Rectal delivery of messenger RNA

Abstract

The present invention provides, inter alia, effective methods and compositions for delivering messenger RNA (mRNA) via rectal delivery. The present invention is based, in part, on the surprising observation that mRNA can be effectively delivered via rectal delivery to the circulation, liver, kidney, colon, and / or rectum despite the presence of many barriers, such as RNases and mucosal layers.

Description

[0001] Cross-reference to related applications

[0002] This application claims the benefit and priority of U.S. Serial No. 62 / 951,844, filed December 20, 2019, the contents of which are incorporated herein by reference. Background Technology

[0003] Delivering nucleic acids, especially messenger RNA (mRNA), to target cells and tissues remains a technical challenge. Various difficulties arise in delivering mRNA to target cells, including physical and chemical barriers. These difficulties are encountered using various delivery methods, such as parenteral and oral delivery routes. Rectal delivery is particularly challenging, at least in part due to the unique composition of the rectum and colon, including the presence of RNases in the rectum. Summary of the Invention

[0004] In particular, this invention provides efficient methods and compositions for delivering messenger RNA (mRNA) via rectal delivery. This invention is based in part on the surprising discovery that, despite numerous barriers such as RNases and mucosal layers, lipid-encapsulated mRNA can be efficiently delivered via mucosal delivery (including rectal delivery) to the circulation, liver, kidneys, intestine, colon, and / or rectum.

[0005] In some aspects, the present invention provides a method for delivering messenger RNA (mRNA) to a subject for the in vivo production of a protein or peptide in the subject, the method comprising administering to the subject by rectal delivery a composition comprising mRNA encoding a protein or peptide and encapsulated within lipid nanoparticles, and wherein administration of the composition results in the expression of the protein or peptide encoded by the mRNA, the protein or peptide encoded by the mRNA being detectable in the subject at least about 24 hours, about 48 hours, about 72 hours, or about 96 hours after administration.

[0006] In some embodiments, the mRNA-encoded protein or peptide can be detected in the circulation of the subject at least about 24 hours, about 48 hours, about 72 hours, or about 96 hours after administration. Therefore, in some embodiments, the mRNA-encoded protein or peptide can be detected in the circulation of the subject at least about 24 hours after administration. In some embodiments, the mRNA-encoded protein or peptide can be detected in the circulation of the subject at least about 48 hours after administration. In some embodiments, the mRNA-encoded protein or peptide can be detected in the circulation of the subject at least about 72 hours after administration. In some embodiments, the mRNA-encoded protein or peptide can be detected in the circulation of the subject at least about 96 hours after administration.

[0007] In some embodiments, the protein or peptide encoded by the mRNA can be detected in the liver of the subject at least about 24 hours, about 48 hours, about 72 hours, or about 96 hours after administration. Thus, in some embodiments, the protein or peptide encoded by the mRNA can be detected in the liver of the subject at least about 24 hours after administration. In some embodiments, the protein or peptide encoded by the mRNA can be detected in the liver of the subject at least about 48 hours after administration. In some embodiments, the protein or peptide encoded by the mRNA can be detected in the liver of the subject at least about 72 hours after administration. In some embodiments, the protein or peptide encoded by the mRNA can be detected in the liver of the subject at least about 96 hours after administration.

[0008] In some embodiments, the protein or peptide encoded by the mRNA can be detected in the kidney of the subject at least about 24 hours, about 48 hours, about 72 hours, or about 96 hours after administration. Thus, in some embodiments, the protein or peptide encoded by the mRNA can be detected in the kidney of the subject at least about 24 hours after administration. In some embodiments, the protein or peptide encoded by the mRNA can be detected in the kidney of the subject at least about 48 hours after administration. In some embodiments, the protein or peptide encoded by the mRNA can be detected in the kidney of the subject at least about 72 hours after administration. In some embodiments, the protein or peptide encoded by the mRNA can be detected in the kidney of the subject at least about 96 hours after administration.

[0009] In some embodiments, the protein or peptide encoded by the mRNA can be detected in the colon of the subject at least about 24 hours, about 48 hours, about 72 hours, or about 96 hours after administration. Thus, in some embodiments, the protein or peptide encoded by the mRNA can be detected in the colon of the subject at least about 24 hours after administration. In some embodiments, the protein or peptide encoded by the mRNA can be detected in the colon of the subject at least about 48 hours after administration. In some embodiments, the protein or peptide encoded by the mRNA can be detected in the colon of the subject at least about 72 hours after administration. In some embodiments, the protein or peptide encoded by the mRNA can be detected in the colon of the subject at least about 96 hours after administration.

[0010] In some embodiments, the protein or peptide encoded by the mRNA is detectable in the rectum of the subject at least about 24 hours, about 48 hours, about 72 hours, or about 96 hours after administration. Thus, in some embodiments, the protein or peptide encoded by the mRNA is detectable in the rectum of the subject at least about 24 hours after administration. In some embodiments, the protein or peptide encoded by the mRNA is detectable in the rectum of the subject at least about 48 hours after administration. In some embodiments, the protein or peptide encoded by the mRNA is detectable in the rectum of the subject at least about 72 hours after administration. In some embodiments, the protein or peptide encoded by the mRNA is detectable in the rectum of the subject at least about 96 hours after administration.

[0011] In some embodiments, the in vivo production of the protein or peptide is in the circulation, liver, kidney, colon, and / or rectum of the subject. Thus, in some embodiments, the in vivo production of the protein or peptide is in the circulation of the subject. In some embodiments, the in vivo production of the protein or peptide is in the liver of the subject. In some embodiments, the in vivo production of the protein or peptide is in the kidney of the subject. In some embodiments, the in vivo production of the protein or peptide is in the colon of the subject. In some embodiments, the in vivo production of the protein or peptide is in the rectum of the subject.

[0012] In some embodiments, the lipid nanoparticle comprises one or more cationic lipids, one or more non-cationic lipids, and one or more PEG-modified lipids. Thus, in some embodiments, the lipid nanoparticle comprises one or more cationic lipids. In some embodiments, the lipid nanoparticle comprises one or more non-cationic lipids. In some embodiments, the lipid nanoparticle comprises one or more PEG-modified lipids.

[0013] In some embodiments, the lipid nanoparticle comprises cholesterol.

[0014] In some embodiments, the rectal delivery is by suppository, enema, catheter, or bulb syringe. Thus, in some embodiments, the rectal delivery is by suppository. In some embodiments, the rectal delivery is by enema. In some embodiments, the rectal delivery is by catheter. In some embodiments, the rectal delivery is by bulb syringe.

[0015] In some embodiments, the rectal delivery is by suppository.

[0016] In some embodiments, the composition does not comprise a lipid-based suppository component.

[0017] In some embodiments, the lipid-based suppository component is cocoa butter, cacao oil, synthetic fat, or synthetic base. Thus, in some embodiments, the lipid-based suppository component is cocoa butter. In some embodiments, the lipid-based suppository component is cacao oil. In some embodiments, the lipid-based suppository component is synthetic fat. In some embodiments, the lipid-based suppository component is synthetic base.

[0018] In some embodiments, the composition comprises a permeation enhancer.

[0019] In some embodiments, the permeation enhancer is selected from a bile salt, a surfactant, a fatty acid and derivative, a glyceride, a chelating agent, a salicylate, or a polymer. Thus, in some embodiments, the permeation enhancer is a bile salt. In some embodiments, the permeation enhancer is a fatty acid and derivative. In some embodiments, the permeation enhancer is a glyceride. In some embodiments, the permeation enhancer is a chelating agent. In some embodiments, the permeation enhancer is a salicylate. In some embodiments, the permeation enhancer is a polymer.

[0020] In some embodiments, the fatty acid and derivative is selected from sorbitan laurate, sodium caprate, sucrose, palmitate, lauroylcholine, sodium myristate, or palmitoyl carnitine. Thus, in some embodiments, the fatty acid and derivative is sorbitan laurate. In some embodiments, the fatty acid and derivative comprises sodium caprate. In some embodiments, the fatty acid and derivative is sucrose. In some embodiments, the fatty acid and derivative is palmitate. In some embodiments, the fatty acid and derivative is lauroylcholine. In some embodiments, the fatty acid and derivative is sodium myristate. In some embodiments, the fatty acid and derivative is palmitoyl carnitine.

[0021] In some embodiments, the permeation enhancer is in the form of a caprate salt.

[0022] In some embodiments, the caprate salt-based permeation enhancer is sodium caprate.

[0023] In some embodiments, the permeation enhancer is

[0024] In some embodiments, the composition comprises a water-based suppository component.

[0025] In some embodiments, the water-based suppository component is selected from glycerol, gelatin, or polyethylene glycol (PEG), or a combination thereof. In some embodiments, the water-based suppository component is glycerol. In some embodiments, the water-based suppository component is gelatin. In some embodiments, the water-based suppository component is polyethylene glycol (PEG).

[0026] In some embodiments, the composition further comprises gelatin.

[0027] In some embodiments, the only water-based suppository component is gelatin.

[0028] In some embodiments, the composition comprises about 5% or more gelatin in water, 10% or more gelatin in water, 20% or more gelatin in water, 30% or more gelatin in water, or 50% or more gelatin in water. Thus, in some embodiments, the composition comprises about 5% or more gelatin in water. For example, in some embodiments, the composition comprises about 5%, 6%, 7%, 8%, or 9% or more gelatin. In some embodiments, the composition comprises about 10% or more gelatin in water. For example, in some embodiments, the composition comprises about 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, or 19% or more gelatin in water. In some embodiments, the composition comprises about 20% or more gelatin in water. For example, in some embodiments, the composition comprises about 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, or 29% or more gelatin in water. In some embodiments, the composition comprises about 30% or more gelatin in water. For example, in some embodiments, the composition comprises about 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, or 49% or more gelatin in water. In some embodiments, the composition comprises about 50% or more gelatin in water. For example, in some embodiments, the composition comprises about 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more gelatin in water.

[0029] In some embodiments, the composition further comprises 0.25 mg / mL or more mRNA, 0.5 mg / mL or more mRNA, 0.75 mg / mL or more mRNA, or 1 mg / mL or more mRNA. Thus, in some embodiments, the composition further comprises 0.25 mg / mL or more mRNA. In some embodiments, the composition comprises 0.5 mg / mL or more mRNA. In some embodiments, the composition comprises 0.75 mg / mL or more mRNA.

[0030] In some embodiments, the composition comprises 1 mg / mL or more mRNA.

[0031] In some embodiments, the composition comprises 0.5 mg or more mRNA, 0.75 mg or more mRNA, 1 mg or more mRNA, 1.25 mg or more mRNA, 1.5 mg or more mRNA, or 1.75 mg or more mRNA. Thus, in some embodiments, the composition comprises 0.5 mg or more mRNA. In some embodiments, the composition comprises 0.75 mg or more mRNA. In some embodiments, the composition comprises 1 mg or more mRNA. In some embodiments, the composition comprises 1.25 mg or more mRNA. In some embodiments, the composition comprises 1.5 mg or more mRNA. In some embodiments, the composition comprises 1.75 mg or more mRNA.

[0032] In some embodiments, the composition is formulated as a suppository of about 3 grams, about 2 grams, or about 1 gram. Thus, in some embodiments, the composition is formulated as a suppository of about 3 grams. In some embodiments, the composition is formulated as a suppository of about 2 grams. In some embodiments, the composition is formulated as a suppository of about 1 gram.

[0033] In some embodiments, the composition is formulated as a suppository having a volume of about 2.0 mL, about 3.5 mL, about 7.5 mL, or about 10.0 mL. Thus, in some embodiments, the composition is formulated as a suppository having a volume of about 2.0 mL. In some embodiments, the composition is formulated as a suppository having a volume of about 3.5 mL. In some embodiments, the composition is formulated as a suppository having a volume of about 7.5 mL. In some embodiments, the composition is formulated as a suppository having a volume of about 10.0 mL.

[0034] In some embodiments, the suppository is refrigerated prior to administration.

[0035] In some embodiments, the subject is first administered a penetration enhancer prior to administration of the composition comprising mRNA.

[0036] In some embodiments, the permeability enhancer is administered to the subject about 30 minutes, about 1 hour, about 2.5 hours, about 5 hours, or about 12 hours prior to administration of the composition comprising mRNA. Thus, in some embodiments, the permeability enhancer is administered to the subject about 30 minutes prior to administration of the composition comprising mRNA. In some embodiments, the permeability enhancer is administered to the subject about 1 hour prior to administration of the composition comprising mRNA. In some embodiments, the permeability enhancer is administered to the subject about 2.5 hours prior to administration of the composition comprising mRNA. In some embodiments, the permeability enhancer is administered to the subject about 5.0 hours prior to administration of the composition comprising mRNA. In some embodiments, the permeability enhancer is administered to the subject about 12 hours prior to administration of the composition comprising mRNA.

[0037] In some aspects, the present application provides a method of delivering messenger RNA (mRNA) to a subject for in vivo production of a protein or peptide in the subject, the method comprising administering to the subject a composition comprising mRNA by mucosal delivery, the mRNA encoding a protein or peptide and being encapsulated within a lipid nanoparticle, and wherein administration of the composition results in expression of the protein or peptide encoded by the mRNA, the protein or peptide encoded by the mRNA being detectable in the subject at least about 24 hours, about 48 hours, about 72 hours, or about 96 hours after administration. Thus, in some embodiments, administration of the composition results in expression of the protein or peptide encoded by the mRNA, the expression being detectable in the subject at least about 24 hours after administration. In some embodiments, administration of the composition results in expression of the protein or peptide encoded by the mRNA, the expression being detectable in the subject at least about 48 hours after administration. In some embodiments, administration of the composition results in expression of the protein or peptide encoded by the mRNA, the expression being detectable in the subject at least about 72 hours after administration. In some embodiments, administration of the composition results in expression of the protein or peptide encoded by the mRNA, the expression being detectable in the subject at least about 96 hours after administration.

[0038] In some embodiments, the mRNA can be detected in the circulation, liver, kidney, colon, and / or rectum of the subject at least about 24 hours, about 48 hours, about 72 hours, or about 96 hours after administration. Thus, in some embodiments, the mRNA can be detected in the circulation of the subject at least about 24 hours after administration. In some embodiments, the mRNA can be detected in the circulation of the subject at least about 48 hours after administration. In some embodiments, the mRNA can be detected in the circulation of the subject at least about 72 hours after administration. In some embodiments, the mRNA can be detected in the circulation of the subject at least about 96 hours after administration. In some embodiments, the mRNA can be detected in the liver of the subject at least about 24 hours after administration. In some embodiments, the mRNA can be detected in the liver of the subject at least about 48 hours after administration. In some embodiments, the mRNA can be detected in the liver of the subject at least about 72 hours after administration. In some embodiments, the mRNA can be detected in the liver of the subject at least about 96 hours after administration. In some embodiments, the mRNA can be detected in the kidney of the subject at least about 24 hours after administration. In some embodiments, the mRNA can be detected in the kidney of the subject at least about 48 hours after administration. In some embodiments, the mRNA can be detected in the kidney of the subject at least about 72 hours after administration. In some embodiments, the mRNA can be detected in the kidney of the subject at least about 96 hours after administration. In some embodiments, the mRNA can be detected in the colon of the subject at least about 24 hours after administration. In some embodiments, the mRNA can be detected in the colon of the subject at least about 48 hours after administration. In some embodiments, the mRNA can be detected in the colon of the subject at least about 72 hours after administration. In some embodiments, the mRNA can be detected in the colon of the subject at least about 96 hours after administration. In some embodiments, the mRNA can be detected in the rectum of the subject at least about 24 hours after administration. In some embodiments, the mRNA can be detected in the rectum of the subject at least about 48 hours after administration. In some embodiments, the mRNA can be detected in the rectum of the subject at least about 72 hours after administration. In some embodiments, the mRNA can be detected in the rectum of the subject at least about 96 hours after administration.

[0039] In some embodiments, the mucosal delivery is rectal, vaginal, ocular, oral, or gastrointestinal. Thus, in some embodiments, the mucosal delivery is rectal. In some embodiments, the mucosal delivery is vaginal. In some embodiments, the mucosal delivery is ocular. In some embodiments, the mucosal delivery is oral. In some embodiments, the mucosal delivery is gastrointestinal.

[0040] In some embodiments, the oral delivery is buccal or sublingual. Thus, in some embodiments, the oral delivery is buccal. In some embodiments, the oral delivery is sublingual.

[0041] In some embodiments, the in vivo production of the protein or peptide is in the circulation, liver, kidney, colon, and / or rectum of the subject. Thus, in some embodiments, the in vivo production of the protein or peptide is in the circulation of the subject. In some embodiments, the in vivo production of the protein or peptide is in the liver of the subject. In some embodiments, the in vivo production of the protein or peptide is in the kidney of the subject. In some embodiments, the in vivo production of the protein or peptide is in the colon of the subject. In some embodiments, the in vivo production of the protein or peptide is in the rectum of the subject.

[0042] In some aspects, the present application provides a suppository for rectal administration of mRNA, the suppository comprising: mRNA encapsulated within a lipid nanoparticle, wherein the mRNA encodes a protein or peptide; and gelatin.

[0043] In some embodiments, the suppository comprises about 5% or more gelatin in water, 10% or more gelatin in water, 20% or more gelatin in water, 30% or more gelatin in water, or 50% or more gelatin in water. Thus, in some embodiments, the suppository comprises about 5% or more gelatin in water. For example, in some embodiments, the suppository comprises about 5%, 6%, 7%, 8%, or 9% or more gelatin. In some embodiments, the suppository comprises about 10% or more gelatin in water. For example, in some embodiments, the suppository comprises about 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, or 19% or more gelatin in water. In some embodiments, the suppository comprises about 20% or more gelatin in water. For example, in some embodiments, the suppository comprises about 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, or 29% or more gelatin in water. In some embodiments, the suppository comprises about 30% or more gelatin in water. For example, in some embodiments, the suppository comprises about 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, or 49% or more gelatin in water. In some embodiments, the suppository comprises about 50% or more gelatin in water. For example, in some embodiments, the suppository comprises about 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more gelatin in water.

[0044] In some embodiments, the suppository does not comprise a lipid-based suppository component.

[0045] In some embodiments, the lipid-based suppository component is cocoa butter, cacao oil, synthetic fat, or synthetic base. Thus, in some embodiments, the lipid-based suppository component is cocoa butter. In some embodiments, the lipid-based suppository component is cacao oil. In some embodiments, the lipid-based suppository component is synthetic fat. In some embodiments, the lipid-based suppository component is synthetic base. In some embodiments, the lipid-based suppository component is cocoa butter, cacao oil, synthetic fat, or synthetic base, or any combination thereof.

[0046] In some embodiments, the suppository comprises a permeation enhancer.

[0047] In some embodiments, the suppository comprises a permeation enhancer selected from a bile salt, a surfactant, a fatty acid and derivative, a glyceride, a chelating agent, a salicylate, or a polymer. Thus, in some embodiments, the permeation enhancer is a bile salt. In some embodiments, the permeation enhancer is a fatty acid and derivative. In some embodiments, the permeation enhancer is a glyceride. In some embodiments, the permeation enhancer is a chelating agent. In some embodiments, the permeation enhancer is a salicylate. In some embodiments, the permeation enhancer is a polymer.

[0048] In some embodiments, the suppository comprises a fatty acid and derivative selected from sorbitan laurate, sodium caprate, sucrose, palmitate, lauroylcholine, sodium myristate, or palmitoyl carnitine. Thus, in some embodiments, the fatty acid and derivative comprises sodium caprate. In some embodiments, the fatty acid and derivative is sucrose. In some embodiments, the fatty acid and derivative is palmitate. In some embodiments, the fatty acid and derivative is lauroylcholine. In some embodiments, the fatty acid and derivative is sodium myristate. In some embodiments, the fatty acid and derivative is palmitoyl carnitine.

[0049] In some embodiments, the suppository comprises a permeation enhancer that is a form of a caprate salt.

[0050] In some embodiments, the caprate salt-based permeation enhancer is sodium caprate.

[0051] In some embodiments, the permeation enhancer is

[0052] In some embodiments, the suppository further comprises glycerol and / or PEG. Thus, in some embodiments, the suppository further comprises glycerol. In some embodiments, the suppository further comprises PEG.

[0053] In some embodiments, the suppository softens or melts at about 36°C to 37°C. Thus, in some embodiments, the suppository softens at about 36.0°C, 36.1°C, 36.2°C, 36.3°C, 36.4°C, 36.5°C, 36.6°C, 36.7°C, 36.8°C, 36.9°C, or 37.0°C.

[0054] In some embodiments, the protein or peptide encoded by the mRNA is detectable in the liver of the subject at least about 24 hours, about 48 hours, about 72 hours, or about 96 hours after administration. BRIEF DESCRIPTION OF DRAWINGS

[0055] The following drawings are for illustrative purposes only and are not meant to be limiting.

[0056] Figure 1 An exemplary suppository comprising mRNA encapsulated within a lipid nanoparticle is depicted. The suppository can be delivered rectally.

[0057] Figure 2A An exemplary imaging of a mouse is depicted 24 hours after rectal administration of saline as a negative control. Figure 2B An exemplary imaging of various tissues is depicted 24 hours after rectal administration of saline as a negative control. No signal was detected when saline was administered rectally.

[0058] Figure 3A An exemplary imaging of a mouse is depicted 24 hours after rectal administration of FFLuc mRNA-LNP. Figure 3B An exemplary imaging of various tissues is depicted 24 hours after rectal administration of FFLuc mRNA-LNP. A signal of luciferase activity was detected in the mouse. The colon showed strong luminescence.

[0059] Figure 4 An exemplary imaging of a mouse is depicted 24 hours after rectal administration of FFLuc mRNA-LNP at a 0.2 mg dose (Group 1) or a 0.05 mg dose (Group 2). The mice in Group 2 were pre-dosed with sodium decanoate prior to administration of the mRNA-LNP.

[0060] Figure 5 An exemplary plot of luminescence detected in a mouse at 24 hours after administration of saline (negative control), a 0.2 mg dose of mRNA-LNP (Group 1), or a 0.05 mg dose of mRNA-LNP and sodium decanoate (Group 2).

[0061] Figure 6A An exemplary imaging of a mouse is depicted 24 hours after rectal administration of a suppository comprising FFLuc mRNA-LNP. Figure 6B An exemplary imaging of various tissues of a mouse is depicted 24 hours after rectal administration of a suppository.

[0062] Figure 7 An exemplary plot of hEPO protein in serum detected in a rat at x hours after administration of a composition.

[0063] Definitions

[0064] To make the present application more readily understood, certain terms are first defined below. Additional definitions for the following terms and other terms are set forth throughout the specification.

[0065] The terms "or more," "at least," "more than," and the like, e.g., "at least one," are understood to include, but not be limited to, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, or 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, or more than the stated value. Any larger number or fraction in between is also included.

[0066] Conversely, the term“no more than” includes every value less than the stated value. For example,“no more than 100 nucleotides” includes 100, 99, 98, 97, 96, 95, 94, 93, 92, 91, 90, 89, 88, 87, 86, 85, 84, 83, 82, 81, 80, 79, 78, 77, 76, 75, 74, 73, 72, 71, 70, 69, 68, 67, 66, 65, 64, 63, 62, 61, 60, 59, 58, 57, 56, 55, 54, 53, 52, 51, 50, 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, and 0 nucleotides. Any smaller number or fraction in between is also included.

