%, about 70%, about 65%, about 60%, about 55%, about 50%, about 40%, about 30%, about 20% or about 10%. In embodiments, the variable x is nothing more than about 65%, about 60%, about 55%, about 50%, about 40%.

[00201] In some embodiments, the variable x, representing the weight percentage of the lipid component (1) (for example, a sterol-based cationic lipid), is: at least about 50%, but less than about 95%; at least about 50%, but less than about 90%; at least about 50%, but less than about 85%; at least about 50%, but less than about 80%; at least about 50%, but less than about 75%; at least about 50%, but less than about 70%; at least about 50%, but less than about 65%; or at least about 50%, but less than about 60%. In the embodiments, the variable x is at least about 50%, but less than about 70%; at least about 50%, but less than about 65%; or at least about 50%, but less than about 60%.

[00202] In some embodiments, the variable z, representing the molar percentage of the lipid component (3) (e.g., a PEG lipid) is no more than about 1%, 2%, 3%, 4%, 5%, 6%, 7%, Petition 870260055239, dated 08 / 06 / 2026, p. 113 / 305 100 / 131 8%, 9%, 10%, 15%, 20% or 25%. In the modalities, the variable z, representing the molar percentage of the lipid component (3) (for example, a PEG lipid) is around 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%. In the embodiments, the variable z, representing the molar percentage of the lipid component (3) (e.g., a PEG lipid) is from about 1% to about 10%, about 2% to about 10%, about 3% to about 10%, about 4% to about 10%, about 1% to about 7.5%, about 2.5% to about 10%, about 2.5% to about 7.5%, about 2.5% to about 5%, about 5% to about 7.5% or about 5% to about 10%.

[00203] In some embodiments, the variable z, representing the weight percentage of the lipid component (3) (e.g., a PEG lipid) is no more than about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20% or 25%. In embodiments, the variable z, representing the weight percentage of the lipid component (3) (e.g., a PEG lipid) is about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%. In the embodiments, the variable z, representing the weight percentage of the lipid component (3) (e.g., a PEG lipid) is from about 1% to about 10%, about 2% to about 10%, about 3% to about 10%, about 4% to about 10%, about 1% to about 7.5%, about 2.5% to about 10%, about 2.5% to about 7.5%, about 2.5% to about 5%, about 5% to about 7.5% or about 5% to about 10%.

[00204] For compositions having three and only three distinct lipid components, the variables x, y, and z may be in any combination, provided that the total of the three variables adds up to 100% of the total lipid content. mRNA synthesis

[00205] mRNAs according to the present invention can be synthesized according to any one of a variety of Petition 870260055239, dated 08 / 06 / 2026, p. 114 / 305 101 / 131 known methods. Several methods are described in Published US Application No. US 2018 / 0258423 and can be used to practice the present invention, all of which are incorporated herein by reference. For example, mRNAs according to the present invention can be synthesized by means of in vitro transcription (IVT). Briefly, IVT is typically performed with a linear or circular DNA template containing a promoter, a set of ribonucleotide triphosphates, a buffer system that may include DTT and magnesium ions, and an appropriate RNA polymerase (e.g., T3, T7, or SP6 RNA polymerase), DNase I, pyrophosphatase, and / or RNase inhibitor. The exact conditions will vary according to the specific application.

[00206] In some embodiments, a suitable mRNA sequence is an mRNA sequence that encodes a protein or a peptide. In some embodiments, a suitable mRNA sequence is codon-optimized for efficient human cell expression. In some embodiments, a suitable mRNA sequence occurs naturally or is a wild-type sequence. In some embodiments, a suitable mRNA sequence encodes a protein or a peptide that contains one or more amino acid sequence mutations.

[00207] The present invention can be used to release mRNAs of a variety of lengths. In some embodiments, the present invention can be used to release mRNA synthesized in vitro of or greater than about 0.5 kb, 1 kb, 1.5 kb, 2 kb, 2.5 kb, 3 kb, 3.5 kb, 4 kb, 4.5 kb, 5 kb, 6 kb, 7 kb, 8 kb, 9 kb, 10 kb, 11 kb, 12 kb, 13 kb, 14 kb, 15 kb, 20 kb, 30 kb, 40 kb, or 50 kb in length. In some embodiments, the present invention can be used to release mRNA synthesized in vitro ranging from about 1 to 20 kb, about 1 to 15 kb, about 1 to 10 kb, about 5 to 20 kb, about 5 to 15 kb, about 5 to 12 kb, about 5 to 10 kb, about 8 to 20 kb, or about Petition 870260055239, dated 08 / 06 / 2026, p. 115 / 305 102 / 131 at 50 kb in length.

[00208] In some embodiments, for the preparation of mRNA according to the invention, a DNA template is transcribed in vitro. A suitable DNA template typically has a promoter, for example, a T3, T7 or SP6r promoter, for in vitro transcription, followed by the desired nucleotide sequence for the mRNA and a thermal signal. Nucleotides

[00209] Various naturally occurring or modified nucleosides can be used to produce mRNA according to the present invention.In some embodiments, an mRNA is or comprises naturally occurring nucleosides (or unmodified nucleotides; for example, adenosine, guanosine, cytidine, uridine); Nucleoside analogs (e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolopyrimidine, 3-methyladenosine, 5-methylcytidine, C-5 propynylcytidine, C5 propynyluridine, 2-aminoadenosine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynyluridine, C5-propynylcytidine, C5-methylcytidine, 2-aminoadenosine, 7-deazadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, O(6)-methylguanine, pseudouridine (e.g., N-1-methylpseudouridine), 2-thiouridine and 2-thiocytidine); chemically modified bases; biologically modified bases (e.g., methylated bases); intercalated bases; Modified sugars (e.g., 2'-fluororibose, ribose, 2'-deoxyribose, arabinose, and hexose); and / or modified phosphate groups (e.g., phosphorothioates and 5'-N-phosphoramidite linkages).

[00210] In some embodiments, a suitable mRNA may contain structural modifications, sugar modifications, and / or base modifications. For example, modified nucleotides may include, but are not limited to, modified purines (adenine (A), guanine (G)) or pyrimidines (thymine (T), cytosine (C), uracil (U)) and as nucleotides Petition 870260055239, dated 08 / 06 / 2026, page 116 / 305 103 / 131 modified analogues or derivatives of purines and pyrimidines, such as, for example, 1-methyladenine, 2-methyladenine, 2-methylthio-N-6-isopentenyladenine, N6-methyladenine, N6-isopentenyladenine, 2-thiocytosine, 3-methylcytosine, 4-acetylcytosine, 5-methylcytosine, 2,6-diaminopurine, 1-methylguanine, 2-methylguanine, 2,2-dimethylguanine, 7-methylguanine, inosine, 1-methylinosine, pseudouracil (5-uracil), dihydrouracil, 2-thiouracil, 4-thiouracil, 5-carboxymethylaminomethyl-2-thiouracil, 5-(carboxyhydroxymethyl)uracil, 5-fluoro-uracil, 5-bromouracil, 5-carboxymethylaminomethyl-uracil, 5-methyl-2-thio-uracil, 5-methyluracil, N-uracil-5-oxyacetic acid methyl ester, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thio-uracil, 5'-methoxycarbonylmethyl-uracil, 5-methoxy-uracil, uracil-5-oxyacetic acid methyl ester, uracil5-oxyacetic acid (v), 1-methyl-pseudouracil, queosin, .beta.-D-mannosylkeosin, wybutoxosin and phosphoramidates, phosphorothioates, peptide nucleotides, methylphosphonates, 7-deazaguanosine, 5-methylcytosine and inosine. The preparation of such analogues is known to a person skilled in the art, for example, from U.S. Pat. No. 4,373,071, U.S. Pat. No. 4,401,796, U.S. Pat. No. 4,415,732, U.S. Pat. No. 4,458,066, U.S. Pat. No. 4,500,707, U.S. Pat. No. 4,668,777, U.S. Pat. No. 4,973,679, U.S. Pat. No. 5,047,524, U.S. Pat. No. 5,132,418, Pat. US No. 5,153,319, Pat. US Nos. 5,262,530 and 5,700,642, disclosures of which are incorporated by reference in their entirety.

