Rectal delivery of messenger RNA

Abstract

In particular, the present invention provides effective methods and compositions for delivering messenger RNA (mRNA) by rectal delivery, which is based in part on the unexpected observation that mRNA can be effectively delivered to the circulation, liver, kidney, colon, and / or rectum by rectal delivery, despite barriers such as RNases and mucus layers.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of and priority to U.S. Provisional Patent Application No. 62 / 951,844, filed December 20, 2019, the contents of which are incorporated herein by reference. [Background technology]

[0002] The delivery of nucleic acids, particularly messenger RNA (mRNA), to target cells and tissues remains a technical challenge. For example, various challenges arise in delivering mRNA to targeted cells, including physical and chemical barriers. These challenges arise using a wide variety of 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 [Means for solving the problem]

[0003] The present invention provides effective methods and compositions for delivering messenger RNA (mRNA), particularly by rectal delivery. The present invention is based, in part, on the surprising discovery that lipid-encapsulated mRNA can be effectively delivered to the circulation, liver, kidney, intestine, colon, and / or rectum by mucosal delivery, including rectal delivery, despite many barriers, such as RNases and mucosal layers.

[0004] In one aspect, the present invention provides a method for delivery of messenger RNA (mRNA) to a subject for in vivo production of a protein or peptide in the subject, comprising administering to the subject by rectal delivery a composition comprising mRNA encoding the protein or peptide and encapsulated in lipid nanoparticles, wherein administration of the composition results in detectable expression of the protein or peptide encoded by the mRNA in the subject at least about 24 hours, about 48 hours, about 72 hours, or about 96 hours after administration.

[0005] In some embodiments, the protein or peptide encoded by the mRNA is detectable in the circulation of a 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 circulation of a subject at least about 24 hours after administration. In some embodiments, the protein or peptide encoded by the mRNA is detectable in the circulation of a subject at least about 48 hours after administration. In some embodiments, the protein or peptide encoded by the mRNA is detectable in the circulation of a subject at least about 72 hours after administration. In some embodiments, the protein or peptide encoded by the mRNA is detectable in the circulation of a subject at least about 96 hours after administration.

[0006] 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. Thus, in some embodiments, the protein or peptide encoded by the mRNA is detectable in the liver 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 liver 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 liver 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 liver of the subject at least about 96 hours after administration.

[0007] In some embodiments, the protein or peptide encoded by the mRNA is detectable 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 is detectable in the kidney 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 kidney 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 kidney 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 kidney of the subject at least about 96 hours after administration.

[0008] In some embodiments, the protein or peptide encoded by the mRNA is detectable 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 is detectable in the colon 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 colon 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 colon 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 colon of the subject at least about 96 hours after administration.

[0009] In some embodiments, the protein or peptide encoded by the mRNA is detectable in the subject's rectum 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 subject's rectum at least about 24 hours after administration. In some embodiments, the protein or peptide encoded by the mRNA is detectable in the subject's rectum at least about 48 hours after administration. In some embodiments, the protein or peptide encoded by the mRNA is detectable in the subject's rectum at least about 72 hours after administration. In some embodiments, the protein or peptide encoded by the mRNA is detectable in the subject's rectum at least about 96 hours after administration.

[0010] In some embodiments, the in vivo production of the protein or peptide is in the circulation, liver, kidney, colon, and / or rectum of a subject. Thus, in some embodiments, the in vivo production of the protein or peptide is in the circulation of a subject. In some embodiments, the in vivo production of the protein or peptide is in the liver of a subject. In some embodiments, the in vivo production of the protein or peptide is in the kidney of a subject. In some embodiments, the in vivo production of the protein or peptide is in the colon of a subject. In some embodiments, the in vivo production of the protein or peptide is in the rectum of a subject.

[0011] In some embodiments, the lipid nanoparticles comprise one or more cationic lipids, one or more non-cationic lipids, and one or more PEG-modified lipids. Thus, in some embodiments, the lipid nanoparticles comprise one or more cationic lipids. In some embodiments, the lipid nanoparticles comprise one or more non-cationic lipids. In some embodiments, the lipid nanoparticles comprise one or more PEG-modified lipids.