[0067] Animal: As used herein, the term“animal” refers to any member of the animal kingdom. In some embodiments,“animal” refers to a human at any stage of development. In some embodiments,“animal” refers to a non-human animal at any stage of development. In certain embodiments, the non-human animal is a mammal (e.g., a rodent, a mouse, a rat, a rabbit, a monkey, a dog, a cat, a sheep, a cow, a primate, and / or a pig). In some embodiments, animals include, but are not limited to, mammals, birds, reptiles, amphibians, fish, insects, and / or worms. In some embodiments, an animal can be a transgenic animal, a genetically engineered animal, and / or a clone.

[0068] Approximately or about: As used herein, the term“approximately” or“about” applied to one or more values of a specified reference value, refers to a value that is similar to the specified 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 either direction (greater than or less than) of the specified reference value, unless otherwise stated or otherwise clear from the context (except that such numerals would exceed 100% of a possible value).

[0069] Comprise: As used herein, the term“comprising” or variations such as“comprises” or“comprising,” will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.

[0070] Delivery: As used herein, the term “delivery” encompasses both local delivery and systemic delivery. For example, delivery of mRNA encompasses a case where mRNA is delivered to a target tissue and the encoded protein is expressed within that target tissue and retained (also referred to as “local distribution” or “local delivery”). Other exemplary cases include a case where mRNA is delivered to a target tissue and the encoded protein is expressed and secreted into the patient’s circulatory system (e.g., serum) and is distributed systemically and taken up by other tissues (also referred to as “systemic distribution” or “systemic delivery”). In other exemplary cases, mRNA is delivered systemically and taken up by multiple cells and tissues in the body. In some exemplary cases, the delivery is intravenous, intramuscular, or subcutaneous.

[0071] Dosing interval: As used herein, in the context of a method for treating a disease, a dosing interval is the frequency of administration of a therapeutic composition (e.g., mRNA composition) at an effective dose of mRNA in a subject (mammal) in need thereof such that one or more symptoms associated with the disease are reduced; or one or more biomarkers associated with the disease are reduced, for at least the time period of the dosing interval. In the present disclosure, dosing frequency and dosing interval can be used interchangeably.

[0072] Efficacy: As used herein, the term “efficacy” or grammatical equivalents refers to an improvement in a biologically relevant endpoint as related to the delivery of mRNA encoding a relevant protein or peptide. In some embodiments, the biological endpoint is protection from ammonium chloride challenge at some time point post administration.

[0073] Encapsulation: As used herein, the term “encapsulation” or grammatical equivalents thereof refers to the process of localizing a nucleic acid molecule within a nanoparticle.

[0074] Expression: As used herein, “expression” of a nucleic acid sequence refers to the translation of mRNA into a polypeptide, assembly of multiple polypeptides (e.g., heavy or light chains of an antibody) into an intact protein (e.g., an antibody), and / or post-translational modification of a polypeptide or fully assembled protein (e.g., an antibody). In the present application, the terms “expression” and “production” and grammatical equivalents thereof are used interchangeably.

[0075] Effective dose: As used herein, an effective dose is a dose of mRNA in a pharmaceutical composition that, when administered to a subject (hereby a mammalian subject) in need thereof according to the methods of the present application, is effective in producing the intended result in the subject, e.g., reduction of symptoms associated with a disease.

[0076] Functional: As used herein, a “functional” biological molecule is a biological molecule in a form in which it exhibits the properties and / or activities that characterize it.

[0077] Improve, increase, or decrease: As used herein, the term “improve,” “increase,” or “decrease,” or grammatical equivalents, indicates a value relative to a baseline measurement, such as a measurement in the same individual prior to initiation of the treatment described herein, or a measurement in a control subject (or subjects) in the absence of the treatment described herein. A “control subject” is a subject having the same form of disease as the subject being treated, which is approximately the same age as the subject being treated.

[0078] In vitro: As used herein, the term “in vitro” refers to events that take place in an artificial environment (e.g., in a test tube or reaction vessel, in a cell culture, etc.) and not within a multicellular organism.

[0079] In vivo: As used herein, the term “in vivo” refers to events that take place within a multicellular organism, such as humans and non-human animals. In the context of cell-based systems, the term can be used to refer to events that take place within a living cell (as opposed to, e.g., an in vitro system).

[0080] Liposome: As used herein, the term “liposome” refers to any lamellar, multilamellar, or solid nanoparticulate vesicle. Typically, liposomes as used herein can be formed by mixing one or more lipids or by mixing one or more lipids and one or more polymers. In some embodiments, liposomes suitable for use in the application contain one or more cationic lipids and optionally one or more non-cationic lipids, optionally one or more cholesterol-based lipids, and / or optionally one or more PEG-modified lipids.

[0081] Messenger RNA (mRNA): As used herein, the term “messenger RNA (mRNA)” refers to a polynucleotide that encodes at least one polypeptide. As used herein, mRNA encompasses modified and unmodified RNA. An mRNA can contain one or more coding and non-coding regions. An mRNA can be purified from a natural source, produced using a recombinant expression system and optionally purified, chemically synthesized, etc. Where appropriate, e.g., in the case of chemically synthesized molecules, an mRNA can comprise nucleotide analogs, such as analogs with chemical modifications of the base or sugar, analogs with modifications of the backbone, etc. Unless otherwise indicated, mRNA sequences are presented in the 5’ to 3’ direction.

[0082] N / P ratio: As used herein, the term “N / P ratio” refers to the molar ratio of positively charged molecular units in a cationic lipid relative to negatively charged molecular units in an mRNA encapsulated within a lipid nanoparticle. As such, the N / P ratio is typically calculated as the ratio of moles of amine groups in a cationic lipid in a lipid nanoparticle relative to moles of phosphate groups in an mRNA encapsulated within the lipid nanoparticle.

[0083] Nucleic acid: As used herein, the term "nucleic acid" refers in its broadest sense 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 phosphodiester bonds. In some embodiments, "nucleic acid" refers to an individual nucleic acid residue (e.g., a nucleotide and / or a nucleoside). 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- and / or double-stranded DNA and / or cDNA. Moreover, the terms "nucleic acid," "DNA," "RNA," and / or similar terms include nucleic acid analogs, i.e., analogs that do not have a phosphodiester backbone. For example, so-called "peptide nucleic acids," which are known in the art and have peptide bonds instead of phosphodiester bonds in the backbone, are considered within the scope of the present application. 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 that encode proteins and / or RNA can include introns. Nucleic acids can be purified from natural sources, produced using recombinant expression systems and optionally purified, chemically synthesized, etc. Where appropriate, e.g., in the case of chemically synthesized molecules, nucleic acids can comprise nucleotide analogs, such as analogs with chemically modified bases or sugars, analogs with backbone modifications, etc. Unless otherwise indicated, nucleic acid sequences are presented in the 5' to 3' direction. 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, pyrrolo-pyrimidine, 3-methyl adenosine, 5- methylcytidine, C-5 propynyl-cytidine, C-5 propynyl-uridine, 2-aminoadenosine, C5- bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynyl-uridine, C5-propynyl- cytidine, C5-methylcytidine, 2-aminoadenosine, 7-deazaadenosine, 7-deazaguanosine, 8- oxoadenosine, 8-oxoguanosine, O(6)-methylguanine, and 2-thiocytidine); chemically modified bases; biologically modified bases (e.g., methylated bases); intercalated bases; modified sugars (e.g., 2'-fluororibose, ribose, 2'-deoxyribose, arabinose, and hexose); and / or modified phosphate groups (e.g., phosphorothioates and 5'-N-phosphoramidite linkages). In some embodiments, the present application specifically relates to "unmodified nucleic acids," meaning nucleic acids (e.g., polynucleotides and residues, including nucleotides and / or nucleosides) that are not chemically modified to facilitate or enable delivery. In some embodiments, the nucleotides T and U are used interchangeably in sequence descriptions.

[0084] Patient: As used herein, the term“patient” or“subject” refers to any organism to which a provided composition can be administered, e.g., 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 particular embodiments, the patient is a human. Humans include pre- and post-natal forms.

[0085] Polypeptide: As used herein, a“polypeptide” is generally a string of at least two amino acids attached to one another by peptide bonds. In some embodiments, a polypeptide can include at least 3-5 amino acids, each attached to other amino acids by at least one peptide bond. Those of ordinary skill in the art will appreciate that a polypeptide sometimes optionally includes“non-natural” amino acids or other entities that can be integrated into the polypeptide chain.

[0086] Protein: As used herein, a“protein” in the term“therapeutic protein” refers to a polypeptide (i.e., a string of at least two amino acids attached to one another by peptide bonds). A protein can include moieties other than amino acids (e.g., can be a glycoprotein, a proteoglycan, etc.) and / or can be otherwise processed or modified. Those of ordinary skill in the art will appreciate that a“protein” can be an entire polypeptide chain produced by a cell (with or without a signal sequence), or can be a characteristic portion thereof. Those of ordinary skill will appreciate that a protein sometimes can include more than one polypeptide chain connected, e.g., by one or more disulfide bonds or otherwise associated. A polypeptide can contain l-amino acids, d-amino acids, or both, and can contain any of a variety of amino acid modifications or analogs known in the art. Useful modifications include, e.g., terminal acetylation, amidation, methylation, etc. In some embodiments, a protein can comprise natural amino acids, non-natural amino acids, synthetic amino acids, and combinations thereof. The term“peptide” is generally used to refer to a polypeptide of less than about 100 amino acids in length, less than about 50 amino acids in length, less than 20 amino acids in length, or less than 10 amino acids in length. In some embodiments, a protein is an antibody, an antibody fragment, a biologically active portion thereof, and / or a characteristic portion thereof.

[0087] Systemic distribution or delivery: As used herein, the term“systemic distribution,”“systemic delivery,” or grammatical equivalents refers to a mechanism or method of delivery or distribution that affects the entire body or entire organism. Typically, systemic distribution or delivery is accomplished via the circulatory system (e.g., blood flow) of the body. In contrast to the definition of“local distribution or delivery.”

[0088] Subject: As used herein, the term“subject” refers to a human or any non-human animal (e.g., a mouse, rat, rabbit, dog, cat, cow, pig, sheep, horse, or primate). Humans include pre- and post-natal forms. In various embodiments, the subject is a human. A subject can be a patient, which refers to a human who visits a medical provider for diagnosis or treatment of a disease. The term“subject” can be used interchangeably with“individual” or“patient” herein. A subject can have or be predisposed to a disease or disorder, but can or can not exhibit symptoms of the disease or disorder.

[0089] Substantially: As used herein, the term“substantially” refers to a qualitative condition of being largely but not wholly that which is specified. As such, a person of ordinary skill in the art will understand that biological and chemical phenomena rarely, if ever, complete and / or proceed to completion or achievement or avoid an absolute result. Thus, the term“substantially” is used herein to capture the potential lack of completion inherent in many biological and chemical phenomena.

[0090] Target tissue: As used herein, the term“target tissue” refers to any tissue affected by a disease to be treated. In some embodiments, target tissues include those tissues exhibiting a pathology, symptom, or characteristic associated with a disease.

[0091] Therapeutically effective amount: As used herein, the term“therapeutically effective amount” of a therapeutic agent means an amount that, when administered to a subject suffering from or susceptible to a disease, disorder, and / or condition, is sufficient to treat, diagnose, prevent, and / or delay the onset of one or more symptoms of the disease, disorder, and / or condition. A person of ordinary skill in the art will appreciate that a therapeutically effective amount is typically administered via a dosing regimen comprising at least one unit dose.

[0092] Treat: As used herein, the terms“treat,”“treatment,” or“treating” refer to any method used to partially or completely alleviate, ameliorate, relieve, inhibit, prevent, delay onset of, reduce severity of, and / or reduce incidence of one or more symptoms or features of a particular disease, disorder, and / or condition. Treatment can be administered to a subject who does not exhibit signs and / or symptoms of a disease for the purpose of decreasing the risk of developing a pathology associated with the disease.

[0093] Unless defined otherwise, all technical and scientific terms and any acronyms used herein have the same meanings as commonly understood by one of ordinary skill in the art in the field of the application, or any acronyms have the same meaning as those set forth in the American Society for Microbiology’s Glossary of Microbiology (1996, American Society for Microbiology, Washington, D.C.) or the Glossary of Biochemistry and Molecular Biology (1997, Oxford University Press, Oxford, U.K.); and the practice of the application employs, unless otherwise indicated, conventional methods of the field to which the application pertains. In the case of conflict between the present specification and the glossaries, the present specification shall control. DETAILED DESCRIPTION

[0094] The present invention provides, among other things, effective methods and compositions for delivering messenger RNA (mRNA) and / or its protein or polypeptide product to a subject via a mucosal route, such as by rectal delivery. The present invention is based, in part, on the surprising finding that mRNA and / or its protein or polypeptide product can be effectively delivered to the circulation, liver, kidney, colon, and / or rectum of a subject via rectal delivery, despite the presence of numerous chemical and physical barriers.

[0095] Various aspects of the present invention are described in detail in the following sections. The use of sections is not meant to limit the present invention. Each section can apply to any aspect of the present invention. In this application, the use of “or” means “and / or” unless otherwise stated.

[0096] Mucosal delivery of lipid encapsulated mRNA

[0097] The present invention provides various methods of delivering mRNA to a target tissue. These methods of delivery include administering lipid encapsulated mRNA across any mucosal tissue. For example, lipid encapsulated mRNA is delivered via rectal, vaginal, ocular, oral, and / or gastrointestinal routes.

[0098] In some aspects, the present invention provides, among other things, a method of delivering messenger RNA (mRNA) to a subject for in vivo production of a protein or peptide in the subject, the method comprising administering to the subject by mucosal delivery a composition comprising mRNA that encodes a protein or peptide and is encapsulated within a lipid nanoparticle, and wherein administration of the composition results in expression of the protein or peptide encoded by the mRNA, the protein or peptide encoded by the mRNA being detectable in the subject at least about 24 hours, about 48 hours, about 72 hours, or about 96 hours after administration.

[0099] In some embodiments, mucosal delivery of lipid encapsulated mRNA is performed via the rectum.

[0100] Rectal delivery

[0101] In some embodiments, the present invention provides a method for rectal delivery of lipid encapsulated mRNA encoding a protein or peptide of interest.

[0102] Advantages of rectal delivery include ease of administration, allowing the patient to remain in the home environment. Rectal delivery does not require the sterile pharmaceutical special preparations needed for intravenous administration and the hospital setting. Therapeutic compositions can be administered rectally via suppositories, enemas, syringes, and catheters. Rectal administration using a specialized rectal catheter can be performed by a clinician in the home. A variety of oral forms of medication can be crushed and suspended in water to be administered via a rectal catheter. Thus, rectal administration is especially safe and convenient for infants and the elderly, and can be used for patients who are averse to needles or have any issues with digestive tract motility that interfere with passage of a drug through the digestive tract (such as difficulty swallowing, ileus, or bowel obstruction). Furthermore, drugs administered rectally generally have a faster onset of action, higher bioavailability, and are less likely to cause nausea compared to oral drug administration. Drugs administered rectally bypass about two-thirds of first-pass metabolism, resulting in less alteration of the drug in the patient’s circulation and higher concentrations. However, delivery of mRNA via the rectal route is extremely challenging. The rectal region (i.e., the rectum and colon) has a high amount of RNases that would immediately degrade mRNA. Furthermore, the mucus layer in the rectum and / or colon would act as an absorption barrier. In addition, fecal impaction can hinder rectal delivery of a drug.

[0103] Despite these challenges, the methods provided herein allow for delivery of messenger RNA (mRNA) via the rectal route. The present invention is based in part on the surprising finding that lipid-encapsulated mRNA can be effectively delivered to the circulation, liver, kidney, colon, and / or rectum of a subject via the rectal route despite the presence of many barriers, such as the presence of RNases, mucus layer, and fecal impaction. Such a non-invasive route of delivery unexpectedly provides an effective way to conveniently deliver lipid-encapsulated therapeutic compositions.

[0104] The present invention provides, among other things, a method of delivering messenger RNA (mRNA) to a subject for in vivo production of a protein or peptide in the subject, the method comprising administering to the subject by rectal delivery a composition comprising mRNA that encodes a protein or peptide and is encapsulated within a lipid nanoparticle, and wherein the administration of the composition results in expression of the protein or peptide encoded by the mRNA, the protein or peptide encoded by the mRNA being detectable in the subject at least about 24 hours, about 48 hours, about 72 hours, or about 96 hours after administration. The method allows for delivery of lipid-encapsulated mRNA via the rectal route, which results in expression of the protein or peptide encoded by the mRNA in various tissues of the recipient subject. For example, the method allows for expression of the protein or peptide encoded by the mRNA in the liver, kidney, circulation, colon, or rectum of the subject.

[0105] The methods described herein are suitable for administering lipid- encapsulated mRNA in a home environment. In some embodiments, rectal delivery is by suppository, enema, catheter, or a bulb syringe. In some embodiments, rectal delivery is by suppository. In some embodiments, rectal delivery is by enema. In some embodiments, rectal delivery is by catheter. Various specialized catheters can be used with the methods disclosed herein, for example, one such specialized catheter is the Macy Catheter. In some embodiments, rectal delivery is by bulb syringe.

[0106] In some embodiments, the compositions of the present application are delivered to various target tissues of a subject. Thus, the present application can be used as a non-invasive means of facilitating delivery of a desired protein or peptide and / or production of a protein or peptide encoded thereby at a target tissue. The methods and compositions described herein can be used to manage and treat a large number of diseases caused by deficiencies in secreted and non-secreted proteins and / or enzymes.

[0107] In some embodiments, rectal delivery of lipid-encapsulated mRNA results in production of a desired protein or peptide encoded by the mRNA in the circulation, liver, kidney, colon, rectum, heart, and / or spleen. Thus, in some embodiments, lipid-encapsulated mRNA results in production of a desired protein or peptide encoded by the mRNA in the circulation. In some embodiments, lipid-encapsulated mRNA results in production of a desired protein or peptide encoded by the mRNA in the liver. In some embodiments, lipid-encapsulated mRNA results in production of a desired protein or peptide encoded by the mRNA in the kidney. In some embodiments, lipid-encapsulated mRNA results in production of a desired protein or peptide encoded by the mRNA in the colon. In some embodiments, lipid-encapsulated mRNA results in production of a desired protein or peptide encoded by the mRNA in the rectum. In some embodiments, lipid-encapsulated mRNA results in production of a desired protein or peptide encoded by the mRNA in the heart. In some embodiments, lipid-encapsulated mRNA results in production of a desired protein or peptide encoded by the mRNA in the spleen.

[0108] In some embodiments, rectal delivery of the lipid encapsulated mRNA results in production of the desired protein or peptide encoded by the mRNA in the circulation, liver, kidney, colon, rectum, heart, and / or spleen. In some embodiments, the protein or peptide encoded by the mRNA is produced in the subject at least about 6 hours, about 12 hours, about 18 hours, about 24 hours, about 36 hours, about 48 hours, about 60 hours, about 72 hours, about 96 hours, about 120 hours, about 144 hours, or about 168 hours after administration. In some embodiments, the protein or peptide encoded by the mRNA is produced in the subject at least about 6 hours after administration. In some embodiments, the protein or peptide encoded by the mRNA is produced in the subject at least about 12 hours after administration. In some embodiments, the protein or peptide encoded by the mRNA is produced in the subject at least about 18 hours after administration. In some embodiments, the protein or peptide encoded by the mRNA is produced in the subject at least about 24 hours after administration. In some embodiments, the protein or peptide encoded by the mRNA is produced in the subject at least about 36 hours after administration. In some embodiments, the protein or peptide encoded by the mRNA is produced in the subject at least about 48 hours after administration. In some embodiments, the protein or peptide encoded by the mRNA is produced in the subject at least about 60 hours after administration. In some embodiments, the protein or peptide encoded by the mRNA is produced in the subject at least about 72 hours after administration. In some embodiments, the protein or peptide encoded by the mRNA is produced in the subject at least about 96 hours after administration. In some embodiments, the protein or peptide encoded by the mRNA is produced in the subject at least about 120 hours after administration. In some embodiments, the protein or peptide encoded by the mRNA is produced in the subject at least about 144 hours after administration. In some embodiments, the protein or peptide encoded by the mRNA is produced in the subject at least about 168 hours after administration.

[0109] In some embodiments, the protein or peptide encoded by the mRNA is produced in the subject at least about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, or 10 days after administration. In some embodiments, the protein or peptide encoded by the mRNA is produced in the subject at least about 1 day after administration. In some embodiments, the protein or peptide encoded by the mRNA is produced in the subject at least about 2 days after administration. In some embodiments, the protein or peptide encoded by the mRNA is produced in the subject at least about 3 days after administration. In some embodiments, the protein or peptide encoded by the mRNA is produced in the subject at least about 4 days after administration. In some embodiments, the protein or peptide encoded by the mRNA is produced in the subject at least about 5 days after administration. In some embodiments, the protein or peptide encoded by the mRNA is produced in the subject at least about 6 days after administration. In some embodiments, the protein or peptide encoded by the mRNA is produced in the subject at least about 7 days after administration. In some embodiments, the protein or peptide encoded by the mRNA is produced in the subject at least about 8 days after administration. In some embodiments, the protein or peptide encoded by the mRNA is produced in the subject at least about 9 days after administration. In some embodiments, the protein or peptide encoded by the mRNA is produced in the subject at least about 10 days after administration.

[0110] In some embodiments, rectal delivery of the lipid encapsulated mRNA results in detection of the desired protein or peptide encoded by the mRNA in the circulation, liver, kidney, colon, rectum, heart, and / or spleen. Thus, in some embodiments, rectal delivery of the lipid encapsulated mRNA results in detection of the desired protein or peptide encoded by the mRNA in the circulation. In some embodiments, rectal delivery of the lipid encapsulated mRNA results in detection of the desired protein or peptide encoded by the mRNA in the liver. In some embodiments, rectal delivery of the lipid encapsulated mRNA results in detection of the desired protein or peptide encoded by the mRNA in the kidney. In some embodiments, rectal delivery of the lipid encapsulated mRNA results in detection of the desired protein or peptide encoded by the mRNA in the colon. In some embodiments, rectal delivery of the lipid encapsulated mRNA results in detection of the desired protein or peptide encoded by the mRNA in the rectum. In some embodiments, rectal delivery of the lipid encapsulated mRNA results in detection of the desired protein or peptide encoded by the mRNA in the heart. In some embodiments, rectal delivery of the lipid encapsulated mRNA results in detection of the desired protein or peptide encoded by the mRNA in the spleen.

[0111] In some embodiments, the protein or peptide encoded by the mRNA can be detectable in the subject at least about 6 hours, about 12 hours, about 18 hours, about 24 hours, about 36 hours, about 48 hours, about 60 hours, about 72 hours, about 96 hours, about 120 hours, about 144 hours, or about 168 hours after administration. In some embodiments, the protein or peptide encoded by the mRNA can be detectable in the subject at least about 6 hours after administration. In some embodiments, the protein or peptide encoded by the mRNA can be detectable in the subject at least about 12 hours after administration. In some embodiments, the protein or peptide encoded by the mRNA can be detectable in the subject at least about 18 hours after administration.