[00211] In some embodiments, mRNA comprises one or more non-standard nucleotide residues. Non-standard nucleotide residues may include, for example, 5-methylcytidine (5mC), pseudouridine (ψU), and / or 2-thiouridine (2sU). See, for example, U.S. Patent No. 8,278,036 or WO 2011 / 012316 for an argument of such residues and their incorporation into mRNA. The mRNA may be mRNA that is defined as RNA in which 25% of the U residues are 2-thiouridine and 25% of the C residues are 5-methylcytidine. Petition 870260055239, dated 08 / 06 / 2026, page 117 / 305 104 / 131 Instructions for the use of RNA are disclosed in US Patent Publication US 2012 / 0195936 and international publication WO 2011 / 012316, both of which are incorporated herein by reference in their entirety. The presence of non-standard nucleotide residues can make an mRNA more stable and / or less immunogenic than a control mRNA with the same sequence but containing only standard residues. In other embodiments, the mRNA may comprise one or more non-standard nucleotide residues selected from isocyosine, pseudoisocytosine, 5-bromouracil, 5-propynyluracil, 6-aminopurine, 2-aminopurine, inosine, diaminopurine, and 2-chloro-6-aminopurine cytosine, as well as combinations of these modifications and other nucleobase modifications. Some embodiments may also include additional modifications to the furanose ring or nucleobase.Additional modifications may include, for example, sugar modifications or substitutions (e.g., one or more 2'-O-alkyl modifications, a blocked nucleic acid (LNA)). In some embodiments, RNAs may be complexed or hybridized with additional polynucleotides and / or peptide polynucleotides (PNA). In some embodiments where the sugar modification is a 2'-O-alkyl modification, such modification may include, but is not limited to, a 2'-deoxy-2-fluoro modification, a 2'-O-methyl modification, a 2'-O-methoxyethyl modification, and a 2'-deoxy modification. In some embodiments, any of these modifications may be present in 0 to 100% of the nucleotides—for example, more than 0%, 1%, 10%, 25%, 50%, 75%, 85%, 90%, 95%, or 100% of the constituent nucleotides individually or in combination.

[00212] In some embodiments, mRNAs may contain modifications to the RNA backbone. Typically, a backbone modification is a modification in which the phosphates Petition 870260055239, dated 08 / 06 / 2026, page 118 / 305 105 / 131 of the RNA backbone of nucleotides are chemically modified. Exemplary backbone modifications typically include, but are not limited to, group modifications consisting of methylphosphonates, methylphosphoramidates, phosphoramidates, phosphorothioates (e.g., cytidine 5'-O-(1-thiophosphate)), boranephosphates, positively charged guanidinium groups, etc., meaning replacing the phosphodiester bond with other anionic, cationic, or neutral groups.

[00213] In some forms, mRNAs may contain sugar modifications. A typical sugar modification is a chemical modification of the sugar of nucleotides containing, including but not limited to, sugar modifications selected from the group consisting of 2'-deoxy-2'-fluoro-oligoribonucleotide (2'-fluoro-2'-deoxycytidine 5'-triphosphate, 2'-fluoro-2'-deoxyuridine 5'-triphosphate), 2'-deoxy-2-deamino-oligoribonucleotide (2'-amino-2'-deoxycytidine 5'-triphosphate, 2'-amino-2'-deoxyuridine 5'-triphosphate), 2'-O-alkyloligoribonucleotide, 2'-deoxy-2'-C-alkyloligoribonucleotide (2'-O-methylcytidine 5'-triphosphate, 2'-methyluridine 5'-triphosphate), 2'-C-alkyloligoribonucleotide, and isomers thereof. (2'-aracitidine 5'-triphosphate, 2'-arauridine 5'-triphosphate) or azidotriphosphates (2-azido-2'-deoxycytidine 5'-triphosphate, 2'-azido-2'deoxyuridine 5'-triphosphate). Post-synthesis processing

[00214] Typically, a 5' cap and / or a 3' end can be added after synthesis. The presence of the cap is important in providing resistance to nucleases found in most eukaryotic cells. The presence of a cap serves to protect the mRNA from exonuclease degradation.

[00215] A 5' cap is typically added as follows: first, a terminal RNA phosphatase removes one of the terminal phosphate groups from the 5' nucleotide, leaving two terminal phosphates; Petition 870260055239, dated 08 / 06 / 2026, page 119 / 305 106 / 131 guanosine triphosphate (GTP) is then added to the terminal phosphates via a guanylyl transferase, producing a 5'5'5 triphosphate linkage; and the 7-nitrogen of guanine is then methylated by a methyltransferase. Examples of cap structures include, but are not limited to, m7G(5')ppp(5'(A),G(5')ppp(5')A and G(5')ppp(5')G. Additional cap structures are described in U.S. Published Application No. 2016 / 0032356 and U.S. Published Application No. 2018 / 0125989, which are incorporated herein by reference.

[00216] Typically, an end structure includes a poly(A) and / or poly(C) end. A poly-A or poly-C 3' terminus of mRNA typically includes at least 50 adenosine or cytosine nucleotides, at least 150 adenosine or cytosine nucleotides, at least 200 adenosine or cytosine nucleotides, at least 250 adenosine or cytosine nucleotides, at least 300 adenosine or cytosine nucleotides, at least 350 adenosine or cytosine nucleotides, at least 400 adenosine or cytosine nucleotides, at least 450 adenosine or cytosine nucleotides, at least 500 adenosine or cytosine nucleotides, at least 550 adenosine or cytosine nucleotides, at least 600 adenosine or cytosine nucleotides, at least 650 adenosine or cytosine nucleotides, at least 700 adenosine or cytosine nucleotides, at least 750 adenosine or cytosine nucleotides, at least 800 adenosine or cytosine nucleotides,at least 850 adenosine or cytosine nucleotides, at least 900 adenosine or cytosine nucleotides, at least 950 adenosine or cytosine nucleotides, or at least 1 kb of adenosine or cytosine nucleotides, respectively. In some embodiments, a poly-A or poly-C terminus may be located about 10 to 800 adenosine or cytosine nucleotides away (e.g., about 10 to 200 adenosine or cytosine nucleotides, about 10 to 300 adenosine or cytosine nucleotides, about 10, Petition 870260055239, dated 08 / 06 / 2026, page 120 / 305 107 / 131 to 400 adenosine or cytosine nucleotides, to approximately 10 to 500 adenosine or cytosine nucleotides, to approximately 10 to 550 adenosine or cytosine nucleotides, to approximately 10 to 600 adenosine or cytosine nucleotides, to approximately 50 to 600 adenosine or cytosine nucleotides, to approximately 100 to 600 adenosine or cytosine nucleotides, to approximately 150 to 600 adenosine or cytosine nucleotides, to approximately 200 to 600 adenosine or cytosine nucleotides, to approximately 250 to 600 adenosine or cytosine nucleotides, approximately 300 to 600 adenosine or cytosine nucleotides, approximately 350 to 600 adenosine or cytosine nucleotides, approximately 400 to 600 adenosine or cytosine nucleotides, approximately 450 to 600 adenosine or cytosine nucleotides, approximately 500 to 600 adenosine or cytosine nucleotides, approximately 10 to 150 adenosine or cytosine nucleotides, approximately 10 to 100 adenosine or cytosine nucleotides, approximately 20 to 70 adenosine or cytosine nucleotides, or approximately 20 to 60 adenosine or cytosine nucleotides, respectively. In some embodiments, an end structure includes a combination of poly(A) and poly(C) tails with various lengths described herein. In some embodiments, an end structure includes at least 50%, 55%, 65%, 70%, 75%, 80%, 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, or 99% adenosine nucleotides. In some embodiments, a fin structure includes at least 50%, 55%, 65%, 70%, 75%, 80%, 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, or 99% cytosine nucleotides.