[0112] In some embodiments, the protein or peptide encoded by the mRNA can be detectable in the subject at least about 24 hours after administration. In some embodiments, the protein or peptide encoded by the mRNA can be detectable in the subject at least about 36 hours after administration. In some embodiments, the protein or peptide encoded by the mRNA can be detectable in the subject at least about 48 hours after administration. In some embodiments, the protein or peptide encoded by the mRNA can be detectable in the subject at least about 60 hours after administration.

[0113] In some embodiments, the protein or peptide encoded by the mRNA can be detectable in the subject at least about 72 hours after administration. In some embodiments, the protein or peptide encoded by the mRNA can be detectable in the subject at least about 96 hours after administration. In some embodiments, the protein or peptide encoded by the mRNA can be detectable in the subject at least about 120 hours after administration. In some embodiments, the protein or peptide encoded by the mRNA can be detectable in the subject at least about 144 hours after administration. In some embodiments, the protein or peptide encoded by the mRNA can be detectable in the subject at least about 168 hours after administration.

[0114] In some embodiments, the protein or peptide encoded by the mRNA is detectable in the subject at least about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, or 10 days after administration. In some embodiments, the protein or peptide encoded by the mRNA is detectable in the subject at least about 1 day after administration. In some embodiments, the protein or peptide encoded by the mRNA is detectable in the subject at least about 2 days after administration. In some embodiments, the protein or peptide encoded by the mRNA is detectable in the subject at least about 3 days after administration. In some embodiments, the protein or peptide encoded by the mRNA is detectable in the subject at least about 4 days after administration. In some embodiments, the protein or peptide encoded by the mRNA is detectable in the subject at least about 5 days after administration. In some embodiments, the protein or peptide encoded by the mRNA is detectable in the subject at least about 6 days after administration. In some embodiments, the protein or peptide encoded by the mRNA is detectable in the subject at least about 7 days after administration. In some embodiments, the protein or peptide encoded by the mRNA is detectable in the subject at least about 8 days after administration. In some embodiments, the protein or peptide encoded by the mRNA is detectable in the subject at least about 9 days after administration. In some embodiments, the protein or peptide encoded by the mRNA is detectable in the subject at least about 10 days after administration.

[0115] In some embodiments, the protein or peptide encoded by the mRNA is detectable in the circulation, liver, kidney, colon, and / or rectum of the subject at least about 6 hours, about 12 hours, about 18 hours, about 24 hours, about 36 hours, about 48 hours, about 60 hours, about 72 hours, about 96 hours, or about 120 hours after administration. In some embodiments, the protein or peptide encoded by the mRNA is detectable in the circulation, liver, kidney, colon, and / or rectum of the subject at least about 6 hours after administration. In some embodiments, the protein or peptide encoded by the mRNA is detectable in the circulation, liver, kidney, colon, and / or rectum of the subject at least about 12 hours after administration. In some embodiments, the protein or peptide encoded by the mRNA is detectable in the circulation, liver, kidney, colon, and / or rectum of the subject at least about 18 hours after administration. In some embodiments, the protein or peptide encoded by the mRNA is detectable in the circulation, liver, kidney, colon, and / or rectum of the subject at least about 24 hours after administration. In some embodiments, the protein or peptide encoded by the mRNA is detectable in the circulation, liver, kidney, colon, and / or rectum of the subject at least about 36 hours after administration. In some embodiments, the protein or peptide encoded by the mRNA is detectable in the circulation, liver, kidney, colon, and / or rectum of the subject at least about 48 hours after administration. In some embodiments, the protein or peptide encoded by the mRNA is detectable in the circulation, liver, kidney, colon, and / or rectum of the subject at least about 60 hours after administration. In some embodiments, the protein or peptide encoded by the mRNA is detectable in the circulation, liver, kidney, colon, and / or rectum of the subject at least about 72 hours after administration. In some embodiments, the protein or peptide encoded by the mRNA is detectable in the circulation, liver, kidney, colon, and / or rectum of the subject at least about 96 hours after administration. In some embodiments, the protein or peptide encoded by the mRNA is detectable in the circulation, liver, kidney, colon, and / or rectum of the subject at least about 120 hours after administration.

[0116] In some embodiments, the protein or peptide encoded by the mRNA can be detected in the circulation, liver, kidney, colon, and / or rectum of the subject at least about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, or 10 days after administration. In some embodiments, the protein or peptide encoded by the mRNA can be detected in the circulation, liver, kidney, colon, and / or rectum of the subject at least about 1 day after administration. In some embodiments, the protein or peptide encoded by the mRNA can be detected in the circulation, liver, kidney, colon, and / or rectum of the subject at least about 2 days after administration. In some embodiments, the protein or peptide encoded by the mRNA can be detected in the circulation, liver, kidney, colon, and / or rectum of the subject at least about 3 days after administration. In some embodiments, the protein or peptide encoded by the mRNA can be detected in the circulation, liver, kidney, colon, and / or rectum of the subject at least about 4 days after administration. In some embodiments, the protein or peptide encoded by the mRNA can be detected in the circulation, liver, kidney, colon, and / or rectum of the subject at least about 5 days after administration. In some embodiments, the protein or peptide encoded by the mRNA can be detected in the circulation, liver, kidney, colon, and / or rectum of the subject at least about 6 days after administration. In some embodiments, the protein or peptide encoded by the mRNA can be detected in the circulation, liver, kidney, colon, and / or rectum of the subject at least about 7 days after administration. In some embodiments, the protein or peptide encoded by the mRNA can be detected in the circulation, liver, kidney, colon, and / or rectum of the subject at least about 8 days after administration. In some embodiments, the protein or peptide encoded by the mRNA can be detected in the circulation, liver, kidney, colon, and / or rectum of the subject at least about 9 days after administration. In some embodiments, the protein or peptide encoded by the mRNA can be detected in the circulation, liver, kidney, colon, and / or rectum of the subject at least about 10 days after administration.

[0117] In some embodiments, the lipid-encapsulated mRNA is capable of being transported to the systemic blood supply and subsequently to different cells or target tissues after rectal delivery (e.g., moved intact by active or passive means).

[0118] Accordingly, in some aspects, the present application provides a method of delivering messenger RNA (mRNA) to a subject for in vivo production of a protein or peptide in the subject, the method comprising administering to the subject by mucosal delivery a composition comprising mRNA, the mRNA encoding a protein or peptide and encapsulated within a lipid nanoparticle, and wherein the mRNA is detectable in the circulation, liver, kidney, colon, and / or rectum of the subject at least about 24 hours, about 48 hours, about 72 hours, or about 96 hours after administration. In some embodiments, in vivo production of the protein or peptide occurs in the circulation, liver, kidney, colon, and / or rectum of the subject. Accordingly, in some embodiments, in vivo production of the rectally delivered, lipid-encapsulated mRNA occurs in the circulation of the subject. In some embodiments, in vivo production of the rectally delivered, lipid-encapsulated mRNA occurs in the liver of the subject. In some embodiments, in vivo production of the rectally delivered, lipid-encapsulated mRNA occurs in the kidney of the subject. In some embodiments, in vivo production of the rectally delivered, lipid-encapsulated mRNA occurs in the colon of the subject. In some embodiments, in vivo production of the rectally delivered, lipid-encapsulated mRNA occurs in the rectum of the subject.

[0119] In some embodiments, the mRNA is detectable in the circulation, liver, kidney, colon, rectum, heart, and / or spleen of the subject. In some embodiments, the mRNA is detectable in the circulation of the subject. In some embodiments, the mRNA is detectable in the liver of the subject. In some embodiments, the mRNA is detectable in the kidney of the subject. In some embodiments, the mRNA is detectable in the colon of the subject. In some embodiments, the mRNA is detectable in the rectum of the subject. In some embodiments, the mRNA is detectable in the heart of the subject. In some embodiments, the mRNA is detectable in the spleen of the subject.

[0120] In some embodiments, the mRNA can be detected in the subject at least about 6 hours, about 12 hours, about 18 hours, about 24 hours, about 36 hours, about 48 hours, about 60 hours, about 72 hours, about 96 hours, or about 120 hours after administration. In some embodiments, the mRNA can be detected in the subject at least about 6 hours after administration. In some embodiments, the mRNA can be detected in the subject at least about 12 hours after administration. In some embodiments, the mRNA can be detected in the subject at least about 18 hours after administration. In some embodiments, the mRNA can be detected in the subject at least about 24 hours after administration. In some embodiments, the mRNA can be detected in the subject at least about 36 hours after administration. In some embodiments, the mRNA can be detected in the subject at least about 48 hours after administration. In some embodiments, the mRNA can be detected in the subject at least about 60 hours after administration. In some embodiments, the mRNA can be detected in the subject at least about 72 hours after administration. In some embodiments, the mRNA can be detected in the subject at least about 96 hours after administration. In some embodiments, the mRNA can be detected in the subject at least about 120 hours after administration.

[0121] In some embodiments, the mRNA can be detected in the subject at least about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, or 10 days after administration. In some embodiments, the mRNA can be detected in the subject at least about 1 day after administration. In some embodiments, the mRNA can be detected in the subject at least about 2 days after administration. In some embodiments, the mRNA can be detected in the subject at least about 3 days after administration. In some embodiments, the mRNA can be detected in the subject at least about 4 days after administration. In some embodiments, the mRNA can be detected in the subject at least about 5 days after administration. In some embodiments, the mRNA can be detected in the subject at least about 6 days after administration. In some embodiments, the mRNA can be detected in the subject at least about 7 days after administration. In some embodiments, the mRNA can be detected in the subject at least about 8 days after administration. In some embodiments, the mRNA can be detected in the subject at least about 9 days after administration. In some embodiments, the mRNA can be detected in the subject at least about 10 days after administration.

[0122] Composition of the formulation

[0123] The present invention provides, inter alia, effective compositions for delivering messenger RNA (mRNA) via rectal delivery. The compositions described herein are suitable for delivering mRNA via mucosal tissue, such as via the rectum.

[0124] In some embodiments, the composition comprises mRNA encoding a protein or peptide, encapsulated within a lipid nanoparticle. In some embodiments, the composition further comprises a suppository component. In some embodiments, the composition further comprises a penetration enhancer.

[0125] suppository

[0126] Compositions for rectal or vaginal (e.g., transvaginal) administration are typically suppositories, which can be prepared by mixing the composition with suitable non-irritating excipients such as cocoa butter, polymeric, hydrogel, glycerin, gelatin, polyethylene glycol, or suppository wax, which are solid at ambient temperature but liquid at body temperature and therefore melt in the rectum or vaginal cavity and release the active component. The type of material used depends on the type of suppository, the type of drug, and the storage conditions of the suppository.

[0127] The present disclosure provides, among other things, a suppository for effective delivery of mRNA encapsulated in a lipid nanoparticle via a mucosal route, such as, for example, rectal, vaginal, ocular, oral, and / or gastrointestinal routes. In some embodiments, the lipid-encapsulated mRNA is delivered via a rectal or vaginal route. The suppositories described herein comprising a lipid nanoparticle and mRNA are solid at room temperature and melt once administered rectally or vaginally. The suppository melts once placed in the rectum or vagina, which allows for effective release of the mRNA-loaded lipid nanoparticle.

[0128] In some embodiments, the suppository is refrigerated prior to administration.

[0129] In some embodiments, the suppository softens or melts at about 30°C to 42°C. In some embodiments, the suppository softens or melts at about 32°C to 40°C. In some embodiments, the suppository softens or melts at about 34°C to 38°C. In some embodiments, the suppository softens or melts at about 36°C to 37°C. In some embodiments, the suppository softens or melts at about 36°C. In some embodiments, the suppository softens or melts at about 37°C.

[0130] In some embodiments, the suppository softens or melts within 30 minutes once administered to a subject. In some embodiments, the suppository softens or melts within 20 minutes once administered to a subject. In some embodiments, the suppository softens or melts within 15 minutes once administered to a subject. In some embodiments, the suppository softens or melts within 10 minutes once administered to a subject. In some embodiments, the suppository softens or melts within 5 minutes once administered to a subject. In some embodiments, the suppository softens or melts within 3 minutes once administered to a subject. In some embodiments, the suppository softens or melts within 1 minute once administered to a subject.

[0131] As non-limiting examples, formulations for rectal and / or vaginal administration can be prepared by mixing the drug with suitable non-irritating excipients which are solids at room temperature but liquid at rectal temperature and which are thereby administered in the rectum or vagina to melt to release the drug. Such materials include cocoa butter and polyethylene glycol.

[0132] Pharmaceutical compositions for rectal or vaginal administration can include at least one inactive ingredient. Any or none of the inactive ingredients used can be approved by the United States Food and Drug Administration (FDA). A non-exhaustive list of inactive ingredients for use in pharmaceutical compositions for rectal or vaginal administration includes methyl cellulose, hydroxypropyl methyl cellulose, hydroxymethyl cellulose, poloxamer, polyvinyl alcohol, polyvinylpyrrolidone, polyacrylamide, polyethylene oxide, modified starch, adipic acid, denatured alcohol, allantoin, anhydrous lactose, apricot kernel oil peg-6 esters, barium sulfate, beeswax, bentonite, benzoic acid, benzyl alcohol, butylated hydroxyanisole, butylated hydroxytoluene, calcium lactate, carbomer 934, carbomer 934p, cellulose, microcrystalline, ceteareth-20, cetearyl alcohol, cetyl alcohol, cetyl esters wax, cetyl palmitate, cholecalciferol, choleth, citric acid, citric acid monohydrate, coconut oil / hydrogenated palm kernel oil glycerides, crospovidone, edetate disodium, ethyl cellulose, ethylene-vinyl acetate copolymer (28% vinyl acetate), ethylene-vinyl acetate copolymer (9% vinyl acetate), fatty alcohols, fd&c yellow 5, gelatin, glutamic acid, dl-glycerin, glyceryl isostearate, glyceryl monostearate, glyceryl stearate, guar gum, high-density polyethylene, hydrogel polymers, hydrogenated palm oil, hypromellose 2208 (15000 mpa.s), hydroxypropyl methylcellulose, isopropyl myristate, lactic acid, lactic acid, dl-lactose, lactose monohydrate, lactose, lactol, lauromacrogol, lauromacrogol, lecithin, lecithin, soybean, light mineral oil, magnesium aluminate silicate, magnesium aluminate silicate hydrate, magnesium stearate, methyl stearate, methyl parahydroxybenzoate, microcrystalline wax, mineral oil, nitric acid, octyldodecanol, peanut oil, peg 6-32 stearate / glycol stearate, peg-100 stearate, peg-120 glyceryl stearate, peg-2 stearate, peg-5 oleate, polyethylene glycol 7 stearate, white petrolatum, phenylmercuric acetate, phospholipon 90g, phosphoric acid, piperazine hexahydrate, dimethylvinyl or dimethylhydroxyl or trimethyl terminated poly(dimethylsiloxane / methylvinylsiloxane / methylhydrogenosiloxane), polycarbophil, polyester, polyethylene glycol 1000, polyethylene glycol 3350, polyethylene glycol 400, polyethylene glycol 4000, polyethylene glycol 6000, polyethylene glycol 8000, polyglyceryl-3 oleate, polyglyceryl-4 oleate, polyoxyl palmitate, polysorbate 20, polysorbate 60, polysorbate 80, polyurethane, potassium alum, potassium hydroxide, povidone k29 / 32, povidone, promulgen d, propylene glycol, propylene glycol monopalmitostearate, propyl parahydroxybenzoate, cis-quaternary ammonium salt-15, silicon dioxide, colloidal silicon dioxide, silicone, sodium bicarbonate, sodium citrate, sodium hydroxide, sodium lauryl sulfate, sodium metabisulfite, anhydrous disodium hydrogen phosphate, anhydrous sodium hydrogen phosphate, sorbic acid, sorbitan monostearate, sorbitol, sorbitol solution, spermaceti, stannous 2-ethylhexanoate, starch, pregelatinized starch 1500, corn starch, stearoyl amidoethyldiethylamine, stearic acid, stearyl alcohol, tartaric acid, dl-tert-butyl hydroquinone, tetrapropyl orthosilicate, triethanolamine, urea, hydrogenated vegetable oil, wecobee fs, white ozokerite, and white wax.

[0133] In some embodiments, a gelatin water system is used in the formulation to keep the lipid nanoparticles intact. The gelatin water solution has mucoadhesive properties and can help the suppository to come in contact with the mucus. Thus, the presence of gelatin will help the mRNA-loaded lipid nanoparticles to be transported into the systemic circulation. Furthermore, the gelatin solution is a gel at room temperature, which in turn prevents the mRNA-loaded nanoparticles from dripping out of the rectum. Gelatin further gradually melts at physiological temperature, enabling the mRNA-loaded lipid nanoparticles to come in contact with the mucosa.

[0134] In some embodiments, the suppository comprises about 1% or more gelatin in water, 3% or more gelatin in water, 5% or more gelatin in water, 10% or more gelatin in water, 15% or more gelatin in water, 20% or more gelatin in water, 30% or more gelatin in water, 40% or more gelatin in water, 50% or more gelatin in water, 60% or more gelatin in water, 70% or more gelatin in water, 80% or more gelatin in water, 90% or more gelatin in water. In some embodiments, the suppository comprises about 1% or more gelatin in water. In some embodiments, the suppository comprises about 3% or more gelatin in water. In some embodiments, the suppository comprises about 5% or more gelatin in water. In some embodiments, the suppository comprises about 10% or more gelatin in water. In some embodiments, the suppository comprises about 15% or more gelatin in water. In some embodiments, the suppository comprises about 20% or more gelatin in water. In some embodiments, the suppository comprises about 30% or more gelatin in water. In some embodiments, the suppository comprises about 40% or more gelatin in water. In some embodiments, the suppository comprises about 50% or more gelatin in water. In some embodiments, the suppository comprises about 60% or more gelatin in water. In some embodiments, the suppository comprises about 70% or more gelatin in water. In some embodiments, the suppository comprises about 80% or more gelatin in water. In some embodiments, the suppository comprises about 90% or more gelatin in water.

[0135] In some embodiments, the suppository does not adversely affect the integrity of the lipid nanoparticle.

[0136] In some embodiments, the composition does not comprise a lipid-based suppository component. In some embodiments, the composition comprises a lipid-based suppository component. In some embodiments, the lipid-based suppository component is cocoa butter, cacao oil, synthetic fat, or synthetic base. In some embodiments, the lipid-based suppository component is cocoa butter, cacao oil, synthetic fat, or synthetic base. In some embodiments, the lipid-based suppository component is cocoa butter. In some embodiments, the lipid-based suppository component is cacao oil. In some embodiments, the lipid-based suppository component is synthetic fat. In some embodiments, the lipid-based suppository component is synthetic base.

[0137] In some embodiments, the composition comprises a water-based suppository component. In some embodiments, the water-based suppository component is selected from glycerol, gelatin, or polyethylene glycol (PEG), or a combination thereof. In some embodiments, the water-based suppository component is glycerol. In some embodiments, the water-based suppository component is gelatin. In some embodiments, the water-based suppository component is polyethylene glycol (PEG). In some embodiments, the only water-based suppository component is gelatin.

[0138] In some embodiments, the suppository comprises glycerol and / or PEG. In some embodiments, the suppository comprises glycerol. In some embodiments, the suppository comprises PEG. In some embodiments, the suppository comprises less than about 10% glycerol. In some embodiments, the suppository comprises less than about 8% glycerol. In some embodiments, the suppository comprises less than about 6% glycerol. In some embodiments, the suppository comprises less than about 4% glycerol. In some embodiments, the suppository comprises less than about 2% glycerol. In some embodiments, the suppository comprises less than about 1% glycerol. In some embodiments, the suppository comprises less than about 0.1% glycerol. In some embodiments, the suppository comprises less than about 10% PEG. In some embodiments, the suppository comprises less than about 8% PEG. In some embodiments, the suppository comprises less than about 6% PEG. In some embodiments, the suppository comprises less than about 4% PEG. In some embodiments, the suppository comprises less than about 2% PEG. In some embodiments, the suppository comprises less than about 1% PEG. In some embodiments, the suppository comprises less than about 0.1% PEG.

[0139] In some embodiments, the suppository further comprises propylene glycol. In some embodiments, the amount of propylene glycol present in the suppository does not disrupt the lipid nanoparticles. In some embodiments, the suppository does not comprise propylene glycol. In some embodiments, the suppository comprises less than about 30% propylene glycol. In some embodiments, the suppository comprises less than about 20% propylene glycol. In some embodiments, the suppository comprises less than about 15% propylene glycol. In some embodiments, the suppository comprises less than about 10% propylene glycol. In some embodiments, the suppository comprises less than about 8% propylene glycol. In some embodiments, the suppository comprises less than about 6% propylene glycol. In some embodiments, the suppository comprises less than about 4% propylene glycol. In some embodiments, the suppository comprises less than about 2% propylene glycol. In some embodiments, the suppository comprises less than about 1% propylene glycol. In some embodiments, the suppository comprises less than about 0.5% propylene glycol. In some embodiments, the suppository comprises less than about 0.1% propylene glycol.

[0140] The present application provides, among other things, a suppository for rectal administration of mRNA. In some embodiments, the suppository comprises mRNA encapsulated within a lipid nanoparticle, wherein the mRNA encodes a protein or peptide and gelatin.

[0141] The suppositories described herein are formulated to contain various concentrations of mRNA and are small enough to allow for convenient and non-invasive administration via rectal or vaginal delivery. This non-invasive delivery route unexpectedly provides an efficient and convenient way to deliver therapeutic compositions.

[0142] In some embodiments, the composition contains 0.25 mg / mL or higher of mRNA, 0.5 mg / mL or higher of mRNA, 0.75 mg / mL or higher of mRNA, or 1 mg / mL or higher of mRNA. In some embodiments, the composition contains 0.1 mg / mL or higher of mRNA. In some embodiments, the composition contains 0.25 mg / mL or higher of mRNA. In some embodiments, the composition contains 0.5 mg / mL or higher of mRNA. In some embodiments, the composition contains 0.75 mg / mL or higher of mRNA. In some embodiments, the composition contains 1 mg / mL or higher of mRNA. In some embodiments, the composition contains 2 mg / mL or higher of mRNA. In some embodiments, the composition contains 2.5 mg / mL or higher of mRNA. In some embodiments, the composition contains 5 mg / mL or higher of mRNA.

[0143] In some embodiments, the composition comprises 0.5 mg or more mRNA, 0.75 mg or more mRNA, 1 mg or more mRNA, 1.25 mg or more mRNA, 1.5 mg or more mRNA, or 1.75 mg or more mRNA. In some embodiments, the composition comprises 0.1 mg or more mRNA. In some embodiments, the composition comprises 0.25 mg or more mRNA. In some embodiments, the composition comprises 0.5 mg or more mRNA. In some embodiments, the composition comprises 0.75 mg or more mRNA. In some embodiments, the composition comprises 1 mg or more mRNA. In some embodiments, the composition comprises 1.25 mg or more mRNA. In some embodiments, the composition comprises 1.5 mg or more mRNA. In some embodiments, the composition comprises 1.75 mg or more mRNA. In some embodiments, the composition comprises 2 mg or more mRNA. In some embodiments, the composition comprises 2.5 mg or more mRNA. In some embodiments, the composition comprises 5 mg or more mRNA.