[00217] As described in this invention, the addition of the 5' cap and / or the 3' end facilitates the detection of aborted transcripts generated during in vitro synthesis because without capping / or tailing, the size of these prematurely aborted mRNA transcripts may be too small to be detected. Thus, in some embodiments, the 5' cap Petition 870260055239, dated 08 / 06 / 2026, p. 121 / 305 108 / 131 and / or the 3' end are added to the synthesized mRNA before the mRNA is tested for purity (e.g., the level of abortive transcripts present in the mRNA). In some embodiments, the 5' cap and / or the 3' end are added to the synthesized mRNA before the mRNA is purified as described in this invention. In other embodiments, the 5' cap and / or the 3' end are added to the synthesized mRNA after the mRNA is purified as described herein.

[00218] The mRNA synthesized according to the present invention can be used without further purification. In particular, the mRNA synthesized according to the present invention can be used without a shortmer removal step. In some embodiments, the mRNA synthesized according to the present invention can be further purified. Several methods can be used to purify the mRNA synthesized according to the present invention. For example, mRNA purification can be performed using centrifugation, filtration and / or chromatographic methods. In some embodiments, the synthesized mRNA is purified by ethanol precipitation or filtration or chromatography, or gel purification or any other suitable means. In some embodiments, the mRNA is purified by HPLC. In some embodiments, the mRNA is extracted in a standard phenol:chloroform:isoamyl alcohol solution, well known to those skilled in the art.In some embodiments, mRNA is purified using tangential flow filtration. Suitable purification methods include those described in US Published Application No. 2016 / 0040154, US Published Application No. 2015 / 0376220, US Published Application No. 2018 / 0251755, US Published Application No. 2018 / 0251754, US Provisional Application No. 62 / 757612 filed on November 8, 2018, and US Provisional Application No. 62 / 891781 filed on August 26, 2019, all of which are... Petition 870260055239, dated 08 / 06 / 2026, p. 122 / 305 109 / 131 are incorporated herein by reference and may be used for the practice of the present invention.

[00219] In some embodiments, mRNA is purified before capping and tailing. In some embodiments, mRNA is purified after capping and tailing. In some embodiments, mRNA is purified before and after capping and tailing.

[00220] In some embodiments, mRNA is purified before or after, or both before and after, capping and tailing, by centrifugation.

[00221] In some embodiments, mRNA is purified before or after, or both before and after, capping and tailing, by filtration.

[00222] In some embodiments, mRNA is purified before or after, or both before and after capping and tailing, by means of Tangential Flow Filtration (TFF).

[00223] In some embodiments, mRNA is purified before or after, or both before and after capping and tailing by chromatography. Characterization of purified mRNA

[00224] The mRNA composition described here is substantially free of contaminants comprising short abortive RNA species, long abortive RNA species, double-stranded RNA (dsRNA), residual plasmid DNA, residual in vitro transcription enzymes, residual solvent and / or residual salt.

[00225] The mRNA composition described here has a purity between approximately 60% and approximately 100%. Consequently, in some embodiments, the purified mRNA has a purity of around 60%. In some embodiments, the purified mRNA has a purity of around 65%. In some embodiments, the purified mRNA has a purity of around 70%. In some embodiments, the mRNA Petition 870260055239, dated 08 / 06 / 2026, p. 123 / 305 110 / 131 purified mRNA has a purity of approximately 75%. In some embodiments, purified mRNA has a purity of approximately 80%. In some embodiments, purified mRNA has a purity of approximately 85%. In some embodiments, purified mRNA has a purity of approximately 90%. In some embodiments, purified mRNA has a purity of approximately 91%. In some embodiments, purified mRNA has a purity of approximately 92%. In some embodiments, purified mRNA has a purity of approximately 93%. In some embodiments, purified mRNA has a purity of approximately 94%. In some embodiments, purified mRNA has a purity of approximately 95%. In some embodiments, purified mRNA has a purity of approximately 96%. In some embodiments, purified mRNA has a purity of approximately 97%. In some embodiments, purified mRNA has a purity of approximately 98%. In some methods, the purified mRNA has a purity of approximately 99%.In some embodiments, the purified mRNA has a purity of around 100%.

[00226] In some embodiments, the mRNA composition described here has less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, less than 1%, less than 0.5% and / or less than 0.1% of impurities in addition to the complete mRNA. Impurities include IVT contaminants, for example, proteins, enzymes, DNA templates, free nucleotides, residual solvent, residual salt, double-stranded RNA (dsRNA), prematurely aborted RNA sequences (shortmers or short abortive RNA species) and / or long abortive RNA species. In some embodiments, the purified mRNA is substantially free of process enzymes.

[00227] In some embodiments, the residual plasmid DNA in the purified mRNA of the present invention is less than about 1 Petition 870260055239, dated 08 / 06 / 2026, p. 124 / 305 111 / 131 pg / mg, less than about 2 pg / mg, less than about 3 pg / mg, less than about 4 pg / mg, less than about 5 pg / mg, less than about 6 pg / mg, less than about 7 pg / mg, less than about 8 pg / mg, less than about 9 pg / mg, less than about 10 pg / mg, less than about 11 pg / mg, or less than about 12 pg / mg. Consequently, the residual plasmid DNA in the purified mRNA is less than about 1 pg / mg. In some embodiments, the residual plasmid DNA in the purified mRNA is less than about 2 pg / mg. In some embodiments, the residual plasmid DNA in the purified mRNA is less than about 3 pg / mg. In some embodiments, the residual plasmid DNA in the purified mRNA is less than about 4 pg / mg. In some embodiments, the residual plasmid DNA in the purified mRNA is less than about 5 pg / mg. In some embodiments, the residual plasmid DNA in the purified mRNA is less than about 6 pg / mg.In some embodiments, the residual plasmid DNA in the purified mRNA is less than about 7 pg / mg. In some embodiments, the residual plasmid DNA in the purified mRNA is less than about 8 pg / mg. In some embodiments, the residual plasmid DNA in the purified mRNA is less than about 9 pg / mg. In some embodiments, the residual plasmid DNA in the purified mRNA is less than about 10 pg / mg. In some embodiments, the residual plasmid DNA in the purified mRNA is less than about 11 pg / mg. In some embodiments, the residual plasmid DNA in the purified mRNA is less than about 12 pg / mg.

[00228] In some embodiments, a method according to the invention removes more than about 90%, 95%, 96%, 97%, 98%, 99% or substantially all prematurely aborted RNA sequences (also known as shortmers). In some embodiments, the mRNA composition is substantially free of Petition 870260055239, dated 08 / 06 / 2026, p. 125 / 305 112 / 131 prematurely aborted RNA sequences. In some embodiments, the mRNA composition contains less than about 5% (e.g., less than about 4%, 3%, 2%, or 1%) of prematurely aborted RNA sequences. In some embodiments, the mRNA composition contains less than about 1% (e.g., less than about 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, or 0.1%) of prematurely aborted RNA sequences. In some embodiments, prematurely aborted RNA sequences are undetectable from the mRNA composition as determined by, for example, high-performance liquid chromatography (HPLC) (e.g., secondary or separate peaks), ethidium bromide, Coomassie staining, capillary electrophoresis, or glyoxal gel electrophoresis (e.g., presence of a separate lower band).As used herein, the terms shortmers, short aborted RNA species, prematurely aborted RNA sequences, or long aborted RNA species refer to any transcripts that are shorter than the total length. In some embodiments, shortmers, short aborted RNA species, or prematurely aborted RNA sequences are less than 100 nucleotides long, less than 90, less than 80, less than 70, less than 60, less than 50, less than 40, less than 30, less than 20, or less than 10 nucleotides long. In some embodiments, shortmers are detected or quantified after the addition of a 5'-cap and / or a 3'-poly A end. In some embodiments, prematurely aborted RNA transcripts comprise fewer than 15 bases (e.g., fewer than 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, or 3 bases). In some embodiments, prematurely aborted RNA transcripts contain approximately 8 to 15, 8 to 14, 8 to 13, 8 to 12, 8 to 11, or 8 to 10 bases.