[0144] In some embodiments, the composition is formulated as a suppository of about 3 grams, about 2 grams, or about 1 gram. In some embodiments, the composition is formulated as a suppository of about 20 grams. In some embodiments, the composition is formulated as a suppository of about 10 grams. In some embodiments, the composition is formulated as a suppository of about 5 grams. In some embodiments, the composition is formulated as a suppository of about 3 grams. In some embodiments, the composition is formulated as a suppository of about 2 grams. In some embodiments, the composition is formulated as a suppository of about 1 gram. In some embodiments, the composition is formulated as a suppository of about 0.5 grams.

[0145] In some embodiments, the composition is formulated as a suppository having a volume of about 2.0 mL, about 3.5 mL, about 7.5 mL, or about 10.0 mL. In some embodiments, the composition is formulated as a suppository having a volume of about 1.0 mL. In some embodiments, the composition is formulated as a suppository having a volume of about 2.0 mL. In some embodiments, the composition is formulated as a suppository having a volume of about 2.5 mL. In some embodiments, the composition is formulated as a suppository having a volume of about 3.0 mL. In some embodiments, the composition is formulated as a suppository having a volume of about 3.5 mL. In some embodiments, the composition is formulated as a suppository having a volume of about 4.0 mL. In some embodiments, the composition is formulated as a suppository having a volume of about 5.0 mL. In some embodiments, the composition is formulated as a suppository having a volume of about 7.5 mL. In some embodiments, the composition is formulated as a suppository having a volume of about 10.0 mL. In some embodiments, the composition is formulated as a suppository having a volume of about 12.5 mL. In some embodiments, the composition is formulated as a suppository having a volume of about 15.0 mL. In some embodiments, the composition is formulated as a suppository having a volume of about 17.5 mL. In some embodiments, the composition is formulated as a suppository having a volume of about 20.0 mL.

[0146] permeation enhancer

[0147] To improve the bioavailability of poorly absorbed drugs across a transmucosal route (e.g., rectal, vaginal, ocular, oral, or gastrointestinal), permeation or permeation enhancers have been used. In some embodiments for rectal or vaginal administration, the suppository further comprises a permeation enhancer. In some embodiments, the suppository does not comprise a permeation enhancer. In some embodiments, the permeation enhancer does not adversely affect the integrity of the lipid nanoparticle.

[0148] In some embodiments, the permeability enhancer is selected from a bile salt, a surfactant, a fatty acid and derivative, a glyceride, a chelating agent, a salicylate, or a polymer. In some embodiments, the permeability enhancer is a bile salt. In some embodiments, the permeability enhancer is a fatty acid and derivative. In some embodiments, the permeability enhancer is a glyceride. In some embodiments, the permeability enhancer is a chelating agent. In some embodiments, the permeability enhancer is a salicylate. In some embodiments, the permeability enhancer is a polymer.

[0149] In some embodiments, the fatty acid and derivative is selected from sorbitan laurate, sodium caprate, sucrose, palmitate, lauroylcholine, sodium myristate, or palmitoyl carnitine. In some embodiments, the fatty acid and derivative includes sorbitan laurate. In some embodiments, the fatty acid and derivative includes sodium caprate. In some embodiments, the fatty acid and derivative is sucrose. In some embodiments, the fatty acid and derivative includes palmitate. In some embodiments, the fatty acid and derivative includes lauroylcholine. In some embodiments, the fatty acid and derivative includes sodium myristate. In some embodiments, the fatty acid and derivative includes palmitoyl carnitine.

[0150] In some embodiments, the permeability enhancer is a form of caprate. In some embodiments, the caprate-based permeability enhancer is sodium caprate.

[0151] In some embodiments, the permeability enhancer includes a cholate. In some embodiments, the permeability enhancer is citric acid. In some embodiments, the permeability enhancer is ethylenediaminetetraacetic acid (EDTA). In some embodiments, the permeability enhancer is oleic acid. In some embodiments, the permeability enhancer is caprate. In some embodiments, the permeability enhancer is a surfactant. In some embodiments, the permeability enhancer is sodium dodecyl sulfate (SDS). In some embodiments, the permeability enhancer is In some embodiments, the permeability enhancer is 80. In some embodiments, the permeability enhancer is In some embodiments, the permeability enhancer is a self-microemulsifying drug delivery system (SMEDDS). In some embodiments, the permeability enhancer is a natural bioenhancer. In some embodiments, the permeability enhancer is allicin. In some embodiments, the permeability enhancer is piperine. In some embodiments, the permeability enhancer is curcumin. In some embodiments, the permeability enhancer is quercetin.

[0152] Several different formulations of lipid-encapsulated mRNA compositions have been designed to facilitate delivery to a subject, including administration of a permeability enhancer prior to administration of a composition comprising mRNA. Administration of a permeability enhancer facilitates the transfer of mRNA-loaded lipid nanoparticles from the colon to systemic circulation.

[0153] In some embodiments, a permeability enhancer is administered to a subject first, prior to administration of a composition comprising mRNA. In some embodiments, a permeability enhancer is administered to a subject about 30 minutes, about 1 hour, about 2.5 hours, about 5 hours, or about 12 hours prior to administration of a composition comprising mRNA. In some embodiments, a permeability enhancer is administered to a subject about 1 minute prior to administration of a composition comprising mRNA. In some embodiments, a permeability enhancer is administered to a subject about 3 minutes prior to administration of a composition comprising mRNA. In some embodiments, a permeability enhancer is administered to a subject about 5 minutes prior to administration of a composition comprising mRNA. In some embodiments, a permeability enhancer is administered to a subject about 10 minutes prior to administration of a composition comprising mRNA. In some embodiments, a permeability enhancer is administered to a subject about 15 minutes prior to administration of a composition comprising mRNA. In some embodiments, a permeability enhancer is administered to a subject about 20 minutes prior to administration of a composition comprising mRNA. In some embodiments, a permeability enhancer is administered to a subject about 25 minutes prior to administration of a composition comprising mRNA. In some embodiments, a permeability enhancer is administered to a subject about 30 minutes prior to administration of a composition comprising mRNA. In some embodiments, a permeability enhancer is administered to a subject about 45 minutes prior to administration of a composition comprising mRNA. In some embodiments, a permeability enhancer is administered to a subject about 1 hour prior to administration of a composition comprising mRNA. In some embodiments, a permeability enhancer is administered to a subject about 1.5 hours prior to administration of a composition comprising mRNA. In some embodiments, a permeability enhancer is administered to a subject about 2 hours prior to administration of a composition comprising mRNA. In some embodiments, a permeability enhancer is administered to a subject about 2.5 hours prior to administration of a composition comprising mRNA. In some embodiments, a permeability enhancer is administered to a subject about 5 hours prior to administration of a composition comprising mRNA. In some embodiments, a permeability enhancer is administered to a subject about 12 hours prior to administration of a composition comprising mRNA. In some embodiments, a permeability enhancer is administered to a subject about 18 hours prior to administration of a composition comprising mRNA. In some embodiments, a permeability enhancer is administered to a subject about 24 hours prior to administration of a composition comprising mRNA.

[0154] In some embodiments, the subject is administered the penetration enhancer and the composition comprising mRNA simultaneously. In some embodiments, the subject is administered the penetration enhancer after administration of the composition comprising mRNA. In some embodiments, the subject is administered the penetration enhancer about 5 minutes after administration of the composition comprising mRNA. In some embodiments, the subject is administered the penetration enhancer about 10 minutes after administration of the composition comprising mRNA. In some embodiments, the subject is administered the penetration enhancer about 15 minutes after administration of the composition comprising mRNA. In some embodiments, the subject is administered the penetration enhancer about 20 minutes after administration of the composition comprising mRNA. In some embodiments, the subject is administered the penetration enhancer about 30 minutes after administration of the composition comprising mRNA. In some embodiments, the subject is administered the penetration enhancer about 45 minutes after administration of the composition comprising mRNA. In some embodiments, the subject is administered the penetration enhancer about 1 hour after administration of the composition comprising mRNA. In some embodiments, the subject is administered the penetration enhancer about 2 hours after administration of the composition comprising mRNA. In some embodiments, the subject is administered the penetration enhancer about 2.5 hours after administration of the composition comprising mRNA. In some embodiments, the subject is administered the penetration enhancer about 5 hours after administration of the composition comprising mRNA. In some embodiments, the subject is administered the penetration enhancer about 12 hours after administration of the composition comprising mRNA. In some embodiments, the subject is administered the penetration enhancer about 18 hours after administration of the composition comprising mRNA. In some embodiments, the subject is administered the penetration enhancer about 24 hours after administration of the composition comprising mRNA.

[0155] mRNA synthesis

[0156] The mRNA according to the present application can be synthesized according to any of a variety of known methods. Various methods are described in published U.S. Application No. US 2018 / 0258423, and can be used to practice the present application, all of which are incorporated herein by reference. For example, the mRNA according to the present application can be synthesized via in vitro transcription (IVT). Briefly, IVT is typically performed with a linear or circular DNA template containing a promoter, a pool of ribonucleotide triphosphates, a buffer system which can 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 depending on the particular application.

[0157] In some embodiments, a suitable mRNA sequence is one that encodes a protein or peptide. In some embodiments, a suitable mRNA sequence is codon-optimized for efficient expression in human cells. In some embodiments, a suitable mRNA sequence is a naturally-occurring or wild-type sequence. In some embodiments, a suitable mRNA sequence encodes a protein or peptide that contains one or more mutations in the amino acid sequence.

[0158] The present application can be used to deliver mRNAs of various lengths. In some embodiments, the present application can be used to deliver in vitro synthesized mRNAs of 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, or greater. In some embodiments, the present application can be used to deliver in vitro synthesized mRNAs of about 1-20 kb, about 1-15 kb, about 1-10 kb, about 5-20 kb, about 5-15 kb, about 5-12 kb, about 5-10 kb, about 8-20 kb, or about 8-50 kb.

[0159] In some embodiments, to prepare an mRNA according to the present application, a DNA template is transcribed in vitro. A suitable DNA template typically has a promoter for in vitro transcription, such as a T3, T7, or SP6 promoter, followed by the desired nucleotide sequence for the desired mRNA and a termination signal.

[0160] Nucleotides

[0161] According to the present application, various naturally occurring or modified nucleosides can be used to produce the mRNA. In some embodiments, the mRNA is or comprises a naturally occurring nucleoside (or unmodified nucleotide; e.g., adenosine, guanosine, cytidine, uridine); a nucleoside analog (e.g., 2- aminoadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine, 3-methyl adenosine, 5- methylcytidine, C-5 propynyl-cytidine, C-5 propynyl-uridine, 2-aminoadenosine, C5-bromo uridine, C5-fluoro uridine, C5-iodo uridine, C5-propynyl-uridine, C5-propynyl-cytidine, C5- methylcytidine, 2-aminoadenosine, 7-deaza-adenosine, 7-deaza-guanosine, 8-oxo- adenosine, 8-oxo-guanosine, O(6)-methylguanine, pseudouridine (e.g., N-l-methyl- pseudouridine), 2-thio-uridine, and 2-thio-cytidine); a chemically modified base; a biologically modified base (e.g., a methylated base); an inserted base; a modified sugar (e.g., 2'-fluoro-ribose, ribose, 2'-deoxyribose, arabinose, and hexose); and / or a modified phosphate group (e.g., phosphorothioate and 5'-N-phosphoramidite linkages).

[0162] In some embodiments, suitable mRNA can contain backbone modifications, sugar modifications, and / or base modifications. For example, modified nucleotides can include, but are not limited to, modified purines (adenine (A), guanine (G)) or pyrimidines (thymine (T), cytosine (C), uracil (U)) and analogs or derivatives of modified nucleotides purines and pyrimidines, such as, for example, 1 -methyl-adenine, 2-methyl-adenine, 2-methylthio-N-6-isopentenyl-adenine, N6-methyl-adenine, N6-isopentenyl-adenine, 2-thio-cytosine, 3-methyl-cytosine, 4-acetyl-cytosine, 5-methyl-cytosine, 2,6-diaminopurine, 1 -methyl-guanine, 2-methyl-guanine, 2,2-dimethyl-guanine, 7-methyl-guanine, inosine, 1 -methyl-inosine, pseudouracil (5-uracil), dihydrouracil, 2-thio-uracil, 4-thio-uracil, 5-carboxymethylaminomethyl-2-thio-uracil, 5-(carboxyhydroxylmethyl)-uracil, 5-fluoro-uracil, 5-bromo-uracil, 5-carboxymethylaminomethyl-uracil, 5-methyl-2-thio-uracil, 5-methyl-uracil, N-uracil-5-oxyacetic acid methyl ester, 5-methylaminomethyl-uracil, 5-methoxyaminomethyl-2-thio-uracil, 5'-methoxycarbonylmethyl-uracil, 5-methoxy-uracil, uracil-5-oxyacetic acid methyl ester, uracil-5-oxyacetic acid (v), 1 -methyl-pseudouracil, queosine, beta-D-mannosyl-queosine, wybutoxosine, and phosphoramidates, phosphorothioates, peptide nucleotides, methylphosphonates, 7-deazaguanosine, 5-methylcytosine, and inosine. One of skill in the art knows of the preparation of such analogs, for example, from U.S. Patent No. 4,373,071, U.S. Patent No. 4,401,796, U.S. Patent No. 4,415,732, U.S. Patent No. 4,458,066, U.S. Patent No. 4,500,707, U.S. Patent No. 4,668,777, U.S. Patent No. 4,973,679, U.S. Patent No. 5,047,524, U.S. Patent No. 5,132,418, U.S. Patent No. 5,153,319, U.S. Patent No. 5,262,530, and 5,700,642, the disclosures of which are incorporated by reference in their entireties.

[0163] In some embodiments, the mRNA comprises one or more non-standard nucleotide residues. Non-standard nucleotide residues can include, for example, 5-methyl-cytidine ("5mC"), pseudouridine ("ψU"), and / or 2-thio-uridine ("2sU"). For discussion of such residues and their incorporation into mRNA, see, e.g., U.S. Patent No. 8,278,036 or WO 2011 / 012316. The mRNA can be RNA defined as RNA in which 25% of the U residues are 2-thio-uridine and 25% of the C residues are 5-methylcytosine. Teachings for using RNA are disclosed in U.S. Patent Publication US 2012 / 0195936 and International Publication WO 2011 / 012316, both of which are hereby incorporated by reference in their entirety. The presence of non-standard nucleotide residues can make the mRNA more stable and / or less immunogenic than a control mRNA having the same sequence but containing only standard residues. In further embodiments, the mRNA can comprise one or more non-standard nucleotide residues selected from the group consisting of isocytosine, pseudoisocytosine, 5-bromouracil, 5-propynyluracil, 6-aminopurine, 2-aminopurine, inosine, diaminopurine, and 2-chloro-6-aminopurine cytosine, and combinations of these modifications and other nucleobase modifications. Some embodiments can further include additional modifications to the furanose ring or nucleobase. Additional modifications can include, for example, sugar modifications or substitutions (e.g., one or more 2'-O-alkyl modifications, locked nucleic acid (LNA)). In some embodiments, the RNA can be complexed or hybridized with additional polynucleotides and / or peptide polynucleotides (PNAs). In some embodiments in which the sugar modification is a 2'-O-alkyl modification, such modifications can include, but are not limited to, 2'-deoxy-2'-fluoro modifications, 2'-O-methyl modifications, 2'-O-methoxyethyl modifications, and 2'-deoxy modifications. In some embodiments, any of these modifications can be present in 0-100% of the nucleotides - e.g., more than 0%, 1%, 10%, 25%, 50%, 75%, 85%, 90%, 95%, or 100% of the constituent nucleotides, either individually or in combination.

[0164] In some embodiments, the mRNA can contain RNA backbone modifications. Typically, backbone modifications are modifications in which the phosphate contained in the nucleotide backbone of the RNA is chemically modified. Exemplary backbone modifications typically include, but are not limited to, modifications from the group consisting of methylphosphonate, methylphosphoramidate, phosphoramidate, phosphorothioate (e.g., cytidine 5'-O-(l-thio-phosphoate)), boranophosphonate, positively charged guanidinium groups, and the like (which means that the phosphodiester linkage is replaced by other anionic, cationic, or neutral groups).

[0165] In some embodiments, the mRNA can contain sugar modifications. Typical sugar modifications are chemical modifications of the sugar of the nucleotides it contains, including but not limited to sugar modifications selected from the group consisting of 2'-deoxy-2'-fluoro-oligonucleotides (2'-fluoro-2'-deoxycytidine 5'-triphosphate, 2'-fluoro-2'-deoxyuridine 5'-triphosphate), 2'-deoxy-2'-deamino-oligoribonucleotides (2'-amino-2'-deoxycytidine 5'-triphosphate, 2'-amino-2'-deoxyuridine 5'-triphosphate), 2'-O-alkyl oligoribonucleotides, 2'-deoxy-2'-C-alkyl oligoribonucleotides (2'-O-methylcytidine 5'-triphosphate, 2'-methyluridine 5'-triphosphate), 2'-C-alkyl oligoribonucleotides, and isomers thereof (2'-aracytidine 5'-triphosphate, 2'-arauridine 5'-triphosphate), or azido triphosphates (2'-azido-2'-deoxycytidine 5'-triphosphate, 2'-azido-2'-deoxyuridine 5'-triphosphate).

[0166] Post-synthesis processing

[0167] Typically, a 5' cap and / or a 3' tail can be added post-synthesis. The presence of a cap is important to provide resistance to nucleases found in most eukaryotic cells. The presence of a "tail" serves to protect the mRNA from exonuclease degradation.

[0168] A 5' cap is typically added as follows: first, an RNA terminal phosphatase removes one terminal phosphate group from the 5' nucleotide, leaving two terminal phosphates; then a guanylyltransferase adds a triphosphate guanosine (GTP) to the terminal phosphates, resulting in a 5'5'5 triphosphate linkage; then a methyltransferase methylates the 7-nitrogen of the guanine. 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 published U.S. Application No. US 2016 / 0032356 and published U.S. Application No. US 2018 / 0125989, which are incorporated herein by reference.

[0169] Typically, the tail structure includes a poly(A) and / or a poly(C) tail. The poly A or poly C tail of the 3' end of the 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. In some embodiments, the poly A or poly C tail can be about 10 to 800 adenosine or cytosine nucleotides (e.g., about 10 to 200 adenosine or cytosine nucleotides, about 10 to 300 adenosine or cytosine nucleotides, about 10 to 400 adenosine or cytosine nucleotides, about 10 to 500 adenosine or cytosine nucleotides, about 10 to 550 adenosine or cytosine nucleotides, about 10 to 600 adenosine or cytosine nucleotides, about 50 to 600 adenosine or cytosine nucleotides, about 100 to 600 adenosine or cytosine nucleotides, about 150 to 600 adenosine or cytosine nucleotides, about 200 to 600 adenosine or cytosine nucleotides, about 250 to 600 adenosine or cytosine nucleotides, about 300 to 600 adenosine or cytosine nucleotides, about 350 to 600 adenosine or cytosine nucleotides, about 400 to 600 adenosine or cytosine nucleotides, about 450 to 600 adenosine or cytosine nucleotides, about 500 to 600 adenosine or cytosine nucleotides, about 10 to 150 adenosine or cytosine nucleotides, about 10 to 100 adenosine or cytosine nucleotides, about 20 to 70 adenosine or cytosine nucleotides, or about 20 to 60 adenosine or cytosine nucleotides), respectively. In some embodiments, the tail structure includes a combination of poly(A) and poly(C) tails having various lengths described herein. In some embodiments, the tail 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, the tail structure includes at least 50%, 55%, 65%, 70%, 75%, 80%, 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, or 99% cytosine nucleotides.

[0170] As described herein, the addition of a 5' cap and / or 3' tail aids in the detection of abortive transcripts generated during in vitro synthesis, as those prematurely aborted mRNA transcripts can be too small to detect if not capped and / or tailed. Thus, in some embodiments, a 5' cap and / or 3' tail is added to the synthesized mRNA prior to testing the purity of the mRNA (e.g., the level of abortive transcripts present in the mRNA). In some embodiments, a 5' cap and / or 3' tail is added to the synthesized mRNA prior to purifying the mRNA as described herein. In other embodiments, a 5' cap and / or 3' tail is added to the synthesized mRNA after purifying the mRNA as described herein.

[0171] The mRNA synthesized according to the present application can be used without further purification. In particular, the mRNA synthesized according to the present application can be used without a step to remove short polymers. In some embodiments, the mRNA synthesized according to the present application can be further purified. Various methods can be used to purify the mRNA synthesized according to the present application. For example, purification of the mRNA can be performed using centrifugation, filtration, and / or chromatography. 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 of skill in the art. In some embodiments, tangential flow filtration is used to purify the mRNA. Suitable purification methods include those described in published U.S. Application No. US 2016 / 0040154, published U.S. Application No. US 2015 / 0376220, published U.S. Application No. US 2018 / 0251755, published U.S. Application No. US 2018 / 0251754, U.S. Provisional Application No. 62 / 757,612, filed November 8, 2018, and U.S. Provisional Application No. 62 / 891,781, filed August 26, 2019, all of which are incorporated by reference herein and can be used to practice the present application.

[0172] In some embodiments, the mRNA is purified prior to capping and tailing. In some embodiments, the mRNA is purified after capping and tailing. In some embodiments, the mRNA is purified both prior to and after capping and tailing.

[0173] In some embodiments, the mRNA is purified by centrifugation prior to or after, or both prior to and after, capping and tailing.

[0174] In some embodiments, the mRNA is purified by filtration prior to or after, or both prior to and after, capping and tailing.

[0175] In some embodiments, the mRNA is purified by tangential flow filtration (TFF) before or after, or both before and after capping and tailing.

[0176] In some embodiments, the mRNA is purified by chromatography before or after, or both before and after capping and tailing.

[0177] Characterization of purified mRNA

[0178] The mRNA compositions described herein are substantially free of contaminants, including short abortive RNA species, long abortive RNA species, double stranded RNA (dsRNA), residual plasmid DNA, residual in vitro transcription enzymes, residual solvents, and / or residual salts.

[0179] The mRNA compositions described herein have a purity of about 60% to about 100%. Thus, in some embodiments, the purified mRNA has a purity of about 60%. In some embodiments, the purified mRNA has a purity of about 65%. In some embodiments, the purified mRNA has a purity of about 70%. In some embodiments, the purified mRNA has a purity of about 75%. In some embodiments, the purified mRNA has a purity of about 80%. In some embodiments, the purified mRNA has a purity of about 85%. In some embodiments, the purified mRNA has a purity of about 90%.

[0180] In some embodiments, the purified mRNA has a purity of about 91%. In some embodiments, the purified mRNA has a purity of about 92%. In some embodiments, the purified mRNA has a purity of about 93%. In some embodiments, the purified mRNA has a purity of about 94%. In some embodiments, the purified mRNA has a purity of about 95%. In some embodiments, the purified mRNA has a purity of about 96%. In some embodiments, the purified mRNA has a purity of about 97%. In some embodiments, the purified mRNA has a purity of about 98%. In some embodiments, the purified mRNA has a purity of about 99%. In some embodiments, the purified mRNA has a purity of about 100%.