[00229] In some embodiments, a purified mRNA of the present Petition 870260055239, dated 08 / 06 / 2026, p. 126 / 305 The invention is substantially free of enzymatic reagents used in in vitro synthesis, including, but not limited to, T7 RNA polymerase, DNase I, pyrophosphatase, and / or RNase inhibitor. In some embodiments, a purified mRNA according to the present invention contains less than about 5% (for example, less than about 4%, 3%, 2%, or 1%) of enzymatic reagents used in in vitro synthesis, inclusive. In some embodiments, a purified mRNA contains less than about 1% (for example, less than about 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, or 0.1%) of enzymatic reagents used in in vitro synthesis, inclusive.In some embodiments, a purified mRNA contains undetectable enzymatic reagents used in in vitro synthesis, including as determined by, for example, silver staining, gel electrophoresis, high-performance liquid chromatography (HPLC), ultra-high-performance liquid chromatography (UPLC) and / or capillary electrophoresis, ethidium bromide and / or Coomassie staining.

[00230] In several embodiments, a purified mRNA of the present invention maintains a high degree of integrity. As used in this invention, the term mRNA integrity generally refers to the quality of the mRNA after purification. mRNA integrity can be determined using methods well known in the art, for example, by agarose RNA gel electrophoresis. In some embodiments, mRNA integrity can be determined by agarose RNA gel electrophoresis band patterns. In some embodiments, a purified mRNA of the present invention shows little or no banding compared to the agarose RNA gel electrophoresis reference band. In some embodiments, a purified mRNA of the present invention has an integrity greater than about 95% (for example, greater than about 96%, 97%, 98%, 99% or more). In some embodiments, a purified mRNA of the Petition 870260055239, dated 08 / 06 / 2026, p. 127 / 305 114 / 131 The present invention has an integrity greater than 98%. In some embodiments, a purified mRNA of the present invention has an integrity greater than 99%. In some embodiments, a purified mRNA of the present invention has an integrity of approximately 100%.

[00231] In some embodiments, the purified mRNA is evaluated for one or more of the following characteristics: appearance, identity, quantity, concentration, presence of impurities, microbiological evaluation, pH level, and activity. In some embodiments, acceptable appearance includes a clear, colorless solution, essentially free of visible particles. In some embodiments, mRNA identity is evaluated by sequencing methods. In some embodiments, concentration is evaluated by a suitable method, such as UV spectrophotometry. In some embodiments, a suitable concentration is between about 90% and 110% nominal (0.9 to 1.1 mg / ml).

[00232] In some embodiments, assessing mRNA purity includes assessing mRNA integrity, assessing residual plasmid DNA, and assessing residual solvent. In some embodiments, acceptable levels of mRNA integrity are assessed by agarose gel electrophoresis. The gels are analyzed to determine if the banding pattern and apparent nucleotide length are consistent with a reference analytical standard. Additional methods for assessing RNA integrity include, for example, assessing purified mRNA using capillary gel electrophoresis (CGE). In some embodiments, acceptable purity of purified mRNA as determined by CGE is that the purified mRNA composition does not contain more than about 55% aborted / degraded long species. In some embodiments, residual plasmid DNA is assessed by methods in the technique, for example, by using qPCR. In some Petition 870260055239, dated 08 / 06 / 2026, page 128 / 305115 / 131 embodiments, less than 10 pg / mg (e.g., less than 10 pg / mg, less than 9 pg / mg, less than 8 pg / mg, less than 7 pg / mg, less than 6 pg / mg, less than 5 pg / mg, less than 4 pg / mg, less than 3 pg / mg, less than 2 pg / mg, or less than 1 pg / mg) is an acceptable level of residual plasmid DNA. In some embodiments, acceptable levels of residual solvent are no greater than 10,000 ppm, 9,000 ppm, 8,000 ppm, 7,000 ppm, 6,000 ppm, 5,000 ppm, 4,000 ppm, 3,000 ppm, 2,000 ppm, 1,000 ppm. Consequently, in some embodiments, acceptable levels of residual solvent are no greater than 10,000 ppm. In some embodiments, acceptable levels of residual solvent are no greater than 9,000 ppm. In some embodiments, acceptable levels of residual solvent are no greater than 8,000 ppm. In some embodiments, acceptable levels of residual solvent are no greater than 7,000 ppm. In some embodiments, acceptable levels of residual solvent are no greater than 6,000 ppm.In some embodiments, acceptable levels of residual solvent are no greater than 5,000 ppm. In some embodiments, acceptable levels of residual solvent are no greater than 4,000 ppm. In some embodiments, acceptable levels of residual solvent are no greater than 3,000 ppm. In some embodiments, acceptable levels of residual solvent are no greater than 2,000 ppm. In some embodiments, acceptable levels of residual solvent are no greater than 1,000 ppm.

[00233] In some embodiments, microbiological tests are performed on the purified mRNA, which include, for example, the evaluation of bacterial endotoxins. In some embodiments, bacterial endotoxins are < 0.5 EU / ml, < 0.4 EU / ml, < 0.3 EU / ml, < 0.2 EU / ml, or < 0.1 EU / ml. Consequently, in some embodiments, bacterial endotoxins in the purified mRNA are < 0.5 EU / ml. In some embodiments, bacterial endotoxins in the mRNA Petition 870260055239, dated 08 / 06 / 2026, page 129 / 305 116 / 131 purified mRNA is < 0.4 EU / ml. In some embodiments, bacterial endotoxins in purified mRNA are < 0.3 EU / ml. In some embodiments, bacterial endotoxins in purified mRNA are < 0.2 EU / ml. In some embodiments, bacterial endotoxins in purified mRNA are < 0.2 EU / ml. In some embodiments, bacterial endotoxins in purified mRNA are < 0.1 EU / ml. In some embodiments, the purified mRNA does not contain more than 1 CFU / 10 ml, 1 CFU / 25 ml, 1 CFU / 50 ml, 1 CFU / 75 ml, or not more than 1 CFU / 100 ml. Consequently, in some embodiments, the purified mRNA does not contain more than 1 CFU / 10 ml. In some embodiments, the purified mRNA does not contain more than 1 CFU / 25 ml. In some embodiments, the purified mRNA has no more than 1 CFU / 50 ml. In some embodiments, the purified mRNA has no more than 1 CFR / 75 ml. In some embodiments, the purified mRNA has 1 CFU / 100 ml.

[00234] In some embodiments, the pH of the purified mRNA is evaluated. In some embodiments, the acceptable pH of the purified mRNA is between 5 and 8. Consequently, in some embodiments, the purified mRNA has a pH of about 5. In some embodiments, the purified mRNA has a pH of about 6. In some embodiments, the purified mRNA has a pH of about 7. In some embodiments, the purified mRNA has a pH of about 7. In some embodiments, the purified mRNA has a pH of about 8.

[00235] In some embodiments, the fidelity of the purified mRNA translation is evaluated. Translation fidelity can be evaluated by several methods and include, for example, transfection and Western blot analysis. Acceptable characteristics of the purified mRNA include a banding pattern on a Western blot that migrates with a molecular weight similar to the reference standard.

[00236] In some embodiments, purified mRNA is evaluated Petition 870260055239, dated 08 / 06 / 2026, p. 130 / 305 117 / 131 regarding conductance. In some embodiments, acceptable characteristics of purified mRNA include conductance between approximately 50% and 150% of a reference standard.

[00237] The purified mRNA is also evaluated for Cap percentage and PolyA end length. In some embodiments, an acceptable Cap percentage includes Cap1, % Area: NLT90. In some embodiments, an acceptable polyA end length is about 100 to 1500 nucleotides (e.g., 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950 and 1000, 1100, 1200, 1300, 1400 or 1500 nucleotides).