[0181] In some embodiments, the mRNA compositions described herein have 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% non-full-length mRNA impurities. These impurities include IVT contaminants such as proteins, enzymes, DNA templates, free nucleotides, residual solvents, residual salts, double-stranded RNA (dsRNA), prematurely aborted RNA sequences (“shorters” or “short aborted RNA species”), and / or long aborted RNA species. In some embodiments, the purified mRNA is substantially free of processing enzymes.

[0182] In some embodiments, the purified mRNA of the present application has less than about 1 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 of residual plasmid DNA. Thus, the purified mRNA has less than about 1 pg / mg of residual plasmid DNA. In some embodiments, the purified mRNA has less than about 2 pg / mg of residual plasmid DNA. In some embodiments, the purified mRNA has less than about 3 pg / mg of residual plasmid DNA. In some embodiments, the purified mRNA has less than about 4 pg / mg of residual plasmid DNA. In some embodiments, the purified mRNA has less than about 5 pg / mg of residual plasmid DNA. In some embodiments, the purified mRNA has less than about 6 pg / mg of residual plasmid DNA. In some embodiments, the purified mRNA has less than about 7 pg / mg of residual plasmid DNA. In some embodiments, the purified mRNA has less than about 8 pg / mg of residual plasmid DNA. In some embodiments, the purified mRNA has less than about 9 pg / mg of residual plasmid DNA. In some embodiments, the purified mRNA has less than about 10 pg / mg of residual plasmid DNA. In some embodiments, the purified mRNA has less than about 11 pg / mg of residual plasmid DNA. In some embodiments, the purified mRNA has less than about 12 pg / mg of residual plasmid DNA.

[0183] In some embodiments, the methods according to the application remove greater than about 90%, 95%, 96%, 97%, 98%, 99%, or substantially all of the prematurely aborted RNA sequences (also referred to as "shorters"). In some embodiments, the mRNA composition is substantially free of 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, the mRNA composition detects no prematurely aborted RNA sequences as determined by, e.g., high performance liquid chromatography (HPLC) (e.g., shoulder peaks or separated peaks), ethidium bromide, Coomassie staining, capillary electrophoresis, or glyoxal gel electrophoresis (e.g., presence of a separate lower band). As used herein, the term "shorter," "short aborted RNA species," "prematurely aborted RNA sequence," or "long aborted RNA species" refers to any transcript that is less than full length. In some embodiments, the "shorter," "short aborted RNA species," or "prematurely aborted RNA sequence" is less than 100 nucleotides in length, 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 in length. In some embodiments, the shorter is detected or quantified after addition of a 5'-cap and / or 3'-poly A tail. In some embodiments, the prematurely aborted RNA transcript comprises less than 15 bases (e.g., less than 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, or 3 bases). In some embodiments, the prematurely aborted RNA transcript contains about 8-15, 8-14, 8-13, 8-12, 8-11, or 8-10 bases.

[0184] In some embodiments, the purified mRNA of the present application is substantially free of in vitro synthesis enzyme reagents, including but not limited to T7 RNA polymerase, DNase I, pyrophosphatase, and / or RNAse inhibitors. In some embodiments, the purified mRNA according to the present application contains less than about 5% (e.g., less than about 4%, 3%, 2%, or 1%) of in vitro synthesis enzyme reagents. In some embodiments, the purified mRNA 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 in vitro synthesis enzyme reagents. In some embodiments, the purified mRNA contains no detectable in vitro synthesis enzyme reagents, including as determined by, e.g., silver staining, gel electrophoresis, high performance liquid chromatography (HPLC), ultra performance liquid chromatography (UPLC), and / or capillary electrophoresis, ethidium bromide, and / or Coomassie staining.

[0185] In various embodiments, the purified mRNA of the present application maintains a high degree of integrity. As used herein, 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, such as by RNA agarose gel electrophoresis. In some embodiments, mRNA integrity can be determined by band pattern of RNA agarose gel electrophoresis. In some embodiments, the purified mRNA of the present application shows little or no bands compared to reference bands of RNA agarose gel electrophoresis. In some embodiments, the purified mRNA of the present application has greater than about 95% (e.g., greater than about 96%, 97%, 98%, 99%, or more) integrity. In some embodiments, the purified mRNA of the present application has greater than 98% integrity. In some embodiments, the purified mRNA of the present application has greater than 99% integrity. In some embodiments, the purified mRNA of the present application has about 100% integrity.

[0186] In some embodiments, the following one or more characteristics of the purified mRNA are assessed: appearance, identity, quantity, concentration, presence of impurities, microbiological assessment, pH level, and activity. In some embodiments, acceptable appearance includes a clear, colorless solution, substantially free of visible particulates. In some embodiments, the identity of the mRNA is assessed by sequencing methods. In some embodiments, the concentration is assessed by suitable methods, such as UV spectrophotometry. In some embodiments, a suitable concentration is about 90% to 110% of the nominal value (0.9-1.1 mg / mL).

[0187] In some embodiments, assessing purity of the mRNA includes assessing mRNA integrity, assessing residual plasmid DNA, and assessing residual solvent. In some embodiments, an acceptable level of mRNA integrity is assessed by agarose gel electrophoresis. The gel is analyzed to determine if the band pattern and apparent nucleotide length are consistent with an analytical reference standard. Additional methods of assessing integrity of the RNA include, for example, assessing purified mRNA using capillary gel electrophoresis (CGE). In some embodiments, an acceptable purity of purified mRNA as determined by CGE is a purified mRNA composition having no more than about 55% long abortive / degraded species. In some embodiments, residual plasmid DNA is assessed by methods in the art, for example, by using qPCR. In some 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, an acceptable level of residual solvent is no more 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. Thus, in some embodiments, an acceptable level of residual solvent is no more than 10,000 ppm. In some embodiments, an acceptable level of residual solvent is no more than 9,000 ppm. In some embodiments, an acceptable level of residual solvent is no more than 8,000 ppm. In some embodiments, an acceptable level of residual solvent is no more than 7,000 ppm. In some embodiments, an acceptable level of residual solvent is no more than 6,000 ppm. In some embodiments, an acceptable level of residual solvent is no more than 5,000 ppm. In some embodiments, an acceptable level of residual solvent is no more than 4,000 ppm. In some embodiments, an acceptable level of residual solvent is no more than 3,000 ppm. In some embodiments, an acceptable level of residual solvent is no more than 2,000 ppm. In some embodiments, an acceptable level of residual solvent is no more than 1,000 ppm.

[0188] In some embodiments, the purified mRNA is tested for microbiological testing, including, for example, assessment of bacterial endotoxin. In some embodiments, the bacterial endotoxin is <0.5 EU / mL, <0.4 EU / mL, <0.3 EU / mL, <0.2 EU / mL, or <0.1 EU / mL. Thus, in some embodiments, the bacterial endotoxin in the purified mRNA is <0.5 EU / mL. In some embodiments, the bacterial endotoxin in the purified mRNA is <0.4 EU / mL. In some embodiments, the bacterial endotoxin in the purified mRNA is <0.3 EU / mL. In some embodiments, the bacterial endotoxin in the purified mRNA is <0.2 EU / mL. In some embodiments, the bacterial endotoxin in the purified mRNA is <0.2 EU / mL. In some embodiments, the bacterial endotoxin in the purified mRNA is <0.1 EU / mL. In some embodiments, the purified mRNA has no more than 1 CFU / 10 mL, 1 CFU / 25 mL, 1 CFU / 50 mL, 1 CFU / 75 mL, or no more than 1 CFU / 100 mL. Thus, in some embodiments, the purified mRNA has no more than 1 CFU / 10 mL. In some embodiments, the purified mRNA has no 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 CFU / 75 mL. In some embodiments, the purified mRNA has 1 CFU / 100 mL.

[0189] In some embodiments, the pH of the purified mRNA is assessed. In some embodiments, an acceptable pH of the purified mRNA is between 5 and 8. Thus, in some embodiments, the pH of the purified mRNA is about 5. In some embodiments, the pH of the purified mRNA is about 6. In some embodiments, the pH of the purified mRNA is about 7. In some embodiments, the pH of the purified mRNA is about 7. In some embodiments, the pH of the purified mRNA is about 8.

[0190] In some embodiments, the translational fidelity of the purified mRNA is assessed. Translational fidelity can be assessed by various methods, and includes, for example, transfection and western blot analysis. An acceptable characteristic of the purified mRNA includes a band pattern on a western blot that migrates at a molecular weight similar to a reference standard.

[0191] In some embodiments, the conductivity of the purified mRNA is assessed. In some embodiments, an acceptable characteristic of the purified mRNA includes a conductivity that is between about 50% and 150% of a reference standard.

[0192] The cap percentage and poly-A tail length of purified mRNA are also assessed. In some embodiments, an acceptable cap percentage includes Cap 1, % area: NLT 90. In some embodiments, an acceptable poly-A tail length is about 100-1500 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).

[0193] In some embodiments, any residual PEG of the purified mRNA is also assessed. In some embodiments, the purified mRNA has less than 10 ng PEG / mg purified mRNA to 1000 ng PEG / mg mRNA. Thus, in some embodiments, the purified mRNA has less than about 10 ng PEG / mg purified mRNA. In some embodiments, the purified mRNA has less than about 100 ng PEG / mg purified mRNA. In some embodiments, the purified mRNA has less than about 250 ng PEG / mg purified mRNA. In some embodiments, the purified mRNA has less than about 500 ng PEG / mg purified mRNA. In some embodiments, the purified mRNA has less than about 750 ng PEG / mg purified mRNA. In some embodiments, the purified mRNA has less than about 1000 ng PEG / mg purified mRNA.

[0194] Various methods of 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 combinations thereof. In some embodiments, the mRNA is first denatured by glyoxal dye prior to gel electrophoresis (“glyoxal gel electrophoresis”). In some embodiments, the synthesized mRNA is characterized prior to capping or tailing. In some embodiments, the synthesized mRNA is characterized after capping and tailing.

[0195] Delivery vehicle

[0196] According to the present application, the mRNA or MCNA encoding a protein or peptide (e.g., a full length, fragment, or portion of a protein or peptide) as described herein can be delivered as naked RNA (unpacked) or via a delivery vehicle. As used herein, the terms “delivery vehicle,” “transfer vehicle,” “nanoparticle,” or grammatical equivalents are used interchangeably.

[0197] The delivery vehicle can be formulated in combination with one or more additional nucleic acids, carriers, targeting ligands, or stabilizing agents, or in a pharmacological composition in admixture with suitable excipients. Techniques for formulation and administration of drugs can be found in the latest edition of "Remington's Pharmaceutical Sciences," Mack Publishing Co., Easton, Pa. Particular delivery vehicles are selected based on their ability to facilitate transfection of nucleic acids into target cells.

[0198] In some embodiments, the mRNA or MCNA encoding at least one protein or peptide can be delivered via a single delivery vehicle. In some embodiments, the mRNA or MCNA encoding at least one protein or peptide can be delivered via one or more delivery vehicles, each having a different composition. In some embodiments, one or more mRNAs and / or MCNAs are encapsulated within the same lipid nanoparticle. In some embodiments, one or more mRNAs are encapsulated within separate lipid nanoparticles.

[0199] 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, natural and synthetically derived exosomes, natural, synthetic and semi-synthetic lamellar bodies, nanoparticles, calcium phosphate-silica nanoparticles, calcium phosphate nanoparticles, silica nanoparticles, nanocrystal particles, semiconductor nanoparticles, poly(D-arginine), sol-gel, nanodendrimers, starch-based delivery systems, micelles, emulsions, non-ionic surfactant vesicles (niosomes), multi-domain block polymers (vinyl polymers, polypropylacrylic acid polymers, dynamic polyconjugates), dry powder formulations, plasmids, viruses, calcium phosphate nucleotides, aptamers, peptides, and other carrier tags. The use of biologic nanocapsules and other viral capsid protein assemblies are also contemplated as suitable transfer vehicles. (Hum. Gene Ther. 2008 Sep; 19(9): 887-95).

[0200] Liposome delivery vehicles

[0201] In some embodiments, a suitable delivery vehicle is a liposomal delivery vehicle, such as a lipid nanoparticle. As used herein, a liposomal delivery vehicle (e.g., a lipid nanoparticle) is generally characterized as a microscopic vesicle having an internal aqueous space that is isolated from the external medium by a membrane of one or more bilayers. The bilayer membrane of a liposome is typically formed from amphiphilic molecules, such as lipids of synthetic or natural origin, which comprise spatially separated hydrophilic and hydrophobic domains (Lasic, Trends Biotechnol. 16:307-321, 1998). The bilayer membrane of a liposome can also be formed from amphiphilic polymers and surfactants (e.g., polymersomes, non-ionic surfactant vesicles, etc.). In the context of the present application, a liposomal delivery vehicle is typically used to transport a desired nucleic acid (e.g., mRNA or MCNA) to a target cell or tissue. In some embodiments, the nanoparticle delivery vehicle is a liposome. In some embodiments, the 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, the liposome comprises no more than three different lipid components. In some embodiments, one of the different lipid components is a sterol-based cationic lipid.

[0202] Cationic lipids

[0203] As used herein, the phrase “cationic lipid” refers to any of a variety of lipid species that have a net positive charge at a selected pH (such as physiological pH).

[0204] Suitable cationic lipids for use in the compositions and methods of the present application include 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 application include a cationic lipid having the following compound structure (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl 4- (dimethylamino)butanoate:

[0205]

[0206] and pharmaceutically acceptable salts thereof.

[0207] Other suitable cationic lipids for use in the compositions and methods of the present application 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 application include a cationic lipid having one of the following formulas:

[0208]

[0209] or a pharmaceutically acceptable salt thereof, wherein R1and R2are each independently selected from the group consisting of hydrogen, optionally substituted, variably saturated or unsaturated C1-C 20 alkyl, and optionally substituted, variably saturated or unsaturated C6-C 20 acyl; wherein L1and L2are each independently selected from the group consisting of hydrogen, optionally substituted C1-C 30 alkyl, optionally substituted, variably unsaturated C1-C 30 alkenyl, and optionally substituted C1-C 30 alkynyl; wherein m and o are each independently selected from the group consisting of zero and any positive integer (e.g., wherein m is three); and wherein n is zero or any positive integer (e.g., wherein n is one). In certain embodiments, the compositions and methods of the present application include a cationic lipid having the following compound structure (15Z, 18Z)-N,N-dimethyl-6-(9Z, 12Z)-octadeca-9, 12-dien-l-yl) tetracosa-15, 18-dien-1-amine (“HGT5000”):

[0210]

[0211] and pharmaceutically acceptable salts thereof. In certain embodiments, the compositions and methods of the present application include a cationic lipid having the following compound structure (15Z, 18Z)-N,N-dimethyl-6-((9Z, 12Z)-octadeca-9, 12-dien-l-yl) tetracosa-4, 15, 18-dien-1-amine (“HGT5001”):

[0212]

[0213] and pharmaceutically acceptable salts thereof. In certain embodiments, the compositions and methods of the present application include a cationic lipid having the following compound structure (15Z, 18Z)-N,N-dimethyl-6-((9Z, 12Z)-octadeca-9, 12-dien-l-yl) tetracosa-5, 15, 18-trien-1-amine (“HGT5002”):

[0214]

[0215] and pharmaceutically acceptable salts thereof.

[0216] Other suitable cationic lipids for use in the compositions and methods of the present application include the cationic lipids described in International Patent Publication WO 2010 / 053572 as amino alcohol-based lipids, which is incorporated herein by reference. In certain embodiments, the compositions and methods of the present application include a cationic lipid having the following compound structure:

[0217]

[0218] and pharmaceutically acceptable salts thereof.

[0219] Other suitable cationic lipids for use in the compositions and methods of the present application include 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 application include a cationic lipid having the following compound structure:

[0220]

[0221] and pharmaceutically acceptable salts thereof.

[0222] Other suitable cationic lipids for use in the compositions and methods of the present application include 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 application include a cationic lipid having the following compound structure:

[0223]

[0224] and pharmaceutically acceptable salts thereof.

[0225] Other suitable cationic lipids for use in the compositions and methods of the present application include cationic lipids having the following formula: 14,25-bistridecyl 15,18,21,24-tetraaza-triacontane, and pharmaceutically acceptable salts thereof.

[0226] Other suitable cationic lipids for use in the compositions and methods of the present application include 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 application include a cationic lipid having the following formula:

[0227]

[0228] or a pharmaceutically acceptable salt thereof, wherein R L each instance of R is independently optionally substituted C6-C 40 In certain embodiments, the compositions and methods of the present application include a cationic lipid having the following compound structure:

[0229]

[0230] and pharmaceutically acceptable salts thereof. In certain embodiments, the compositions and methods of the present application include a cationic lipid having the following compound structure:

[0231]

[0232] and pharmaceutically acceptable salts thereof. In certain embodiments, the compositions and methods of the present application include a cationic lipid having the following compound structure:

[0233]

[0234] and pharmaceutically acceptable salts thereof. In certain embodiments, the compositions and methods of the present application include a cationic lipid having the following compound structure:

[0235]

[0236] and pharmaceutically acceptable salts thereof.

[0237] Other suitable cationic lipids for use in the compositions and methods of the present application include cationic lipids as described in International Patent Publication WO 2015 / 184256, which is incorporated herein by reference. In some embodiments, the compositions and methods of the present application include a cationic lipid having the following formula:

[0238]

[0239] or a pharmaceutically acceptable salt thereof, wherein each X is independently O or S; each Y is independently O or S; each m is independently 0 to 20; each n is independently 1 to 6; each R A 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 3-14 membered heterocyclyl, optionally substituted C6-14 aryl, optionally substituted 5-14 membered heteroaryl, or halogen; and each R B 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 3-14 membered heterocyclyl, optionally substituted C6-14 aryl, optionally substituted 5-14 membered heteroaryl, or halogen. In certain embodiments, the compositions and methods of the present application include a cationic lipid “Target 23” having the following compound structure:

[0240]

[0241] and pharmaceutically acceptable salts thereof.

[0242] Other suitable cationic lipids for use in the compositions and methods of the present application include 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 application include a cationic lipid having the following compound structure:

[0243]

[0244] or a pharmaceutically acceptable salt thereof. In some embodiments, the compositions and methods of the present application include a cationic lipid having the following compound structure:

[0245]

[0246] or a pharmaceutically acceptable salt thereof. In some embodiments, the compositions and methods of the present application include a cationic lipid having the following compound structure:

[0247]

[0248] or a pharmaceutically acceptable salt thereof.

[0249] Other suitable cationic lipids for use in the compositions and methods of the present application include cationic lipids as described in U.S. Provisional Patent Application Serial No. 62 / 758,179, which is incorporated herein by reference. In some embodiments, the compositions and methods of the present application include a cationic lipid having the following formula:

[0250]

[0251] or a pharmaceutically acceptable salt thereof, wherein each R 1 and R 2 is independently H or C1-C6 aliphatic; each m is independently an integer having a value of 1 to 4; each A is independently a covalent bond or an arylene; each L 1 is independently an ester, thioester, disulfide, or anhydride group; each L 2 is independently C2-C 10 aliphatic; each X 1 is independently H or OH; and each R 3 is independently C6-C 20 aliphatic. In some embodiments, the compositions and methods of the present application include a cationic lipid having the following formula:

[0252]

[0253] or a pharmaceutically acceptable salt thereof. In some embodiments, the compositions and methods of the present application include a cationic lipid having the following formula:

[0254]

[0255] or pharmaceutically acceptable salts thereof. In some embodiments, the compositions and methods of the present application include a cationic lipid having the following compound structure:

[0256]

[0257] or pharmaceutically acceptable salts thereof.

[0258] Other suitable cationic lipids for use in the compositions and methods of the present application include cationic lipids as described in J. McClellan, M. C. King, Cell 2010, 141, 210-217 and Whitehead et al., Nature Communications (2014) 5:4277, which are incorporated herein by reference. In certain embodiments, the cationic lipids in the compositions and methods of the present application include cationic lipids having the following compound structure:

[0259]

[0260] and pharmaceutically acceptable salts thereof.

[0261] Other suitable cationic lipids for use in the compositions and methods of the present application include 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 application include a cationic lipid having the following compound structure:

[0262]

[0263] and pharmaceutically acceptable salts thereof. In some embodiments, the compositions and methods of the present application include a cationic lipid having the following compound structure:

[0264]

[0265] and pharmaceutically acceptable salts thereof. In some embodiments, the compositions and methods of the present application include a cationic lipid having the following compound structure:

[0266]

[0267] and pharmaceutically acceptable salts thereof. In some embodiments, the compositions and methods of the present application include a cationic lipid having the following compound structure:

[0268]

[0269] and pharmaceutically acceptable salts thereof. In some embodiments, the compositions and methods of the application include a cationic lipid having the following compound structure:

[0270]

[0271] and pharmaceutically acceptable salts thereof. In some embodiments, the compositions and methods of the application include a cationic lipid having the following compound structure:

[0272]

[0273] and pharmaceutically acceptable salts thereof. In some embodiments, the compositions and methods of the application include a cationic lipid having the following compound structure:

[0274]

[0275] and pharmaceutically acceptable salts thereof. In some embodiments, the compositions and methods of the application include a cationic lipid having the following compound structure:

[0276]

[0277] and pharmaceutically acceptable salts thereof. In some embodiments, the compositions and methods of the application include a cationic lipid having the following compound structure:

[0278]

[0279] and pharmaceutically acceptable salts thereof. In some embodiments, the compositions and methods of the application include a cationic lipid having the following compound structure:

[0280]

[0281] and pharmaceutically acceptable salts thereof. In some embodiments, the compositions and methods of the application include a cationic lipid having the following compound structure:

[0282]

[0283] and pharmaceutically acceptable salts thereof. In some embodiments, the compositions and methods of the application include a cationic lipid having the following compound structure:

[0284]

[0285] and pharmaceutically acceptable salts thereof. In some embodiments, the compositions and methods of the application include a cationic lipid having the following compound structure:

[0286]

[0287] and pharmaceutically acceptable salts thereof.

[0288] Other suitable cationic lipids for use in the compositions and methods of the present application include 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 application include a cationic lipid having the following compound structure:

[0289]

[0290] and pharmaceutically acceptable salts thereof. In some embodiments, the compositions and methods of the present application include a cationic lipid having the following compound structure:

[0291]

[0292] and pharmaceutically acceptable salts thereof. In some embodiments, the compositions and methods of the present application include a cationic lipid having the following compound structure:

[0293]

[0294] and pharmaceutically acceptable salts thereof. In some embodiments, the compositions and methods of the present application include a cationic lipid having the following compound structure:

[0295]

[0296] and pharmaceutically acceptable salts thereof. In some embodiments, the compositions and methods of the present application include a cationic lipid having the following compound structure:

[0297]

[0298] and pharmaceutically acceptable salts thereof. In some embodiments, the compositions and methods of the present application include a cationic lipid having the following compound structure:

[0299]

[0300] and pharmaceutically acceptable salts thereof. In some embodiments, the compositions and methods of the present application include a cationic lipid having the following compound structure:

[0301]

[0302] and pharmaceutically acceptable salts thereof. In some embodiments, the compositions and methods of the present application include a cationic lipid having the following compound structure:

[0303]

[0304] and pharmaceutically acceptable salts thereof. In some embodiments, the compositions and methods of the application include a cationic lipid having the following compound structure:

[0305]

[0306] and pharmaceutically acceptable salts thereof. In some embodiments, the compositions and methods of the application include a cationic lipid having the following compound structure:

[0307]

[0308] and pharmaceutically acceptable salts thereof. In some embodiments, the compositions and methods of the application include a cationic lipid having the following compound structure:

[0309]

[0310] and pharmaceutically acceptable salts thereof. In some embodiments, the compositions and methods of the application include a cationic lipid having the following compound structure:

[0311]

[0312] and pharmaceutically acceptable salts thereof. In some embodiments, the compositions and methods of the application include a cationic lipid having the following compound structure:

[0313]

[0314] and pharmaceutically acceptable salts thereof. In some embodiments, the compositions and methods of the application include a cationic lipid having the following compound structure:

[0315]

[0316] and pharmaceutically acceptable salts thereof. In some embodiments, the compositions and methods of the application include a cationic lipid having the following compound structure:

[0317]

[0318] and pharmaceutically acceptable salts thereof. In some embodiments, the compositions and methods of the application include a cationic lipid having the following compound structure:

[0319]

[0320] and pharmaceutically acceptable salts thereof. In some embodiments, the compositions and methods of the application include a cationic lipid having the following compound structure:

[0321]

[0322] and pharmaceutically acceptable salts thereof.

[0323] Other suitable cationic lipids for use in the compositions and methods of the present application include 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 application include a cationic lipid having the formula:

[0324]

[0325] or a pharmaceutically acceptable salt thereof, wherein L 1 or L 2 one of L x , -S-S-, -C(=O)S-, -SC(=O)-, -NR a C(=O)-, -C(=O)NR a -, NR a C(=O)NR a -, -OC(=O)NR a -, or -NR a C(=O)O-; and the other of L 1 or L 2 one of L x , -S-S-, -C(=O)S-, SC(=O)-, -NR a C(=O)-, -C(=O)NR a -, NR a C(=O)NR a -, -OC(=O)NR a -, or -NR a C(=O)O- or a direct bond; G 1 and G 2 are each independently unsubstituted C1-C 12 alkylene or C1-C 12 alkenylene; G 3 is C1-C 24 alkylene, C1-C 24 alkenylene, C3-C8cycloalkylene, C3-C8cycloalkenylene; R a is H or C1-C 12 alkyl; R 1 and R 2 are each independently C6-C 24 alkyl or C6-C 24 alkenyl; R 3 is H, OR5 CN, -C(=0)OR 4 -OC(=0)R 4 or -NR 5 C(=0)R 4 ; R 4 is Ci-C 12 alkyl; R 5 is H or Ci-C6alkyl; and x is 0, 1, or 2.

[0326] Other suitable cationic lipids for use in the compositions and methods of the present application include 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 application include a cationic lipid having the following compound structure:

[0327]

[0328] and pharmaceutically acceptable salts thereof. In some embodiments, the compositions and methods of the present application include a cationic lipid having the following compound structure:

[0329]

[0330] and pharmaceutically acceptable salts thereof. In some embodiments, the compositions and methods of the present application include a cationic lipid having the following compound structure:

[0331]

[0332] and pharmaceutically acceptable salts thereof.

[0333] Other suitable cationic lipids for use in the compositions and methods of the present application include cationic lipids as described in International Patent Publication WO 2017 / 049245, which is incorporated herein by reference. In some embodiments, the cationic lipids in the compositions and methods of the present application include a compound having one of the following formulas:

[0334]

[0335]

[0336] and pharmaceutically acceptable salts thereof. For any of these four formulas, R4is independently selected from -(CH2) n Q and -(CH2) n CHQR; Q is selected from the group consisting of -OR, -OH, -0(CH2) nN(R)2, -OC(0)R, -CX3, -CN, -N(R)C(0)R, -N(H)C(0)R, -N(R)S(0)2R, -N(H)S(0)2R, -N(R)C(0)N(R)2, -N(H)C(0)N(R)2, -N(H)C(0)N(H)(R), -N(R)C(S)N(R)2, -N(H)C(S)N(R)2, -N(H)C(S)N(H)(R), and heterocycle; and n is 1, 2, or 3. In certain embodiments, the compositions and methods of the present application include a cationic lipid having the following compound structure:

[0337]

[0338] and pharmaceutically acceptable salts thereof. In certain embodiments, the compositions and methods of the present application include a cationic lipid having the following compound structure:

[0339]

[0340] and pharmaceutically acceptable salts thereof. In certain embodiments, the compositions and methods of the present application include a cationic lipid having the following compound structure:

[0341]

[0342] and pharmaceutically acceptable salts thereof. In certain embodiments, the compositions and methods of the present application include a cationic lipid having the following compound structure:

[0343]

[0344] and pharmaceutically acceptable salts thereof.

[0345] Other suitable cationic lipids for use in the compositions and methods of the present application include cationic lipids as described in International Patent Publications WO 2017 / 173054 and WO 2015 / 095340, each incorporated herein by reference. In certain embodiments, the compositions and methods of the present application include a cationic lipid having the following compound structure:

[0346]

[0347] and pharmaceutically acceptable salts thereof. In certain embodiments, the compositions and methods of the present application include a cationic lipid having the following compound structure:

[0348]

[0349] and pharmaceutically acceptable salts thereof. In certain embodiments, the compositions and methods of the present application include a cationic lipid having the following compound structure:

[0350]

[0351] and pharmaceutically acceptable salts thereof. In certain embodiments, the compositions and methods of the present application include a cationic lipid having the following compound structure:

[0352]

[0353] and pharmaceutically acceptable salts thereof.

[0354] Other suitable cationic lipids for use in the compositions and methods of the present application 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 application include a cationic lipid having the following formula:

[0355]

[0356] wherein R1is selected from the group consisting of imidazole, guanidinium, amino, imine, enamine, optionally substituted alkylamino (e.g., alkylamino such as dimethylamino), and pyridyl; wherein R2is selected from the group consisting of one of the following two formulas:

[0357]

[0358] and wherein R3and R4are each independently selected from the group consisting of optionally substituted, variably saturated or unsaturated C6-C 20 alkyl and optionally substituted, variably saturated or unsaturated C6-C 20 acyl; 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 greater). In certain embodiments, the compositions and methods of the present application include a cationic lipid "HGT4001" having the following compound structure:

[0359]

[0360] and pharmaceutically acceptable salts thereof. In certain embodiments, the compositions and methods of the present application include a cationic lipid "HGT4002" having the following compound structure:

[0361]

[0362] and pharmaceutically acceptable salts thereof. In certain embodiments, the compositions and methods of the present application include a cationic lipid "HGT4003" having the following compound structure:

[0363]

[0364] and pharmaceutically acceptable salts thereof. In certain embodiments, the compositions and methods of the present application include a cationic lipid “HGT4004” having the following compound structure:

[0365]

[0366] and pharmaceutically acceptable salts thereof. In certain embodiments, the compositions and methods of the present application include a cationic lipid “HGT4005” having the following compound structure:

[0367]

[0368] and pharmaceutically acceptable salts thereof.

[0369] Other suitable cationic lipids for use in the compositions and methods of the present application include cleavable cationic lipids as described in International Application No. PCT / US 2019 / 032522, which is incorporated herein by reference. In certain embodiments, the compositions and methods of the present application include a cationic lipid that is any of the general formulae or structures (la)-(2la) and (lb)-(2lb) and (22)-(237) described in International Application No. PCT / US 2019 / 032522. In certain embodiments, the compositions and methods of the present application include a cationic lipid having a structure according to Formula (I’),

[0370]

[0371] wherein:

[0372] R X is independently -H, -L 1 -R 1 , or -L 5A -L 5B -B’;

[0373] L 1 , L 2 , and L 3 are each independently a covalent bond, -C(O)-, -C(O)O-, -C(O)S-, or -C(O)NR L -;

[0374] each L 4A and L 5A is independently -C(O)-, -C(O)O-, or -C(O)NR L -;

[0375] each L 4B and L 5B is independently C1-C 20 alkylene, C2-C 20 alkenylene, or C2-C 20 alkynylene;

[0376] each B and B’ is NR 4 R 5 or 5- to 10-membered nitrogen-containing heteroaryl;

[0377] each R 1 , R 2 , and R 3 is independently C6-C 30 alkyl, C6-C 30 alkenyl, or C6-C 30 alkynyl;

[0378] each R 4 and R 5 is independently hydrogen, C1-C 10 alkyl, C2-C 10 alkenyl, or C2-C 10 alkynyl; and

[0379] each R L is independently hydrogen, C1-C 20 alkyl, C2-C 20 alkenyl, or C2-C 20 alkynyl.

[0380] In certain embodiments, the compositions and methods of the present application include a cationic lipid that is compound (139) of International Application No. PCT / US 2019 / 032522, having the following compound structure:

[0381] (“18:1 carbon tail-ribo-lipid”).

[0382] In some embodiments, the compositions and methods of the present application include the cationic lipid N-[l-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride ("DOTMA"). (Feigner et al. Proc. Nat'l Acad. Sci. 84, 7413 (1987); U.S. Patent No. 4,897,355, which documents are incorporated herein by reference). Other suitable cationic lipids suitable for use in the compositions and methods of the present application include, for example, 5-carboxyamido glycine dioctadecylamide ("DOGS"); 2,3-dioleyloxy-N-[2(spermine- formylamido)ethyl]-N,N-dimethyl-l-propanaminium ("DOSPA") (Behr et al. Proc. Nat.'l Acad. Sci. 86, 6982 (1989), U.S. Patent No. 5,171,678; U.S. Patent No. 5,334,761); l,2-dioleoyl-3-dimethylammonium-propane ("DODAP"); l,2-dioleoyl-3- trimethylammonium-propane ("DOTAP").

[0383] Additional exemplary cationic lipids suitable for use in the compositions and methods of the present application also include: l,2-distearyloxy-N,N-dimethyl-3-aminopropane ("DSDMA"); 1,2-dioleyloxy-N,N-dimethyl-3-aminopropane ("DODMA"); 1,2-dilinoleloxy-N,N-dimethyl-3-aminopropane ("DLinDMA"); l,2-dilinoleloxy-N,N-dimethyl-3-aminopropane ("DLenDMA"); N-dioleyl-N,N-dimethylammonium chloride ("DODAC"); N,N-distearyl-N,N-dimethylammonium bromide ("DDAB"); N-(l,2-dimyristyloxyprop-3-yl)-N,N-dimethyl-N-hydroxyethylammonium bromide ("DMRIE"); 3-dimethylamino-2-(cholesteryl-5-en-3-β-oxybutane-4-oxy)-l-(cis,cis-9,12-octadecadienyloxy)propane ("CLinDMA"); 2-[5'-(cholesteryl-5-en-3-β-oxy)-3'-oxapentoxy)-3-dimethyl-l-l-(cis,cis-9',l-2'-octadecadienyloxy)propane ("CpLinDMA"); N,N-dimethyl-3,4-dioleyloxybenzylamine ("DMOBA"); 1,2-N,N'-dioleylcarbamoyl-3-dimethylaminopropane ("DOcarbDAP"); 2,3-dilinoleloxy-Ν,Ν-dimethylpropanamine ("DLinDAP"); l,2-N,N'-dilinoleylcarbamoyl-3-dimethylaminopropane ("DLincarbDAP"); l,2-dilinoleylcarbamoyl-3-dimethylaminopropane ("DLinCDAP"); 2,2-dilinoleyl-4-dimethylaminomethyl-[l,3]-dioxolane ("DLin-K-DMA"); 2-((8-[(3P)-cholesteryl-5-en-3-yl oxy]octyl)oxy)-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-l-yloxy]propan-l-amine ("Octyl-CLinDMA"); (2R)-2-((8-[(3β)-cholesteryl-5-en-3-yl oxy]octyl)oxy)-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-l-yloxy]propan-l-amine ("Octyl-CLinDMA(2R)"); (2S)-2-((8-[(3P)-cholesteryl-5-en-3-yl oxy]octyl)oxy)-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-l-yloxy]propan-l-amine ("Octyl-CLinDMA(2S)");2,2-dilinoleyl-4-dimethylaminoethyl-[l,3]-dioxolane ("DLin-K-XTC2-DMA"); and 2-(2,2-bis((9Z,12Z)-octadeca-9,12-dien-1-yl)-l,3-dioxolan-4-yl)-N,N-dimethylethanamine ("DLin-KC2-DMA") (see WO 2010 / 042877, incorporated herein by reference; Semple et 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, the one or more cationic lipids comprise at least one of an imidazole, a dialkylamino, or a guanidinium moiety.

[0384] In some embodiments, the one or more cationic lipids suitable for use in the compositions and methods of the application include 2,2-dilinoleyl-4-dimethylaminoethyl-[l,3]-dioxolane ("XTC"); (3aR,5s,6aS)-N,N-dimethyl-2,2-bis((9Z,12Z)-octadeca-9,12-dienyl)tetrahydro-3aH-cyclopenta[d][l,3]dioxol-5-amine ("ALNY-100") and / or 4,7,13-tris(3-oxo-3-(undecylamino)propyl)-N1,N16-bisundecyl-4,7,10,13-tetraazahexadecane-l,16-diamide ("NC98-5").

[0385] In some embodiments, the compositions of the application include one or more cationic lipids that comprise at least about 5%, 10%, 20%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, or 70% of the total lipid content of the composition (e.g., lipid nanoparticle) by weight. In some embodiments, the compositions of the application include one or more cationic lipids that comprise at least about 5%, 10%, 20%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, or 70% of the total lipid content of the composition (e.g., lipid nanoparticle) by mol%. In some embodiments, the compositions of the application include one or more cationic lipids that comprise about 30-70% (e.g., about 30-65%, about 30-60%, about 30-55%, about 30-50%, about 30-45%, about 30-40%, about 35-50%, about 35-45%, or about 35-40%) of the total lipid content of the composition (e.g., lipid nanoparticle) by weight. In some embodiments, the compositions of the application include one or more cationic lipids that comprise about 30-70% (e.g., about 30-65%, about 30-60%, about 30-55%, about 30-50%, about 30-45%, about 30-40%, about 35-50%, about 35-45%, or about 35-40%) of the total lipid content of the composition (e.g., lipid nanoparticle) by mol%.

[0386] Non-cationic / Helper lipids

[0387] In some embodiments, the liposome contains one or more non-cationic ("helper") lipids. As used herein, the phrase "non-cationic lipid" refers to any neutral lipid, amphipathic ionic lipid, or anionic lipid. As used herein, the phrase "anionic lipid" refers to any of a variety of 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), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoyl-phosphatidylethanolamine (POPE), dioleoyl-phosphatidylethanolamine 4-(N-maleimidomethyl)- cyclohexane-l-carboxylate (DOPE-mal), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoyl-phosphatidyl-ethanolamine (DSPE), phosphatidylserine, sphingolipids, cerebrosides, gangliosides, 16-O- monomethyl PE, 16-O-dimethyl PE, 18-l-trans PE, l-stearoyl-2-oleoyl-phosphatidylethanolamine (SOPE), or mixtures thereof.

[0388] In some embodiments, the non-cationic lipid is a neutral lipid, i.e., a lipid that does not carry a net charge under the conditions in which the composition is formulated and / or administered.

[0389] In some embodiments, such non-cationic lipids can be used alone, but are preferably used in combination with other lipids (e.g., cationic lipids).

[0390] In some embodiments, the non-cationic lipid can be present in a molar ratio (mol%) 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 lipid present in the composition. In some embodiments, the total non-cationic lipid can be present in a molar ratio (mol%) 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 lipid present in the composition. In some embodiments, the percentage of non-cationic lipid in the liposome can be greater than about 5 mol%, greater than about 10 mol%, greater than about 20 mol%, greater than about 30 mol%, or greater than about 40 mol%. In some embodiments, the percentage of total non-cationic lipid in the liposome can be greater than about 5 mol%, greater than about 10 mol%, greater than about 20 mol%, greater than about 30 mol%, or greater than about 40 mol%. In some embodiments, the percentage of non-cationic lipid in the liposome is no more than about 5 mol%, no more than about 10 mol%, no more than about 20 mol%, no more than about 30 mol%, or no more than about 40 mol%. In some embodiments, the percentage of total non-cationic lipid in the liposome can be no more than about 5 mol%, no more than about 10 mol%, no more than about 20 mol%, no more than about 30 mol%, or no more than about 40 mol%.

[0391] In some embodiments, the non-cationic lipid can be present in a weight ratio (wt%) 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 lipid present in the composition. In some embodiments, the total non-cationic lipid can be present in a weight ratio (wt%) 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 lipid present in the composition. In some embodiments, the percentage of non-cationic lipid in the liposome can be greater than about 5 wt%, greater than about 10 wt%, greater than about 20 wt%, greater than about 30 wt%, or greater than about 40 wt%. In some embodiments, the percentage of total non-cationic lipid in the liposome can be greater than about 5 wt%, greater than about 10 wt%, greater than about 20 wt%, greater than about 30 wt%, or greater than about 40 wt%. In some embodiments, the percentage of non-cationic lipid in the liposome is no more than about 5 wt%, no more than about 10 wt%, no more than about 20 wt%, no more than about 30 wt%, or no more than about 40 wt%. In some embodiments, the percentage of total non-cationic lipid in the liposome can be no more than about 5 wt%, no more than about 10 wt%, no more than about 20 wt%, no more than about 30 wt%, or no more than about 40 wt%.

[0392] Cholesterol-based lipids

[0393] In some embodiments, the liposome comprises one or more cholesterol-based lipids. For example, suitable cholesterol-based cationic lipids include, for example, DC-Choi (N,N- dimethyl-N-ethylformamidocholine cholesterol), l,4-bis(3-N-oleylamino-propyl)piperazine (Gao et al. Biochem. Biophys. Res. Comm. 179, 280 (1991); Wolf et al. BioTechniques 23, 139 (1997); U.S. Patent No. 5,744,335), or imidazol cholesterol ester (ICE) having the following structure:

[0394]

[0395] In embodiments, the cholesterol-based lipid is cholesterol.

[0396] In some embodiments, the cholesterol-based lipid can be present in a molar ratio (mol%) of about 1% to about 30%, or about 5% to about 20%, of the total lipid present in the liposome. In some embodiments, the percentage of cholesterol-based lipid in the lipid nanoparticle can be greater than about 5 mol%, greater than about 10 mol%, greater than about 20 mol%, greater than about 30 mol%, or greater than about 40 mol%. In some embodiments, the percentage of cholesterol-based lipid in the lipid nanoparticle can be no more than about 5 mol%, no more than about 10 mol%, no more than about 20 mol%, no more than about 30 mol%, or no more than about 40 mol%.

[0397] In some embodiments, the cholesterol-based lipid can be present in a weight ratio (wt%) of about 1% to about 30%, or about 5% to about 20%, of the total lipid present in the liposome. In some embodiments, the percentage of cholesterol-based lipid in the lipid nanoparticle can be greater than about 5 wt%, greater than about 10 wt%, greater than about 20 wt%, greater than about 30 wt%, or greater than about 40 wt%. In some embodiments, the percentage of cholesterol-based lipid in the lipid nanoparticle can be no more than about 5 wt%, no more than about 10 wt%, no more than about 20 wt%, no more than about 30 wt%, or no more than about 40 wt%.

[0398] PEG-modified lipids

[0399] In some embodiments, the liposome comprises one or more PEGylated lipids.

[0400] For example, the present application also contemplates the use of polyethylene glycol (PEG)-modified phospholipids and derivatized lipids, such as derivatized ceramides (PEG-CER), including N-octanoyl-sphingosine-1 -[succinyl(methoxypolyethylene glycol)-2000] (C8 PEG-2000 ceramide), alone or preferably in combination with other lipid formulations comprising transfer vehicles (e.g., lipid nanoparticles).

[0401] PEG-modified lipids contemplated include, but are not limited to, PEG- conjugated phospholipids having C6-C 20PEG chains up to 5 kDa in length covalently attached to one or more alkyl chains of the lipid. In some embodiments, the PEG-modified or PEGylated lipid is PEGylated cholesterol or PEG-2K. The addition of such components can prevent complex aggregation and can also provide a means for increasing circulation lifetime and enhancing delivery of the lipid-nucleic acid composition to target tissues (Klibanov et al. (1990) FEBS Letters, 268(1): 235-237), or they can be selected to be rapidly exchanged out of the formulation in vivo (see U.S. Patent No. 5,885,613). Particularly useful exchangeable lipids are PEG-ceramides with shorter acyl chains (e.g., C 14 or C 18 ) PEG-ceramides.

[0402] The PEG-modified phospholipids and derivatized lipids of the present application can comprise about 0% to about 20%, about 0.5% to about 20%, about 1% to about 15%, about 4% to about 10%, or about 2% by molar ratio of the total lipid present in the liposomal transfer vehicle. In some embodiments, the one or more PEG-modified lipids comprise about 4% by molar ratio of the total lipid. In some embodiments, the one or more PEG-modified lipids comprise about 5% by molar ratio of the total lipid. In some embodiments, the one or more PEG-modified lipids comprise about 6% by molar ratio of the total lipid.

[0403] Amphiphilic block copolymers

[0404] In some embodiments, a suitable delivery vehicle contains an amphiphilic block copolymer (e.g., a poloxamer).

[0405] Various amphiphilic block copolymers can be used in the practice of the present application. In some embodiments, the amphiphilic block copolymers are also referred to as surfactants or non-ionic surfactants.

[0406] In some embodiments, the amphiphilic polymers suitable for use in the present application are selected from poloxamers poloxamines polyethylene glycol sorbitan alkyl esters (polysorbates) and polyvinylpyrrolidone (PVP).

[0407] poloxamers

[0408] In some embodiments, a suitable amphiphilic polymer is a poloxamer. For example, a suitable poloxamer has the following structure:

[0409]

[0410] wherein a is an integer from 10 to 150, and b is an integer from 20 to 60. For example, a is about 12 and b is about 20, or a is about 80 and b is about 27, or a is about 64 and b is about 37, or a is about 141 and b is about 44, or a is about 101 and b is about 56.

[0411] In some embodiments, the poloxamer useful in the present application has from about 10 to about 150 ethylene oxide units. In some embodiments, the poloxamer has from about 10 to about 100 ethylene oxide units.

[0412] 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 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 embodiments, a suitable poloxamer is poloxamer 217. 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.

[0413] In some embodiments, the suitable poloxamer has an average molecular weight of about 4,000 g / mol to about 20,000 g / mol. In some embodiments, the suitable poloxamer has an average molecular weight of about 1,000 g / mol to about 50,000 g / mol. In some embodiments, the suitable poloxamer has an average molecular weight of about 1,000 g / mol. In some embodiments, the suitable poloxamer has an average molecular weight of about 2,000 g / mol. In some embodiments, the suitable poloxamer has an average molecular weight of about 3,000 g / mol. In some embodiments, the suitable poloxamer has an average molecular weight of about 4,000 g / mol. In some embodiments, the suitable poloxamer has an average molecular weight of about 5,000 g / mol. In some embodiments, the suitable poloxamer has an average molecular weight of about 6,000 g / mol. In some embodiments, the suitable poloxamer has an average molecular weight of about 7,000 g / mol. In some embodiments, the suitable poloxamer has an average molecular weight of about 8,000 g / mol. In some embodiments, the suitable poloxamer has an average molecular weight of about 9,000 g / mol. In some embodiments, the suitable poloxamer has an average molecular weight of about 10,000 g / mol. In some embodiments, the suitable poloxamer has an average molecular weight of about 20,000 g / mol. In some embodiments, the suitable poloxamer has an average molecular weight of about 25,000 g / mol. In some embodiments, the suitable poloxamer has an average molecular weight of about 30,000 g / mol. In some embodiments, the suitable poloxamer has an average molecular weight of about 40,000 g / mol. In some embodiments, the suitable poloxamer has an average molecular weight of about 50,000 g / mol.