[00238] In some embodiments, the purified mRNA is also evaluated for any residual PEG. In some embodiments, the purified mRNA has less than 10 ng PEG / mg of purified mRNA and 1000 ng PEG / mg of mRNA. Consequently, in some embodiments, the purified mRNA has less than about 10 ng PEG / mg of purified mRNA. In some embodiments, the purified mRNA has less than about 100 ng PEG / mg of purified mRNA. In some embodiments, the purified mRNA has less than about 250 ng PEG / mg of purified mRNA. In some embodiments, the purified mRNA has less than about 500 ng PEG / mg of purified mRNA. In some embodiments, the purified mRNA has less than about 750 ng PEG / mg of purified mRNA. In some embodiments, the purified mRNA has less than about 1000 ng PEG / mg of purified mRNA.

[00239] Several methods for detecting and quantifying mRNA purity are known in the art. For example, such methods include blotting, capillary electrophoresis, chromatography, fluorescence, gel electrophoresis, HPLC, silver staining, spectroscopy, ultraviolet (UV) or UPLC, or a combination thereof. In some embodiments, the Petition 870260055239, dated 08 / 06 / 2026, page 131 / 305 118 / 131 mRNA is first denatured by a glyoxal dye before gel electrophoresis (glyoxal gel electrophoresis). In some embodiments, the synthesized mRNA is characterized before capping or tailing. In some embodiments, the synthesized mRNA is characterized after capping and tailing. Therapeutic Use of the Compositions

[00240] To facilitate mRNA expression in vivo, delivery vehicles such as liposomes may be formulated in combination with one or more additional nucleic acids, carriers, targeting ligands or stabilizing reagents, or in pharmaceutical compositions where it is mixed with suitable excipients. Techniques for drug formulation and administration can be found in Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa., latest edition.

[00241] In some embodiments, a composition comprises mRNA encapsulated or complexed with a delivery vehicle. In some embodiments, the delivery vehicle is selected from the group consisting of liposomes, lipid nanoparticles, solid lipid nanoparticles, polymers, viruses, sol-gels, and nanogels.

[00242] mRNA-laden nanoparticles and compositions containing them may be administered and dosed according to current medical practice, taking into account the individual's clinical condition, the site and method of administration, the administration schedule, the individual's age, sex, body weight, and other factors relevant to clinicians skilled in the art. The effective amount for the purposes of this invention may be determined by such relevant considerations as known to those skilled in experimental, pharmacological, clinical, and medical arts research. In some embodiments, the amount administered is effective to achieve by Petition 870260055239, dated 08 / 06 / 2026, page 132 / 305 119 / 131 less any stabilization, improvement, or elimination of symptoms and other indicators as selected as appropriate measures of disease progress, regression, or improvement by those skilled in the art. For example, an adequate amount and dosage regimen is one that causes at least transient protein (e.g., enzyme) production.

[00243] The present invention provides methods of administering mRNA for in vivo protein production, comprising administering mRNA to an individual requiring administration. In some embodiments, the mRNA is administered via a route of administration selected from the group consisting of intravenous administration, subcutaneous administration, oral administration, subdermal administration, ocular administration, pulmonary administration by intratracheal injection (e.g., nebulization), intramuscular administration, intrathecal administration, or intra-articular administration.

[00244] Suitable routes of administration include, for example, oral, rectal, vaginal, transmucosal, pulmonary including intratracheal or inhaled, or intestinal administration; parenteral administration, including intradermal, transdermal (topical), intramuscular, subcutaneous, intramedullary, as well as intrathecal, direct intraventricular, intravenous, intraperitoneal or intranasal injections. In some embodiments, intramuscular administration is into a muscle selected from the group consisting of skeletal muscle, smooth muscle and cardiac muscle. In some embodiments, administration results in the administration of mRNA to a muscle cell. In some embodiments, administration results in the administration of mRNA to a hepatocyte (i.e., liver cell). In one particular embodiment, intramuscular administration results in the administration of mRNA to a muscle cell.

[00245] Additional teachings on pulmonary administration and Petition 870260055239, dated 08 / 06 / 2026, p. 133 / 305 120 / 131 nebulization are described in U.S. Published Application No. U.S. 2018 / 0125989 and U.S. Published Application No. U.S. 2018 / 0333457, each of which is incorporated by reference in its entirety.

[00246] Alternatively or additionally, mRNA-laden nanoparticles and compositions of the invention can be administered locally rather than systemically, for example, by injecting the pharmaceutical composition directly into a target tissue, preferably in a controlled-release formulation. Local administration can be affected in several ways depending on the tissue to be targeted.For example, aerosols containing compositions of the present invention can be inhaled (for nasal, tracheal, or bronchial administration); the compositions of the present invention can be injected at the site of injury, disease manifestation, or pain, for example; the compositions can be supplied in lozenges for oral, tracheal, or esophageal application; they can be supplied in liquid, tablet, or capsule form for administration into the stomach or intestines; they can be supplied in suppository form for rectal or vaginal application; or they can be administered into the eye by means of creams, drops, or even injection. Formulations containing compositions supplied complexed with therapeutic molecules or ligands can even be administered surgically, for example, in association with a polymer or other structure or substance that can allow the compositions to diffuse from the implantation site to the surrounding cells.Alternatively, they can be applied surgically without the use of polymers or supports.

[00247] The methods provided in the present invention contemplate single and multiple administrations of a therapeutically effective amount of the therapeutic agents (e.g., mRNA) described herein. Therapeutic agents may be administered at regular intervals, Petition 870260055239, dated 08 / 06 / 2026, page 134 / 305 121 / 131 depending on the nature, severity and extent of the individual's condition. In some embodiments, a therapeutically effective amount of the therapeutic agents (e.g., mRNA) of the present invention may be administered intrathecally periodically at regular intervals (e.g., once a year, once every six months, once every five months, once every three months, bimonthly (once every two months), monthly (once a month), fortnightly (once every two weeks), twice a month, once every 30 days, once every 28 days, once every 14 days, once every 10 days, once every 7 days, weekly, twice a week, daily or continuously).

[00248] In some embodiments, the liposomes and / or compositions provided are formulated in such a way that they are suitable for prolonged release of the mRNA contained therein. Such prolonged-release compositions can be conveniently administered to an individual at prolonged dosing intervals. For example, in one embodiment, the compositions of the present invention are administered to an individual twice daily, daily or on alternate days.In a preferred embodiment, the compositions of the present invention are administered to an individual twice a week, once a week, once every 7 days, once every 10 days, once every 14 days, once every 28 days, once every 30 days, once every two weeks, once every three weeks, or more preferably once every four weeks, once a month, twice a month, once every six weeks, once every eight weeks, once every two months, once every three months, once every four months, once every six months, once every eight months, once every nine months, or annually. Also contemplated are compositions and liposomes that are formulated for long-acting administration (e.g., intramuscularly). Petition 870260055239, dated 08 / 06 / 2026, p. 135 / 305 122 / 131 subcutaneous, intravitreal) to administer or release a therapeutic agent (e.g., mRNA) over long periods of time. Preferably, the sustained-release media employed are combined with modifications made to the mRNA to increase stability.

[00249] As used herein, the term therapeutically effective amount is broadly defined based on the total amount of the therapeutic agent contained in the pharmaceutical compositions of the present invention. Generally, a therapeutically effective amount is sufficient to achieve a significant benefit for the individual (e.g., to treat, modulate, cure, prevent, and / or improve a disease or disorder). For example, a therapeutically effective amount may be an amount sufficient to achieve a desired therapeutic and / or prophylactic effect. Generally, the amount of a therapeutic agent (e.g., mRNA) administered to an individual with their need will depend on the individual's characteristics. Such characteristics include the individual's condition, disease severity, general health, age, sex, and body weight.A person with practical skill in the technique will be easily able to determine the appropriate dosages, depending on these and other related factors. Furthermore, both objective and subjective assays can optionally be employed to identify the ideal dosage ranges.

[00250] A therapeutically effective amount is commonly administered in a dosing regimen that may comprise multiple unit doses. For any particular therapeutic protein, a therapeutically effective amount (and / or an appropriate unit dose within an effective dosing regimen) may vary, for example, depending on the route of administration, in combination with other pharmaceutical agents. Furthermore, the specific amount Petition 870260055239, dated 08 / 06 / 2026, page 136 / 305 The therapeutically effective (and / or unit dose) for any particular patient may depend on a variety of factors, including the disorder being treated and the severity of the disorder; the activity of the specific pharmaceutical agent employed; the specific composition employed; the age, body weight, general health, sex, and diet of the patient; the timing of administration, route of administration, and / or rate of excretion or metabolism of the specific protein employed; the duration of treatment; and similar factors, as is well known in medical techniques.

[00251] In some embodiments, the therapeutically effective dose ranges from about 0.005 mg / kg of body weight to 500 mg / kg of body weight, for example, from about 0.005 mg / kg of body weight to 400 mg / kg of body weight, from about 0.005 mg / kg of body weight to 300 mg / kg of body weight, from about 0.005 mg / kg of body weight to 200 mg / kg of body weight, from about 0.005 mg / kg of body weight to 100 mg / kg of body weight, from about 0.005 mg / kg of body weight to 90 mg / kg of body weight, from about 0.005 mg / kg of body weight to 80 mg / kg of body weight, from about 0.005 mg / kg of body weight to 70 mg / kg of body weight, from about 0.005 mg / kg body weight to 60 mg / kg body weight, from about 0.005 mg / kg body weight to 50 mg / kg body weight, from about 0.005 mg / kg body weight to 40 mg / kg body weight, from about 0.005 mg / kg body weight to 30 mg / kg body weight, from about 0.005 mg / kg body weight to 25 mg / kg of body weight,from about 0.005 mg / kg of body weight to 20 mg / kg of body weight, from about 0.005 mg / kg of body weight to 15 mg / kg of body weight, from about 0.005 mg / kg of body weight to 10 mg / kg of body weight.,

[00252] In some modalities, the therapeutically effective dose is greater than about 0.1 mg / kg of body weight, greater than about 0.5 mg / kg of body weight, greater than about 1.0 mg / kg of Petition 870260055239, dated 08 / 06 / 2026, page 137 / 305 124 / 131 body weight, greater than about 3 mg / kg of body weight, greater than about 5 mg / kg of body weight, greater than about 10 mg / kg of body weight, greater than about 15 mg / kg of body weight, greater than about 20 mg / kg of body weight, greater than about 30 mg / kg of body weight, greater than about 40 mg / kg of body weight, greater than about 50 mg / kg of body weight, greater than about 60 mg / kg of body weight, greater than about 70 mg / kg of body weight, greater than about 80 mg / kg of body weight, greater than about 90 mg / kg of body weight, greater than about 100 mg / kg of body weight, greater than about 150 mg / kg of body weight, greater than about 200 mg / kg of body weight, greater than about 250 mg / kg of body weight, greater than approximately 300 mg / kg of body weight, greater than approximately 350 mg / kg of body weight, greater than approximately 400 mg / kg of body weight,greater than approximately 450 mg / kg of body weight, greater than approximately 500 mg / kg of body weight. In one particular embodiment, the therapeutically effective dose is 1.0 mg / kg. In some embodiments, the therapeutically effective dose of 1.0 mg / kg is administered intramuscularly or intravenously.

[00253] Also contemplated in this invention are lyophilized pharmaceutical compositions comprising one or more of the liposomes disclosed herein and related methods for the use of such compositions as disclosed, for example, in U.S. Provisional Application No. 61 / 494,882, filed June 8, 2011, the teachings of which are incorporated herein by reference in their entirety. For example, the lyophilized pharmaceutical compositions according to the invention may be reconstituted prior to administration or may be reconstituted in vivo. For example, a lyophilized pharmaceutical composition may be formulated in an appropriate dosage form (e.g., a dosage form). Petition 870260055239, dated 08 / 06 / 2026, page 138 / 305 125 / 131 intradermal, such as a disc, rod or membrane) and administered in such a way that the dosage form is rehydrated over time in vivo by the individual's body fluids.

[00254] The supplied liposomes and compositions can be delivered to any desired tissue. In some embodiments, the mRNA released by the supplied liposomes or compositions is expressed in the tissue to which the liposomes and / or compositions were delivered. In some embodiments, the released mRNA is expressed in a tissue different from the tissue to which the liposomes and / or compositions were delivered. Exemplary tissues in which the released mRNA can be delivered and / or expressed include, but are not limited to, liver, kidney, heart, spleen, serum, brain, skeletal muscle, lymph nodes, skin, and / or cerebrospinal fluid.

[00255] In some embodiments, administering the provided composition results in an increased mRNA expression level in a biological sample from an individual compared to a baseline expression level before treatment. Typically, the baseline level is measured immediately before treatment. Biological samples include, for example, whole blood, serum, plasma, urine, and tissue samples (e.g., muscle, liver, skin fibroblasts). In some embodiments, administration of the provided composition results in an increase in mRNA expression level by at least approximately 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% as compared to the baseline level immediately before treatment. In some embodiments, administering the provided composition results in an increased mRNA expression level compared to an mRNA expression level in untreated individuals.

[00256] According to various modalities, the appropriate timing of expression of the released mRNA can be adjusted to meet a Petition 870260055239, dated 08 / 06 / 2026, page 139 / 305 126 / 131 specific medical need. In some embodiments, expression of the protein encoded by the released mRNA is detectable 1, 2, 3, 6, 12, 24, 48, 72 and / or 96 hours after administration of the supplied liposomes and / or compositions. In some embodiments, expression of the protein encoded by the released mRNA is detectable one week, two weeks and / or one month after administration.

[00257] The present invention also provides for the release of a composition having mRNA molecules encoding a peptide or polypeptide of interest for use in the treatment of an individual, for example, a human being or a cell of a human being or a cell that is treated and released to a human individual. EXAMPLES

[00258] Although certain compounds, compositions and methods of the present invention have been described specifically according to certain embodiments, the following examples serve only to illustrate the invention and are not intended to limit it. Although certain compounds, compositions and methods of the present invention have been described specifically according to certain embodiments, the following examples serve only to illustrate the invention and are not intended to limit it. Example 1. Formulation of Lipid Nanoparticles with a High Concentration of Citrate

[00259] This example illustrates that mRNA-LNPs formed by Process A with a high concentration of citrate (i.e., > 10 mM) in an mRNA solution without heating the lipid and mRNA solutions before mixing have encapsulation efficiency lower than about 60%.

[00260] As used herein, Process A refers to a conventional method of encapsulating mRNA by mixing the mRNA with a lipid mixture, without first pre-forming the Petition 870260055239, dated 08 / 06 / 2026, page 140 / 305 127 / 131 Lipids in lipid nanoparticles. In summary, in this process, a lipid mixture solution (i.e., cationic lipids, auxiliary lipids, PEG-modified lipids, cholesterol lipids, etc.) was prepared by dissolving lipids in ethanol. The mRNA solution was prepared by dissolving the mRNA in citrate buffer. The lipid solution and the mRNA solution were kept at room temperature without heating. Then, these two solutions were mixed using a pump system. Typically, after mixing, the solution comprising mRNA encapsulated within LNPs was incubated for 60 to 90 minutes before purification by diafiltration with a TFF process.