[0414] Other amphiphilic polymers

[0415] In some embodiments, the amphiphilic polymer is a poloxamine, such as tetronic 304 or tetronic 904.

[0416] In some embodiments, the amphiphilic polymer is polyvinylpyrrolidone (PVP), such as PVP with a molecular weight of 3 kDa, 10 kDa, or 29 kDa.

[0417] In some embodiments, the amphiphilic polymer is a polyethylene glycol ether (Brij), a polysorbate, sorbitol, and derivatives thereof. In some embodiments, the amphiphilic polymer is a polysorbate, such as PS 20.

[0418] In some embodiments, the amphiphilic polymer is a polyethylene glycol ether (Brij), a poloxamer, a polysorbate, sorbitol, or a derivative thereof.

[0419] In some embodiments, the amphiphilic polymer is a polyethylene glycol ether. In some embodiments, a suitable polyethylene glycol ether is a compound of Formula (S-l):

[0420]

[0421] or a salt or isomer thereof, wherein:

[0422] t is an integer from 1 to 100;

[0423] R 1BRIJ is independently C 10-40 alkyl, C 10-40 alkenyl, or C 10-40 alkynyl; and optionally, one or more methylene groups of R 5PEG are independently replaced by C 3-10 carbocyclylenyl, 4- to 10-membered heterocyclylenyl, C 6-10 arylenyl, 4- to 10-membered heteroarylenyl, -N(R N )-, -O-, -S-, -C(O)-, -C(O)N(R N )-, -NR N C(O)-, -NR C(O)N(R)-, -C(O)O-, -OC(O)-, -OC(O)O-, -OC(O)N(R N )-, -NR N C(O)O-, -C(O)S-, -SC(O)-, -C(=NR N )-, —C(=NR)N(R)—, -NRNC(=NR N )-, -NR N C(=NR N )N(R N )-, -C(S)-, -C(S)N(R N )-, -NR N C(S)-, -NR N C(S)N(R N )-, -S(O)-, -OS(O)-, -S(O)O-, -OS(O)O-, -OS(O)2-, -S(O)2O-, -OS(O)2O-, -N(R N )S(O)-, -S(O)N(R N )-, -N(R N )S(O)N(R N )-, -OS(O)N(R N )-, -N(R N )S(O)0-, -S(O)2-, -N(R N )S(O)2-, -S(O)2N(R N)-, -N(R N )S(O)2N(R N )-, -OS(O)2N(R N )-, or -N(R N )S(O)2O-; and

[0424] R N is independently hydrogen, C 1-6 alkyl, or a nitrogen protecting group.

[0425] In some embodiments, R 1BRIJ is C is alkyl. For example, a suitable polyethylene glycol ether is a compound of formula (S-la):

[0426]

[0427] or a salt or isomer thereof, wherein s is an integer from 1 to 100.

[0428] In some embodiments, R 1BRIJ is C is alkenyl. For example, a suitable polyethylene glycol ether is a compound of formula (S-lb):

[0429]

[0430] or a salt or isomer thereof, wherein s is an integer from 1 to 100.

[0431] Typically, the amphiphilic polymer (e.g., poloxamer) is present in the formulation in an amount less than its critical micelle concentration (CMC). In some embodiments, the amphiphilic polymer (e.g., poloxamer) is present in the mixture in an amount that is less than its CMC by 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%. In some embodiments, the amphiphilic polymer (e.g., poloxamer) is present in the mixture in an amount that is less than its CMC by about 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%. In some embodiments, the amphiphilic polymer (e.g., poloxamer) is present in the mixture in an amount that is less than its CMC by about 55%, 60%, 65%, 70%, 75%, 80%, 90%, or 95%.

[0432] 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) remains in the formulation after removal. In some embodiments, an amount of amphiphilic polymer (e.g., poloxamer) remains in the formulation after removal. As used herein, an amount means the amount remaining after removal of substantially all of the material (amphiphilic polymer described herein, such as poloxamer) in the composition. The amount can be detected qualitatively or quantitatively using known techniques. The amount can not be detectable using known techniques.

[0433] In some embodiments, a suitable delivery vehicle contains less than 5% amphiphilic block copolymer (e.g., poloxamer). In some embodiments, a suitable delivery vehicle contains less than 3% amphiphilic block copolymer (e.g., poloxamer). In some embodiments, a suitable delivery vehicle contains less than 2.5% amphiphilic block copolymer (e.g., poloxamer). In some embodiments, a suitable delivery vehicle contains less than 2% amphiphilic block copolymer (e.g., poloxamer). In some embodiments, a suitable delivery vehicle contains less than 1.5% amphiphilic block copolymer (e.g., poloxamer). In some embodiments, a suitable delivery vehicle contains less than 1% amphiphilic block copolymer (e.g., poloxamer). In some embodiments, a suitable delivery vehicle contains less than 0.5% (e.g., less than 0.4%, 0.3%, 0.2%, 0.1%) amphiphilic block copolymer (e.g., poloxamer). In some embodiments, a suitable delivery vehicle contains less than 0.09%, 0.08%, 0.07%, 0.06%, 0.05%, 0.04%, 0.03%, 0.02%, or 0.01% amphiphilic block copolymer (e.g., poloxamer). In some embodiments, a suitable delivery vehicle contains less than 0.01% amphiphilic block copolymer (e.g., poloxamer). In some embodiments, a suitable delivery vehicle contains an amount of amphiphilic polymer (e.g., poloxamer). As used herein, an amount means the amount remaining after removal of substantially all of the material (amphiphilic polymer described herein, such as poloxamer) in the composition. The amount can be detected qualitatively or quantitatively using known techniques. The amount can not be detectable using known techniques.

[0434] Polymers

[0435] In some embodiments, suitable delivery vehicles are formulated using polymers as carriers, either alone or in combination with other carriers including the various lipids described herein. Thus, in some embodiments, liposome delivery vehicles as used herein also encompass nanoparticles comprising polymers. Suitable polymers can include, for example, polyacrylates, polyalkyl cyanoacrylates, polylactides, polylactide-polyglycolide copolymers, polycaprolactones, dextran, albumin, gelatin, alginate, collagen, chitosan, cyclodextrins, protamine, PEGylated protamine, PLL, PEGylated PLL, and polyethylenimine (PEI). When PEI is present, it can be a branched PEI with a molecular weight range of 10 to 40 kDa, for example, a branched PEI of 25 kDa (Sigma Cat. No. 408727).

[0436] According to various embodiments, the selection of the following is based on the characteristics of the selected one or more lipids, the nature of the intended target cell, the characteristics of the nucleic acid to be delivered: cationic lipids, non-cationic lipids, PEG-modified lipids, cholesterol-based lipids, and / or amphipathic block copolymers (comprising the lipid nanoparticles), and the relative molar ratios of such components (lipids) to each other. Additional considerations include, for example, the saturation of the alkyl chains and the size, charge, pH, pKa, fusogenicity, and toxicity of the selected one or more lipids. The molar ratios can thus be adjusted accordingly.

[0437] Ratios of different lipid components

[0438] Suitable liposomes for use in the present application can include one or more of the following: any of the cationic lipids, non-cationic lipids, cholesterol lipids, PEG-modified lipids, amphipathic block copolymers, and / or polymers described herein in various ratios. In some embodiments, the lipid nanoparticles comprise five and no more than five different nanoparticle components. In some embodiments, the lipid nanoparticles comprise four and no more than four different nanoparticle components. In some embodiments, the lipid nanoparticles comprise three and no more than three different nanoparticle components. As non-limiting examples, suitable liposome formulations can include combinations selected from: cKK-E12, 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.

[0439] In various embodiments, the cationic lipid (e.g., cKK-E12, C12-200, ICE, and / or HGT4003) comprises about 30-60% (e.g., about 30-55%, about 30-50%, about 30-45%, about 30-40%, about 35-50%, about 35-45%, or about 35-40%) of the liposome on a molar ratio basis. In some embodiments, the percentage of cationic lipid (e.g., cKK-E12, C12-200, ICE, and / or HGT4003) is about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, or about 60% of the liposome on a molar ratio basis.

[0440] In some embodiments, the ratio of one or more cationic lipids to one or more non-cationic lipids to one or more cholesterol-based lipids to one or more PEG-modified lipids can be about 30-60:25-35:20-30:1-15, respectively. In some embodiments, the ratio of one or more cationic lipids to one or more non-cationic lipids to one or more cholesterol-based lipids to one or more PEG-modified lipids is about 40:30:20:10, respectively. In some embodiments, the ratio of one or more cationic lipids to one or more non-cationic lipids to one or more cholesterol-based lipids to one or more PEG-modified lipids is about 40:30:25:5, respectively. In some embodiments, the ratio of one or more cationic lipids to one or more non-cationic lipids to one or more cholesterol-based lipids to one or more PEG-modified lipids is about 40:32:25:3, respectively. In some embodiments, the ratio of one or more cationic lipids to one or more non-cationic lipids to one or more cholesterol-based lipids to one or more PEG-modified lipids is about 50:25:20:5.

[0441] In embodiments in which the lipid nanoparticle comprises three and no more than three different lipid components, the ratio of the total lipid content (i.e., the ratio of lipid component (1):lipid component (2):lipid component (3)) can be represented as x:y:z, where

[0442] (y+z) = 100 - x.

[0443] In some embodiments, each of“x,”“y,” and“z” represents a molar percentage of the three different components of the lipid, and the ratio is a molar ratio.

[0444] In some embodiments, each of“x,”“y,” and“z” represents a weight percentage of the three different components of the lipid, and the ratio is a weight ratio.

[0445] In some embodiments, the lipid component (1) represented by the variable “x” is a sterol-based cationic lipid.

[0446] In some embodiments, the lipid component (2) represented by the variable “y” is a helper lipid.

[0447] In some embodiments, the lipid component (3) represented by the variable “z” is a PEG lipid.

[0448] In some embodiments, the variable “x” representing the mole percent of the lipid component (1) (e.g., 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%.

[0449] In some embodiments, the variable “x” representing the mole percent of the lipid component (1) (e.g., 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 no more than about 65%, about 60%, about 55%, about 50%, about 40%.

[0450] In some embodiments, the variable “x” representing the mole percent of the lipid component (1) (e.g., 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 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%.

[0451] In some embodiments, the variable “x” representing the weight percent of the lipid component (1) (e.g., 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%.

[0452] In some embodiments, the variable "x" representing the weight percent of lipid component (1) (e.g., 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 no more than about 65%, about 60%, about 55%, about 50%, about 40%.

[0453] In some embodiments, the variable "x" representing the weight percent of lipid component (1) (e.g., 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 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%.

[0454] In some embodiments, the variable "z" representing the mole percent of lipid component (3) (e.g., 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 mole percent of lipid component (3) (e.g., PEG lipid) is about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%. In embodiments, the variable "z" representing the mole percent of lipid component (3) (e.g., PEG lipid) is 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%.

[0455] In some embodiments, the variable "z" representing the weight percent of lipid component (3) (e.g., 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 percent of lipid component (3) (e.g., PEG lipid) is about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%. In embodiments, the variable "z" representing the weight percent of lipid component (3) (e.g., PEG lipid) is 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%.

[0456] For compositions having three and only three different lipid components, the variables "x", "y", and "z" can be any combination, as long as the sum of the three variables is 100% of the total lipid content.

[0457] Formation of mRNA-encapsulating liposomes

[0458] Liposome transfer vehicles for use in the compositions of the present application can be prepared by a variety of techniques currently known in the art. For example, multilamellar vesicles (MLVs) can be prepared according to conventional techniques, such as by depositing the selected lipids on the inside wall of a suitable container or vessel by dissolving the lipids in an appropriate solvent, then evaporating the solvent to leave a thin film on the inside of the vessel; or by spray-drying. An aqueous phase can then be added to the vessel with concomitant vortexing, which results in the formation of MLVs. Unilamellar vesicles (ULVs) can then be formed by homogenization, sonication, or extrusion of the multilamellar vesicles. In addition, unilamellar vesicles can be formed by detergent removal techniques.

[0459] Various methods are described in published U.S. Application No. US 2011 / 0244026, published U.S. Application No. US 2016 / 0038432, published U.S. Application No. US 2018 / 0153822, published U.S. Application No. US 2018 / 0125989, and U.S. Provisional Application No. 62 / 877,597, filed July 23, 2019, and can be used to practice the present application, all of which are incorporated herein by reference. As used herein, Process A refers to a conventional method of encapsulating mRNA by mixing the mRNA with a mixture of lipids as described in US 2016 / 0038432, which does not require first pre-forming the lipids into lipid nanoparticles. As used herein, Process B refers to a process of encapsulating messenger RNA (mRNA) by mixing pre-formed lipid nanoparticles with mRNA as described in US 2018 / 0153822.

[0460] Briefly, the process of preparing a mRNA or MCNA loaded lipid liposome includes the step of heating (i.e., applying heat from a heat source to the solution) one or more solutions to a temperature greater than ambient temperature (or maintaining a temperature), the one or more solutions being a solution comprising preformed lipid nanoparticles, a solution comprising mRNA, and a mixed solution comprising lipid nanoparticle encapsulated mRNA. In some embodiments, the process includes the step of heating one or both of the mRNA solution and the preformed lipid nanoparticle solution prior to the mixing step. In some embodiments, the process includes heating one or more of the one or more solutions comprising preformed lipid nanoparticles, the solution comprising mRNA, and the solution comprising lipid nanoparticle encapsulated mRNA during the mixing step. In some embodiments, the process includes the step of heating the lipid nanoparticle encapsulated mRNA after the mixing step. In some embodiments, the one or more solutions are heated to (or the one or more solutions are maintained at) a temperature of or greater than about 30°C, 37°C, 40°C, 45°C, 50°C, 55°C, 60°C, 65°C, or 70°C. In some embodiments, the one or more solutions are heated to a temperature range of about 25°C-70°C, about 30°C-70°C, about 35°C-70°C, about 40°C-70°C, about 45°C-70°C, about 50°C-70°C, or about 60°C-70°C. In some embodiments, the one or more solutions are heated to a temperature greater than ambient temperature of about 65°C.

[0461] Various methods can be used to prepare an mRNA solution suitable for use in the present application. In some embodiments, mRNA can be directly dissolved in the buffer solutions described herein. In some embodiments, an mRNA solution can be generated by mixing an mRNA stock solution with a buffer solution prior to 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 prior to mixing with a lipid solution for encapsulation. In some embodiments, a suitable mRNA stock solution can contain mRNA in water at a concentration of 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, or 1.6 mg / ml, 2.0 mg / ml, 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.

[0462] In some embodiments, a pump is used to mix the mRNA stock solution with the buffer solution. Exemplary pumps include, but are not limited to, gear pumps, peristaltic pumps, and centrifugal pumps.

[0463] Typically, the mixing rate of the buffer solution is greater than the mixing rate of the mRNA stock solution. For example, the mixing rate of the buffer solution can be at least 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, or 20-fold greater than the mixing rate of the mRNA stock solution. In some embodiments, the buffer solution is mixed at a flow rate ranging from about 100-6000 ml / min (e.g., about 100-300 ml / min, 300-600 ml / min, 600-1200 ml / min, 1200-2400 ml / min, 2400-3600 ml / min, 3600-4800 ml / min, 4800-6000 ml / min, or 60-420 ml / min). In some embodiments, the buffer solution is mixed at a flow rate of about 60 ml / min, 100 ml / min, 140 ml / min, 180 ml / min, 220 ml / min, 260 ml / min, 300 ml / min, 340 ml / min, 380 ml / min, 420 ml / min, 480 ml / min, 540 ml / min, 600 ml / min, 1200 ml / min, 2400 ml / min, 3600 ml / min, 4800 ml / min, or 6000 ml / min, or greater.

[0464] In some embodiments, the mRNA stock solution is mixed at a flow rate ranging from about 10-600 ml / min (e.g., about 5-50 ml / min, about 10-30 ml / min, about 30-60 ml / min, about 60-120 ml / min, about 120-240 ml / min, about 240-360 ml / min, about 360-480 ml / min, or about 480-600 ml / min). In some embodiments, the mRNA stock solution is mixed at a flow rate of about 5 ml / min, 10 ml / min, 15 ml / min, 20 ml / min, 25 ml / min, 30 ml / min, 35 ml / min, 40 ml / min, 45 ml / min, 50 ml / min, 60 ml / min, 80 ml / min, 100 ml / min, 200 ml / min, 300 ml / min, 400 ml / min, 500 ml / min, or 600 ml / min, or greater.

[0465] According to the present application, the lipid solution contains a mixture of lipids suitable for forming lipid nanoparticles for encapsulating mRNA. In some embodiments, a suitable lipid solution is ethanol-based. For example, a suitable lipid solution can contain a mixture of desired lipids dissolved in pure ethanol (i.e., 100% ethanol). In another embodiment, a suitable lipid solution is isopropanol-based. In another embodiment, a suitable lipid solution is dimethyl sulfoxide-based. In another embodiment, a suitable lipid solution is a mixture of suitable solvents including, but not limited to, ethanol, isopropanol, and dimethyl sulfoxide.

[0466] A suitable lipid solution can contain a mixture of desired lipids at various concentrations. For example, a suitable lipid solution can contain a mixture of desired lipids at a total concentration of about 0.1 mg / ml, 0.5 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, or greater. In some embodiments, a suitable lipid solution can contain a mixture of desired lipids at a total concentration ranging from about 0.1-100 mg / ml, 0.5-90 mg / ml, 1.0-80 mg / ml, 1.0-70 mg / ml, 1.0-60 mg / ml, 1.0-50 mg / ml, 1.0-40 mg / ml, 1.0-30 mg / ml, 1.0-20 mg / ml, 1.0-15 mg / ml, 1.0-10 mg / ml, 1.0-9 mg / ml, 1.0-8 mg / ml, 1.0-7 mg / ml, 1.0-6 mg / ml, or 1.0-5 mg / ml. In some embodiments, a suitable lipid solution can 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.

[0467] Any desired lipids can be mixed in any ratio suitable for encapsulating mRNA. In some embodiments, a suitable lipid solution contains a mixture of desired lipids, including a cationic lipid, a helper lipid (e.g., a non-cationic lipid and / or a cholesterol lipid), an amphiphilic block copolymer (e.g., a poloxamer), and / or a PEGylated lipid. In some embodiments, a suitable lipid solution contains a mixture of desired lipids, including one or more cationic lipids, one or more helper lipids (e.g., a non-cationic lipid and / or a cholesterol lipid), and one or more PEGylated lipids.

[0468] In certain embodiments, the provided compositions comprise liposomes in which mRNA is bound to the surface of the liposome and encapsulated within the same liposome. For example, during preparation of the compositions of the application, cationic liposomes can bind to mRNA or MCNA through electrostatic interactions.

[0469] In some embodiments, the compositions and methods of the application comprise mRNA encapsulated in liposomes. In some embodiments, one or more mRNA species can be encapsulated in the same liposome. In some embodiments, one or more mRNA species can be encapsulated in different liposomes. In some embodiments, mRNA is encapsulated in one or more liposomes that differ in their lipid composition, molar ratio of lipid components, size, charge (zeta potential), targeting ligand, and / or combinations thereof. In some embodiments, one or more liposomes can have different compositions of sterol-based cationic lipids, neutral lipids, PEG-modified lipids, and / or combinations thereof. In some embodiments, one or more liposomes can have different molar ratios of cholesterol-based cationic lipids, neutral lipids, and PEG-modified lipids used to generate the liposome.

[0470] The process of incorporating a desired nucleic acid (e.g., mRNA or MCNA) into a liposome is often referred to as "loading." Exemplary methods are described in Lasic et al. FEBS Lett. 312:255-258, 1992, which is incorporated herein by reference. Nucleic acids incorporated into liposomes can be located entirely or partially within the interior space of the liposome, within the bilayer membrane of the liposome, or bound to the outer surface of the liposome membrane. Incorporation of nucleic acids into liposomes is also referred to herein as "encapsulation," in which the nucleic acid is entirely contained within the interior space of the liposome. The purpose of incorporating mRNA into a transfer vehicle, such as a liposome, is generally to protect the nucleic acid from the environment, which can contain enzymes or chemicals that degrade the nucleic acid and / or systems or receptors that cause rapid excretion of the nucleic acid. Thus, in some embodiments, a suitable delivery vehicle is capable of enhancing the stability of the mRNA contained therein and / or facilitating delivery of the therapeutic (e.g., mRNA or MCNA) to the target cell or tissue.

[0471] Suitable liposomes according to the present application can be made in a variety of sizes. In some embodiments, the liposomes provided can be made smaller than previously known liposomes. In some embodiments, the reduced liposome size is associated with more efficient delivery of the therapeutic agent (e.g., mRNA or MCNA). The selection of a suitable liposome size can take into account the location of the target cell or tissue, and to some extent, the application for which the liposome is being prepared.

[0472] In some embodiments, the liposome is selected to be of an appropriate size to facilitate systemic distribution of the antibody encoded by the mRNA. In some embodiments, it can be desirable to limit transfection of the mRNA to certain cells or tissues. For example, to target hepatocytes, the size of the liposome can be adjusted to be smaller than the fenestrations of the endothelial layer lining the sinusoids of the liver; in such a case, the liposome can readily penetrate such endothelial fenestrations to reach the target hepatocytes.

[0473] Alternatively or additionally, the size of the liposome can be adjusted so that the liposome is of a sufficient diameter to limit or specifically avoid distribution into certain cells or tissues.

[0474] Various alternative methods known in the art can be used to size the population of liposomes. One such method of sizing is described in U.S. Patent No. 4,737,323, which is incorporated herein by reference. Gradual size reduction to small ULVs of less than about 0.05 microns in diameter can be achieved by water bath or probe sonication of the liposome suspension. Homogenization is another method that relies on shear energy to fragment large liposomes into smaller ones. In a typical homogenization procedure, MLVs are recirculated through a standard emulsification homogenizer until a selected liposome size, typically about 0.1 to 0.5 microns, is observed. The size of the liposomes can be determined by quasi-elastic light scattering (QELS) as described in Bloomfield, Ann. Rev. Biophys. Bioeng., 10:421-450 (1981), which is incorporated herein by reference. The average liposome diameter can be reduced by subjecting the formed liposomes to sonication. Intermittent sonication cycles can be alternated with QELS evaluation to guide efficient liposome synthesis.

[0475] Provided are nanoparticles encapsulating mRNA

[0476] In some embodiments, a majority of the purified nanoparticles in the composition (i.e., greater than about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of the nanoparticles) are about 150 nm in size (e.g., 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, or about 80 nm). In some embodiments, substantially all of the purified nanoparticles are about 150 nm in size (e.g., 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, or about 80 nm).