[00261] The effect of various citrate concentrations in mRNA solution was studied. Table 1 shows exemplary encapsulation efficiencies for mRNA-LNPs prepared with mRNA solution comprising 10 mM, 20 mM, or 40 mM citrate. All other variables, including batch size, flow rate, temperature, pH, and salt concentration, were kept constant. Encapsulation efficiencies for the lipid nanoparticle formulation with high citrate concentration (> 10 mM) were approximately 60%. Table 1. Encapsulation efficiencies for lipid nanoparticle formulations with various citrate concentrations. Formulation Citrate Concentration Encapsulation 1 10 mM 58.2% 2 20 mM 60.9% 3 40 mM 57.8% Example 2. Formulation of lipid nanoparticles with low citrate concentration.

[00262] This example illustrates that mRNA-LNPs prepared with low citrate concentration (i.e., < 5 mM) in the mRNA solution have high encapsulation efficiencies of about 60% or more. Petition 870260055239, dated 08 / 06 / 2026, page 141 / 305 128 / 131 High encapsulation efficiencies were observed even when the process did not include a heating step of the mRNA and / or lipid solutions prior to the mixing step.

[00263] 50 mg of mRNA were encapsulated within lipid nanoparticles by Process A with various concentrations of citrate in the mRNA solution. After mixing, the mRNA-LNPs were incubated at 30 °C for 90 minutes. FIG. 1 shows encapsulation efficiencies for mRNA-LNPs prepared with 0, 1.5, 2.0, 2.5, 3, 5, 7.5, and 10 mM citrate, before and after incubation at 30 °C. As shown in FIG. 1, mRNA-LNPs prepared with 5 mM or less of citrate resulted in final encapsulation efficiencies above 70%. It is also noteworthy that the change in pH (3.0 to 4.5) did not impact the encapsulation efficiency. Example 3. Formulation of lipid nanoparticles with various concentrations of sodium chloride.

[00264] This example illustrates that the concentration of sodium chloride (NaCl) in mRNA solutions does not have a significant impact on the encapsulation efficiencies of mRNA-LNPs.

[00265] 50 mg of mRNA were encapsulated within lipid nanoparticles by Process A with various concentrations of NaCl in the mRNA solution with 2.5 mM citrate and a pH of 4.5. After mixing, the mRNA-LNPs were incubated at 30°C for 90 minutes. FIG. 2 shows encapsulation efficiencies for mRNA-LNPs prepared with 0, 37.5, 75, 150, and 300 mM NaCl, before and after incubation at 30°C. As shown in FIG. 2, mRNA-LNPs prepared with 37.5 to 300 mM NaCl resulted in final encapsulation efficiencies above 70%. It is also noteworthy that the change in pH (3.0 to 4.5) did not impact the encapsulation efficiency (data not shown).

[00266] To confirm the above results, 50 mg of mRNA were Petition 870260055239, dated 08 / 06 / 2026, p. 142 / 305 129 / 131 encapsulated within lipid nanoparticles under the following conditions: i) 2.5 mM citrate + 150 mM NaCl, ii) 2.5 mM citrate + 300 mM NaCl, iii) 3.0 mM citrate + 150 mM NaCl, or iv) 3.0 mM citrate + 300 mM NaCl. The results are shown in FIG. 3. No significant difference was observed between 2.5 mM and 3.0 mM citrate concentrations with 150 mM or 300 mM NaCl. All four conditions resulted in final encapsulation efficiencies greater than 70%. Example 4. Lipid Nanoparticle Formulation with Various mRNA:lipid (v / v) Ratios

[00267] This example illustrates the effect of mRNA:lipid ratio and flow rate during mixing on mRNA-LNP encapsulation efficiencies.

[00268] 20 mg of mRNAs were encapsulated in lipid nanoparticles using an mRNA solution comprising 10 mM citrate, 150 mM NaCl, and a pH of 4.5. Different lipid concentrations in the lipid solution, mRNA concentrations in the mRNA solution, and flow rates during the mixing step were studied. The volume of mRNA or lipid solution was decreased or increased to achieve higher or lower concentrations, respectively. Table 2. Encapsulation efficiencies for the formulation of lipid nanoparticles with various mRNA / lipid concentrations and flow rates. Conditions Volume of mRNA solution Volume of lipid solution Ratio of mRNA flow rate: lipid flow rate Formulation 1 EE% Formulation 2 EE% A (Control) 100% 100% 4:1 46 43 B 100% 133% 3:1 50 51 C 100% 80% 5:1 39 39 D 75% 100% 3:1 48 53 E 125% 100% 5:1 37 40 Petition 870260055239, dated 08 / 06 / 2026, page 143 / 305 130 / 131

[00269] The results in Table 2 show that a relatively higher concentration of mRNA (i.e., smaller volume of mRNA solution; condition D) and a relatively lower concentration of lipids (i.e., larger volume of lipid solution; condition B) correlate with greater encapsulation efficiency compared to a control (condition A).

[00270] To confirm the above results, different mRNA:lipid solution ratios (v / v) were studied. 50 mg of mRNAs were encapsulated in lipid nanoparticles using an mRNA solution comprising 2.5 mM citrate and 150 mM NaCl and pH 4.5. As shown in FIG. 4, an mRNA:lipid ratio greater than 3:1 resulted in a final encapsulation efficiency of more than about 75%. Notably, a 4:1 ratio resulted in encapsulation efficiency greater than 70% before and after the incubation step.

[00271] Next, the effect of flow rates on the encapsulation efficiency of mRNA-LNPs was studied. 50 mg of mRNAs were encapsulated in lipid nanoparticles using an mRNA solution comprising 2.5 mM citrate and 150 mM NaCl and pH 4.5. Several combined flow rates (mRNA flow rate + lipid flow rate) ranging from 200 ml / min to 500 ml / min were tested. As shown in FIG. 5, no significant change in encapsulation efficiency was observed with different flow rates. High encapsulation efficiencies were achieved with Process A when a low citrate concentration (< 5 mM) was used, regardless of flow rate, pH, and NaCl concentration. EQUIVALENTS

[00272] Those skilled in the art will recognize, or be able to determine using no more than routine experimentation, many equivalents to the specific embodiments of the invention. Petition 870260055239, dated 08 / 06 / 2026, p. 144 / 305 131 / 131 described herein. The scope of the present invention is not intended to be limited by the above description, but rather as set forth in the following claims. Petition 870260055239, dated 08 / 06 / 2026, p. 145 / 305

Claims

1 / 7 CLAIMS 1. A process for encapsulating messenger RNA (mRNA) in lipid nanoparticles (LNPs), characterized in that it comprises a mixing step of (a) an mRNA solution comprising one or more mRNAs with (b) a lipid solution comprising one or more cationic lipids, one or more non-cationic lipids and one or more PEG-modified lipids, to form mRNA encapsulated within LNPs (mRNA-LNPs) in an LNP-forming solution, wherein the mRNA solution comprises less than 5 mM citrate and wherein the mRNA-LNPs have an encapsulation efficiency greater than 60%.

2. Process according to claim 1, characterized in that the mRNA solution and / or the lipid solution are at room temperature before mixing, optionally wherein: (i) the room temperature is less than about 35°C, less than about 30°C, less than about 26°C, less than about 23°C, less than about 21°C, less than about 20°C or less than about 18°C; and / or (ii) the room temperature ranges from about 18 to 32°C, from about 21 to 26°C or from about 23 to 25°C.

3. Process, according to claim 1 or 2, characterized in that: (i) the mRNA solution comprises less than about 4.0 mM citrate, less than about 3.0 mM citrate, less than about 2.5 mM citrate, less than about 2.0 mM citrate, less than about 1.5 mM citrate, less than about 1.25 mM citrate, less than about 1.0 mM citrate or less than about 0.5 mM citrate; and / or (ii) the mRNA solution comprises about 3.0 mM citrate, about 2.5 mM citrate, about 2.0 mM citrate, about Petition 870260055239, dated 08 / 06 / 2026, page. 146 / 305 2 / 7 1.5 mM citrate, about 1.25 mM citrate, about 1.0 mM citrate, about 0.5 mM citrate or 0 mM citrate.

4. Messenger RNA (mRNA) encapsulation process in lipid nanoparticles (LNPs), characterized in that it comprises a mixing step of (a) an mRNA solution comprising one or more mRNAs with (b) a lipid solution comprising one or more cationic lipids, one or more non-cationic lipids and one or more PEG-modified lipids, to form an mRNA encapsulated within LNPs (mRNA-LNPs) in an LNP-forming solution, wherein the mRNA solution comprises between 0.1 mM and 5 mM citrate, and wherein the mRNA-LNPs have an encapsulation efficiency greater than 60%.

5. Process according to claim 1 or 2, characterized in that the mRNA solution comprises between about 1 mM and 4 mM of citrate.

6. Process according to claim 5, characterized in that the mRNA solution comprises between about 1 mM and 3 mM of citrate, optionally wherein the mRNA solution comprises between about 1 mM and 2 mM of citrate or between about 2 mM and 3 mM of citrate.

7. Process according to claim 5, characterized in that the mRNA solution comprises about 1 mM citrate, about 2 mM citrate, or about 3 mM citrate.

8. Process according to any one of claims 1 to 7, characterized in that: (i) the mRNA solution further comprises trehalose; and / or (ii) the process does not require a heating step of the mRNA solution and the lipid solution prior to the mixing step.

9. Process, according to any one of claims 1 to 8, characterized in that the mRNA solution Petition 870260055239, dated 08 / 06 / 2026, p. 147 / 305 3 / 7 comprises more than about 1 g of mRNA per 12 L of mRNA solution, optionally where: the mRNA solution comprises about 1 g of mRNA per 8 L of mRNA solution, about 1 g of mRNA per 4 L of mRNA solution, or about 1 g of mRNA per 2 L of mRNA solution; or the concentration of mRNA in the mRNA solution is greater than about 0.125 mg / ml, greater than about 0.25 mg / ml, greater than about 0.5 mg / ml or greater than about 1.0 mg / ml.

10. Process according to any one of claims 1 to 9, characterized in that the mRNA solution and the lipid solution are mixed in a ratio (v / v) between 2:1 and 6:1, optionally wherein: (i) the mRNA solution and the lipid solution are mixed in a ratio (v / v) greater than about 3:1; or (ii) the mRNA solution and the lipid solution are mixed in a ratio (v / v) of about 4:

1.

11. Process, according to any one of claims 1 to 10, characterized in that: (i) the mRNA solution has a pH between 3.0 and 5.0, optionally wherein the mRNA solution has a pH of about 3.5, 4.0 or 4.5; and / or (ii) the mRNA solution comprises about 37.5 mM to 300 mM of NaCl, optionally wherein the mRNA solution comprises about 37.5 mM, about 75 mM, about 100 mM, about 150 mM or about 300 mM of NaCl.

12. Process according to claim 1 or 2, characterized in that the mRNA solution comprises about 2.5 mM citrate, about 150 mM NaCl and a pH of about 4.

5.

13. Process, according to any of the claims 1 to 12 of Petition 870260055239, dated 08 / 06 / 2026, p. 148 / 305 4 / 7, characterized in that the process further comprises an incubation step of the mRNA-LNPs, optionally wherein: (i) the mRNA-LNPs are incubated at a temperature between 21 °C and 65 °C, optionally wherein the mRNA-LNPs are incubated at a temperature of about 26 °C, about 30 °C or about 65 °C; and / or (ii) mRNA-LNPs are incubated for more than about 20 minutes, about 30 minutes, about 60 minutes, about 90 minutes or about 120 minutes, optionally wherein mRNA-LNPs are incubated for about 60 minutes.

14. Process according to any one of claims 1 to 13, characterized in that: (i) the lipid solution comprises less than 50%, less than 25%, less than 20%, less than 10%, less than 5% of non-aqueous solvent, such as ethanol; (ii) the lipid solution further comprises one or more cholesterol-based lipids; (iii) the mRNA-LNPs are purified by Tangential Flow Filtration; (iv) the mRNA-LNPs have an average size of less than 150 nm, less than 100 nm, less than 80 nm, less than 60 nm or less than 40 nm, optionally wherein the mRNA-LNPs have an average size ranging from 40 to 70 nm; (v) the lipid nanoparticles have a PDI less than about 0.3, less than about 0.2, less than about 0.18, less than about 0.15, less than about 0.1; (vi) the mRNA-LNPs have an N / P ratio between 1 and 10, optionally wherein the mRNA-LNPs have an N / P ratio between Petition 870260055239, dated 08 / 06 / 2026, page.149 / 305 5 / 7 2 to 6, optionally where the mRNA-LNPs have an N / P ratio of about 4; and / or (vii) the mRNA solution and the lipid solution are mixed by a pulse-free flow pump, optionally where the pump is a gear pump, optionally where the pump is a centrifugal pump.

15. Process, according to any one of claims 1 to 14, characterized in that the encapsulation efficiency of mRNA-LNPs is greater than about 65%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%.

16. Process, according to any one of claims 1 to 15, characterized in that 5 g or more, 10 g or more, 20 g or more, 50 g or more, 100 g or more, or 1 kg or more of mRNA is encapsulated in lipid nanoparticles in a single batch.

17. Process according to any one of claims 1 to 16, characterized in that: (i) the mRNA solution is mixed at a flow rate ranging from about 150 to 250 ml / minute, 250 to 500 ml / minute, 500 to 1000 ml / minute, 1000 to 2000 ml / minute, 2000 to 3000 ml / minute, 3000 to 4000 ml / minute or 4000 to 5000 ml / minute, optionally wherein the mRNA solution is mixed at a flow rate of about 800 ml / minute, about 1000 ml / minute or about 1200 ml / minute; and / or (ii) the lipid solution is mixed at a flow rate ranging from about 25 to 75 ml / minute, about 75 to 200 ml / minute, about 200 to 350 ml / minute, about 350 to 500 ml / minute, about 500 to 650 ml / minute, about 650 to 850 ml / minute or about 850 to 1000 ml / minute, optionally wherein the lipid solution is mixed in Petition 870260055239, dated 08 / 06 / 2026, p.150 / 305 6 / 7 a flow rate of approximately 100 ml / minute, approximately 150 ml / minute, approximately 200 ml / minute, approximately 250 ml / minute, approximately 300 ml / minute, approximately 350 ml / minute, optionally wherein the flow rate of the mRNA solution is 2 times, 4 times or 6 times greater than the flow rate of the lipid solution.

18. Process, according to any one of claims 1 to 17, characterized in that the encapsulation efficiency of mRNA-LNP is at least 10% higher compared to an mRNA-LNP formed from mRNA solution mixed with lipid solution under the same conditions, except with the mRNA solution having 10 mM citrate.

19. Composition, characterized in that it comprises mRNA encapsulated in lipid nanoparticles prepared by the process as defined in any one of claims 1 to 18.

20. Composition according to claim 19, characterized in that it comprises 5 g or more, 10 g or more, 20 g or more, 50 g or more, 100 g or more, or 1 kg or more of mRNA.

21. Composition according to claim 19 or 20, characterized in that: (i) the mRNA comprises one or more modified nucleotides, or the mRNA is not modified; and / or (ii) the mRNA is larger than about 0.5 kb, 1 kb, 2 kb, 3 kb, 4 kb, 5 kb, 8 kb, 10 kb, 20 kb, 30 kb or 40 kb.

22. Use of an mRNA encapsulated in lipid nanoparticles, obtainable by the process as defined in any one of claims 1 to 18, characterized in that it is for the preparation of a composition or medicament to treat a disease in an individual requiring therapy related to the production of the protein encoded by said mRNA. Petition 870260055239, dated 08 / 06 / 2026, p. 151 / 305 7 / 7 23. Messenger RNA (mRNA) encapsulated in lipid nanoparticles (LNPs), characterized in that it is obtained by a process as defined in any one of claims 1 to 18. Petition 870260055239, dated 08 / 06 / 2026, p. 152 / 305

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