[0477] In some embodiments, the average size of the lipid nanoparticle is less than 150 nm. In some embodiments, the average size of the lipid nanoparticle is less than 120 nm. In some embodiments, the average size of the lipid nanoparticle is less than 100 nm. In some embodiments, the average size of the lipid nanoparticle is less than 90 nm. In some embodiments, the average size of the lipid nanoparticle is less than 80 nm. In some embodiments, the average size of the lipid nanoparticle is less than 70 nm. In some embodiments, the average size of the lipid nanoparticle is less than 60 nm. In some embodiments, the average size of the lipid nanoparticle is less than 50 nm. In some embodiments, the average size of the lipid nanoparticle is less than 30 nm. In some embodiments, the average size of the lipid nanoparticle is less than 20 nm.

[0478] In some embodiments, the present application provides a composition in which the measure of the dispersion of the nanoparticles, or the amount of molecular size heterogeneity (PDI), is less than about 0.5. In some embodiments, the PDI of the lipid nanoparticle is less than about 0.5. In some embodiments, the PDI of the lipid nanoparticle is less than about 0.4. In some embodiments, the PDI of the lipid nanoparticle is less than about 0.3. In some embodiments, the PDI of the lipid nanoparticle is less than about 0.28. In some embodiments, the PDI of the lipid nanoparticle is less than about 0.25. In some embodiments, the PDI of the lipid nanoparticle is less than about 0.23. In some embodiments, the PDI of the lipid nanoparticle is less than about 0.20. In some embodiments, the PDI of the lipid nanoparticle is less than about 0.18. In some embodiments, the PDI of the lipid nanoparticle is less than about 0.16. In some embodiments, the PDI of the lipid nanoparticle is less than about 0.14. In some embodiments, the PDI of the lipid nanoparticle is less than about 0.12. In some embodiments, the PDI of the lipid nanoparticle is less than about 0.10. In some embodiments, the PDI of the lipid nanoparticle is less than about 0.08.

[0479] In some embodiments, greater than about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of the purified lipid nanoparticles in the composition encapsulate mRNA within each individual particle. In some embodiments, substantially all of the purified lipid nanoparticles in the composition encapsulate mRNA within each individual particle. In some embodiments, the encapsulation efficiency of the lipid nanoparticles is from 50% to 99%. In some embodiments, the encapsulation efficiency of the lipid nanoparticles is greater than about 60%. In some embodiments, the encapsulation efficiency of the lipid nanoparticles is greater than about 65%. In some embodiments, the encapsulation efficiency of the lipid nanoparticles is greater than about 70%. In some embodiments, the encapsulation efficiency of the lipid nanoparticles is greater than about 75%. In some embodiments, the encapsulation efficiency of the lipid nanoparticles is greater than about 80%. In some embodiments, the encapsulation efficiency of the lipid nanoparticles is greater than about 85%. In some embodiments, the encapsulation efficiency of the lipid nanoparticles is greater than about 90%. In some embodiments, the encapsulation efficiency of the lipid nanoparticles is greater than about 92%. In some embodiments, the encapsulation efficiency of the lipid nanoparticles is greater than about 95%. In some embodiments, the encapsulation efficiency of the lipid nanoparticles is greater than about 98%. In some embodiments, the encapsulation efficiency of the lipid nanoparticles is greater than about 99%.

[0480] In some embodiments, the N / P ratio of the lipid nanoparticles is from 1 to 10. As used herein, the term "N / P ratio" refers to the molar ratio of positively charged molecular units in the cationic lipids in the lipid nanoparticle relative to the negatively charged molecular units in the mRNA encapsulated within the lipid nanoparticle. As such, the N / P ratio is typically calculated as the ratio of moles of amine groups in the cationic lipids in the lipid nanoparticle relative to the moles of phosphate groups in the mRNA encapsulated within the lipid nanoparticle. In some embodiments, the N / P ratio of the lipid nanoparticles is greater than 1. In some embodiments, the N / P ratio of the lipid nanoparticles is about 1. In some embodiments, the N / P ratio of the lipid nanoparticles is about 2. In some embodiments, the N / P ratio of the lipid nanoparticles is about 3. In some embodiments, the N / P ratio of the lipid nanoparticles is about 4. In some embodiments, the N / P ratio of the lipid nanoparticles is about 5. In some embodiments, the N / P ratio of the lipid nanoparticles is about 6. In some embodiments, the N / P ratio of the lipid nanoparticles is about 7. In some embodiments, the N / P ratio of the lipid nanoparticles is about 8.

[0481] In some embodiments, the compositions according to the application contain at least about 0.5 mg, 1 mg, 5 mg, 10 mg, 100 mg, 500 mg, or 1000 mg of encapsulated mRNA. In some embodiments, the compositions contain about 0.1 mg to 1000 mg of encapsulated mRNA. In some embodiments, the compositions contain at least about 0.5 mg of encapsulated mRNA. In some embodiments, the compositions contain at least about 0.8 mg of encapsulated mRNA. In some embodiments, the compositions contain at least about 1 mg of encapsulated mRNA. In some embodiments, the compositions contain at least about 5 mg of encapsulated mRNA. In some embodiments, the compositions contain at least about 8 mg of encapsulated mRNA. In some embodiments, the compositions contain at least about 10 mg of encapsulated mRNA. In some embodiments, the compositions contain at least about 50 mg of encapsulated mRNA. In some embodiments, the compositions contain at least about 100 mg of encapsulated mRNA. In some embodiments, the compositions contain at least about 500 mg of encapsulated mRNA. In some embodiments, the compositions contain at least about 1000 mg of encapsulated mRNA.

[0482] Therapeutic uses of the compositions

[0483] To facilitate expression of the mRNA in vivo, the delivery vehicle (e.g., liposome) can be formulated in combination with one or more additional nucleic acids, carriers, targeting ligands, or stabilizing agents, or in a pharmacological composition in admixture with suitable excipients. Techniques for formulation and administration of pharmaceuticals can be found in the latest edition of “Remington’s Pharmaceutical Sciences,” Mack Publishing Co., Easton, Pa.

[0484] In some embodiments, the compositions comprise mRNA encapsulated or complexed with a delivery vehicle. In some embodiments, the delivery vehicle is selected from the group consisting of a liposome, a lipid nanoparticle, a solid lipid nanoparticle, a polymer, a virus, a sol-gel, and a nanogel.

[0485] The mRNA-loaded nanoparticles and compositions containing the same provided can be administered and dosed according to current medical practice, taking into account the clinical condition of the subject, the site and method of administration, the scheduling of administration, the age, sex, weight, and medical condition of the subject, and other factors relevant to the particular patient and to the medical arts. An “effective amount” for purposes herein can be determined by such relevant considerations as are known to one of ordinary skill in the experimental clinical research, pharmacological, clinical, and medical arts. In some embodiments, the amount administered is effective to achieve at least some stabilization, amelioration, or elimination of symptoms and other indicators selected by those of skill in the art as appropriate measures of disease progression, regression, or amelioration. For example, a suitable amount and dosing regimen is one that results in at least transient protein (e.g., enzyme) production.

[0486] The present disclosure provides methods of delivering mRNA for in vivo protein production, the methods comprising administering mRNA to a subject in need of delivery. In some embodiments, the mRNA is administered via a delivery route selected from the group consisting of intravenous delivery, subcutaneous delivery, oral delivery, subdermal delivery, ocular delivery, intratracheal injection pulmonary delivery (e.g., aerosolization), intramuscular delivery, intrathecal delivery, or intra-articular delivery.

[0487] Suitable routes of administration include, e.g., oral, rectal, vaginal, transmucosal, pulmonary including intratracheal or inhalation, or intestinal administration; parenteral delivery, including intradermal, transdermal (topical), intramuscular, subcutaneous, intramedullary injection, and intrathecal, direct intracerebroventricular, intravenous, intraperitoneal, or intranasal. In some embodiments, intramuscular administration is to a muscle selected from the group consisting of skeletal muscle, smooth muscle, and cardiac muscle. In some embodiments, the administration results in delivery of the mRNA to a muscle cell. In some embodiments, the administration results in delivery of the mRNA to a hepatocyte (i.e., liver cell). In particular embodiments, intramuscular administration results in delivery of the mRNA to a muscle cell.

[0488] Additional teachings of pulmonary delivery and aerosolization are described in published U.S. Application No. US 2018 / 0125989 and published U.S. Application No. US 2018 / 0333457, each incorporated by reference in its entirety.

[0489] Alternatively or additionally, the mRNA-loaded nanoparticles and compositions of the application can be administered in a local rather than systemic manner, for example, via injection of the pharmaceutical composition directly into a target tissue, preferably in a sustained release formulation. Local delivery can be achieved in a variety of ways depending on the tissue to be targeted. For example, an aerosol containing the compositions of the application can be inhaled (for nasal, tracheal, or bronchial delivery); for example, the compositions of the application can be injected into a site of injury, disease manifestation, or pain; the compositions can be provided in lozenge form for oral, tracheal, or esophageal application; can be provided in liquid, tablet, or capsule form for administration to the stomach or intestines; can be provided in suppository form for rectal or vaginal application; or can even be delivered to the eye by use of a cream, drop, or even an injectable solution. Formulations containing the provided compositions complexed with a therapeutic molecule or ligand can even be administered surgically, for example, in conjunction with a polymer or other structure or substance that is capable of allowing the composition to diffuse from the site of implantation to surrounding cells. Alternatively, they can be applied surgically without the use of a polymer or support.

[0490] The methods provided herein contemplate single and multiple administrations of a therapeutically effective amount of a therapeutic agent (e.g., mRNA) described herein. Depending on the nature, severity, and extent of the subject’s condition, the therapeutic agent can be administered at regular intervals. In some embodiments, a therapeutically effective amount of a therapeutic agent (e.g., mRNA) of the application can be administered intrathecally periodically at regular intervals (e.g., once a year, once every six months, once every five months, once every three months, once every two months (every two months), once a month (every month), once every two weeks (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, once a week, twice a week, every day, or continuously).

[0491] In some embodiments, the liposomes and / or compositions provided are formulated so that they are suitable for extended release of the mRNA contained therein. Such extended release compositions can be conveniently administered to a subject at extended dosing intervals. For example, in one embodiment, the compositions of the application are administered to a subject twice daily, daily, or every other day. In preferred embodiments, the compositions of the application are administered to a subject twice weekly, once weekly, 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 other month, once every three months, once every four months, once every six months, once every eight months, once every nine months, or once a year. Also contemplated are compositions and liposomes formulated for depot administration (e.g., intramuscular, subcutaneous, intravitreal) to deliver or release a therapeutic agent (e.g., mRNA) over an extended period of time. Preferably, the extended release means employed is combined with a modification that enhances the stability of the mRNA.

[0492] As used herein, the term "therapeutically effective amount" is determined primarily based on the total amount of the therapeutic agent contained in the pharmaceutical compositions of the application. Generally, a therapeutically effective amount is sufficient to effect a meaningful benefit (e.g., treatment, modulation, cure, prevention, and / or alleviation of a disease or disorder) in a subject. For example, a therapeutically effective amount can be an amount sufficient to achieve a desired therapeutic and / or prophylactic effect. Generally, the amount of the therapeutic agent (e.g., mRNA) administered to a subject in need will depend on the characteristics of that subject. Such characteristics include the subject's condition, disease severity, general health, age, sex, and body weight. One of ordinary skill in the art will be able to determine appropriate dosages depending on these and other relevant factors.

[0493] A therapeutically effective amount is generally administered in a regimen that can comprise multiple unit doses. The therapeutically effective amount (and / or appropriate unit dose in an effective regimen) can vary for any particular therapeutic protein, e.g., depending on the route of administration, depending on the combination with other agents. Furthermore, the specific therapeutically effective amount (and / or unit dose) for any particular patient can depend on a variety of factors including the disorder being treated and the severity of the disorder; the activity of the specific agent employed; the specific composition employed; the patient's age, body weight, general health, sex, and diet; the time of administration, route of administration, and / or rate of excretion or metabolism of the specific protein employed; the duration of the treatment; and like factors well known in the medical arts.

[0494] In some embodiments, the effective therapeutic dose ranges from about 0.005 mg / kg body weight to 500 mg / kg body weight, for example, from about 0.005 mg / kg body weight to 400 mg / kg body weight, from about 0.005 mg / kg body weight to 300 mg / kg body weight, from about 0.005 mg / kg body weight to 200 mg / kg body weight, from about 0.005 mg / kg body weight to 100 mg / kg body weight, from about 0.005 mg / kg body weight to 90 mg / kg body weight, from about 0.005 mg / kg body weight to 80 mg / kg body weight, and from about 0.005 mg / kg body weight to 500 mg / kg body weight. From body weight to 70 mg / kg 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 body weight, from about 0.005 mg / kg body weight to 20 mg / kg body weight, from about 0.005 mg / kg body weight to 15 mg / kg body weight, from about 0.005 mg / kg body weight to 10 mg / kg body weight.

[0495] In some embodiments, the therapeutically effective dose is greater than about 0.1 mg / kg body weight, greater than about 0.5 mg / kg body weight, greater than about 1.0 mg / kg body weight, greater than about 3 mg / kg body weight, greater than about 5 mg / kg body weight, greater than about 10 mg / kg body weight, greater than about 15 mg / kg body weight, greater than about 20 mg / kg body weight, greater than about 30 mg / kg body weight, greater than about 40 mg / kg body weight, greater than about 50 mg / kg body weight, greater than about 60 mg / kg body weight, greater than about 70 mg / kg body weight, greater than about 80 mg / kg body weight, greater than about 90 mg / kg body weight, greater than about 100 mg / kg body weight, greater than about 150 mg / kg body weight, greater than about 200 mg / kg body weight, greater than about 250 mg / kg body weight, greater than about 300 mg / kg body weight, greater than about 350 mg / kg body weight, greater than about 400 mg / kg body weight, greater than about 450 mg / kg body weight, and greater than about 500 mg / kg body weight. In certain embodiments, 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.

[0496] Also contemplated herein are lyophilized pharmaceutical compositions comprising one or more liposomes disclosed herein and related methods of using 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, a lyophilized pharmaceutical composition according to the present application can be reconstituted prior to administration or can be reconstituted in vivo. For example, a lyophilized pharmaceutical composition can be formulated into an appropriate dosage form (e.g., an intradermal dosage form, such as a disc, a rod, or a film) and administered such that the dosage form is rehydrated by the bodily fluids of the individual over time in vivo.

[0497] The provided liposomes and compositions can be administered to any desired tissue. In some embodiments, the mRNA delivered by the provided liposomes or compositions is expressed in the tissue to which the liposomes and / or compositions are administered. In some embodiments, the mRNA delivered is expressed in a tissue different from the tissue to which the liposomes and / or compositions are administered. Exemplary tissues in which the delivered mRNA can be delivered and / or expressed include, but are not limited to, liver, kidney, heart, spleen, serum, brain, skeletal muscle, lymph node, skin, and / or cerebrospinal fluid.

[0498] In some embodiments, administration of the provided compositions results in an increase in mRNA expression levels in a biological sample from a subject as compared to baseline expression levels prior to treatment. Typically, baseline levels are measured immediately prior to 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 compositions results in an increase in mRNA expression levels of at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% as compared to baseline expression levels immediately prior to treatment. In some embodiments, administration of the provided compositions results in an increase in mRNA expression levels of at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% as compared to mRNA expression levels in an untreated subject.

[0499] According to various embodiments, the timing of expression of the delivered mRNA can be adjusted to suit particular medical needs. In some embodiments, expression of a protein encoded by the delivered mRNA is detectable 1, 2, 3, 6, 12, 24, 48, 72, and / or 96 hours after administration of the provided liposomes and / or compositions. In some embodiments, expression of a protein encoded by the delivered mRNA is detectable one week, two weeks, and / or one month after administration.

[0500] The present application also provides compositions that deliver mRNA molecules having a coding for a peptide or polypeptide of interest for use in treating a subject, e.g., a human subject, or a cell of a human subject, or a cell treated and delivered to a human subject.

[0501] Examples

[0502] While certain compounds, compositions, and methods of the application have been described with specificity in terms of certain embodiments, the following examples are merely illustrative of the compounds of the application and are not intended to limit the same.

[0503] Example 1. Formulation of mRNA-LNP compositions for rectal delivery

[0504] This example illustrates an exemplary process for making a composition comprising mRNA encapsulated within a lipid nanoparticle (LNP) suitable for rectal delivery.

[0505] Messenger RNA was encapsulated within a lipid nanoparticle comprising ML-2:DOPE:Cholesterol:DMG-PEG (40:30:25:5) using Process B. As used herein, Process B refers to a process for encapsulating mRNA by mixing preformed lipid nanoparticles with mRNA as described in US 2018 / 0153822, which is incorporated herein in its entirety. Suppositories were prepared in LNP with a 10% w / v concentration of gelatin. Briefly, gelatin was dissolved directly in LNP at 65 °C over 5 minutes and poured into disposable molds. These molds were then frozen at -80 °C. Suppositories comprising LNP encapsulating mRNA remained intact. Exemplary suppositories comprising mRNA-LNP are shown in Figure 1 .

[0506] A Labrasol (permeation enhancer) solution was prepared by dissolving 153 mg Labrasol in 1 mL of water.

[0507] Different amounts of gelatin can be used to control the melting rate of the suppository, and thus the release time of mRNA encapsulated within LNP once administered to a subject. Additionally, viscosity-modulating excipients can be added to control the release time.

[0508] Example 2. Rectal delivery of FFLuc mRNA-LNP to mice

[0509] This example illustrates the successful rectal delivery of mRNA in a lipid nanoparticle.

[0510] Firefly luciferase (FFLuc) mRNA encapsulated within a lipid nanoparticle (0.2 mg / animal) or saline was administered rectally in mice. Whole body and individual tissues were imaged 24 hours after rectal administration, as shown in FIGS. 2 and 3.

[0511] As Figure 2A and Figure 2BAs expected, mice dosed with saline rectally showed no luminescence. Mice dosed with FFLuc mRNA-LNP rectally exhibited luminescence, especially high luminescence at the rectal region Figure 3A ). In tissues, the clones showed strong luminescence, as Figure 3B illustrated.

[0512] This example demonstrates that mRNA-LNP can be successfully delivered rectally for protein expression in vivo. Expression was detected in the rectum and colon.

[0513] Example 3. Rectal delivery of FFLuc mRNA-LNP and a permeability enhancer

[0514] This example demonstrates the successful delivery of mRNA in a lipid nanoparticle and sodium caprate, a permeability enhancer. Rectal administration of mRNA-LNP and sodium caprate significantly increased protein expression in vivo.

[0515] A group of mice was dosed with 0.2 mg FFLuc mRNA-LNP (Group 1) as described in Example 2. A second group of mice was pre-dosed with sodium caprate (200 mg / ml solution - 50 ult injection) prior to rectal administration of 0.05 mg of FFLuc mRNA-LNP (Group 2). Whole body and individual tissues were imaged 24 hours after rectal administration.

[0516] As shown in Figure 4 , mice dosed with 0.05 mg FFLuc mRNA-LNP and sodium caprate rectally showed significantly higher signal compared to mice dosed with 0.2 mg FFLuc mRNA-LNP. Figure 5 It is shown that despite the dose of mRNA-LNP being 25% of the dose in Group 1, the luminescence of Group 2 was almost doubled.

[0517] This example demonstrates that a permeability enhancer can increase the expression of protein delivered by mRNA-LNP.

[0518] Example 4. Rectal delivery of FFLuc mRNA-LNP in suppositories to mice and rats

[0519] This example demonstrates the successful rectal delivery of mRNA-LNP in a suppository formulation. Rectal administration of mRNA-LNP in a suppository significantly increased protein expression in vivo and protein expression was detected in various tissues.

[0520] Mice or rats were dosed rectally with 30 μΐ of Labrasol solution (153 mg / ml). After 30 minutes, a suppository formulation of FFLuc mRNA-LNP was administered rectally. Whole body and individual tissues were imaged 24 hours after rectal administration.

[0521] Figure 6A Mice rectally administered FFLuc mRNA-LNP in a suppository showed significantly higher luminescence compared to mice rectally administered mRNA-LNP without a suppository. In addition, strong luminescence signals were observed in different tissues (e.g. rectal colon, liver, kidney, etc.). As shown in Figure 6B Two out of four rats showed luminescence in the liver, colon, and rectum. Smaller variability was observed in mice compared to rats.

[0522] This example demonstrates that when mRNA-LNP is delivered in the form of a suppository, a significant increase in protein expression is observed. This example also supports that a suppository can help mRNA-LNP survive in RNase and mucus barriers and can also control the release of mRNA-LNP in a subject's body once delivered. In addition, in vivo protein expression was detected in various tissues including the kidney. This is significant because it is known that targeted delivery of agents into the mouse kidney is laborious and can require laparotomy surgery. Additionally, expression of FFL in the liver indicates uptake of LNP in systemic circulation.

[0523] Example 5. Rectal delivery of EPO mRNA-LNP in a suppository to rats

[0524] This example demonstrates the successful rectal delivery of mRNA-LNP in a suppository against a secreted protein.

[0525] Rats were dosed rectally with 30 μΐ of Labrasol solution (153 mg / ml). After 30 minutes, a suppository formulation of hEPO mRNA-LNP was administered rectally. hEPO levels in serum were measured 24 hours after administration. As shown in Figure 7 Rats rectally dosed with hEPO mRNA-LNP in a suppository showed detectable levels of hEPO in serum. These levels were much higher than normal physiological levels of EPO.

[0526] This example shows that rectal delivery of mRNA-LNP in the form of a suppository can successfully provide expressed protein in systemic circulation.

[0527] Equivalents and Ranges

[0528] Those skilled in the art will appreciate or, using no more than routine experimentation, be able to determine many equivalents of the specific examples of the application described herein. It is therefore not intended that the application be limited, except as presented in the following claims.

Claims

1. A combination of a permeation enhancer and a suppository composition, wherein the permeation enhancer comprises sodium caprate or Labrasol® and the suppository comprises: a. mRNA encapsulated within a lipid nanoparticle, wherein the mRNA encodes a protein or peptide; and b. gelatin; wherein the suppository is formulated for rectal administration.

2. The composition of claim 1, wherein the suppository comprises 5% or more of the gelatin in water.

3. The composition of claim 2, wherein the suppository comprises 10% or more of the gelatin in water.

4. The composition of claim 3, wherein the suppository comprises 20% or more of the gelatin in water.

5. The composition of claim 4, wherein the suppository comprises 30% or more of the gelatin in water.

6. The composition of claim 5, wherein the suppository comprises 50% or more of the gelatin in water.

7. The composition of claim 1, wherein the suppository does not comprise a lipid-based suppository component.

8. The composition of claim 7, wherein the lipid-based suppository component is cocoa butter, cacao oil, or synthetic fat.

9. The composition of any one of claims 1-8, wherein the permeation enhancer is present in the suppository.

10. The composition of any one of claims 1-8, wherein the permeation enhancer is separate from the suppository.

11. The composition of any one of claims 1-8, wherein the permeation enhancer is sodium caprate.

12. The composition of any one of claims 1-8, wherein the permeation enhancer is Labrasol®.

13. The composition of any one of claims 1-8, wherein the suppository further comprises glycerol and / or PEG.

14. The composition of any one of claims 1-8, wherein the suppository softens or melts at 36°C to 37°C. ​ ​ ​

Images