[0012] In certain embodiments, the lipid nanoparticles comprise cholesterol.

[0013] In some embodiments, rectal delivery is by suppository, enema, catheter, or bulb syringe. Thus, in some embodiments, rectal delivery is by suppository. In some embodiments, rectal delivery is by enema. In some embodiments, rectal delivery is by catheter. In some embodiments, rectal delivery is by bulb syringe.

[0014] In certain embodiments, rectal delivery is by suppository.

[0015] In certain embodiments, the composition does not include a lipid-based suppository component.

[0016] In some embodiments, the lipid-based suppository component is cocoa butter, theobroma oil, a synthetic fat, or a synthetic base. Thus, in some embodiments, the lipid-based suppository component is cocoa butter. In some embodiments, the lipid-based suppository component is theobroma oil. In some embodiments, the lipid-based suppository component is a synthetic fat. In some embodiments, the lipid-based suppository component is a synthetic base.

[0017] In certain embodiments, the composition comprises a permeation enhancer.

[0018] In certain embodiments, the permeation enhancer is selected from bile salts, surfactants, fatty acids and derivatives, glycerides, chelating agents, salicylates, or polymers. Thus, in certain embodiments, the permeation enhancer is a bile salt. In certain embodiments, the permeation enhancer is a fatty acid and derivatives. In certain embodiments, the permeation enhancer is a glyceride. In certain embodiments, the permeation enhancer is a chelating agent. In certain embodiments, the permeation enhancer is a salicylate. In certain embodiments, the permeation enhancer is a polymer.

[0019] In some embodiments, the fatty acids and derivatives are selected from sorbitan laurate, sodium caprate, sucrose, palmitate, lauroylcholine, sodium myristate, or palmitoylcarnitine. Thus, in some embodiments, the fatty acids and derivatives are sorbitan laurate. In some embodiments, the fatty acids and derivatives include sodium caprate. In some embodiments, the fatty acids and derivatives are sucrose. In some embodiments, the fatty acids and derivatives are palmitate. In some embodiments, the fatty acids and derivatives are lauroylcholine. In some embodiments, the fatty acids and derivatives are sodium myristate. In some embodiments, the fatty acids and derivatives are palmitoylcarnitine.

[0020] In certain embodiments, the permeation enhancer is in the form of caprate.

[0021] In certain embodiments, the caprate-based permeation enhancer is sodium caprate.

[0022] In one embodiment, the permeation enhancer is Labrasol®.

[0023] In certain embodiments, the composition comprises an aqueous suppository component.

[0024] In some embodiments, the aqueous suppository component is selected from glycerin, gelatin, or polyethylene glycol (PEG), or a combination thereof. In some embodiments, the aqueous suppository component is glycerin. In some embodiments, the aqueous suppository component is gelatin. In some embodiments, the aqueous suppository component is polyethylene glycol (PEG).

[0025] In some embodiments, the composition further comprises gelatin.

[0026] In some embodiments, the only aqueous suppository component is gelatin.

[0027] In some embodiments, the composition contains 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 contains about 5% or more gelatin in water. For example, in some embodiments, the composition contains about 5%, 6%, 7%, 8%, or 9% or more gelatin. In some embodiments, the composition contains about 10% or more gelatin in water. For example, in some embodiments, the composition contains about 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, or 19% or more gelatin in water. In some embodiments, the composition contains about 20% or more gelatin in water. For example, in some embodiments, the composition contains 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 certain 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.

[0028] In some embodiments, the composition further comprises 0.25 mg / mL or more of mRNA, 0.5 mg / mL or more of mRNA, 0.75 mg / mL or more of mRNA, or 1 mg / mL or more of mRNA. Thus, in some embodiments, the composition further comprises 0.25 mg / mL or more of mRNA. In some embodiments, the composition comprises 0.5 mg / mL or more of mRNA. In some embodiments, the composition comprises 0.75 mg / mL or more of mRNA. In some embodiments, the composition comprises 1 mg / mL or more of mRNA.

[0029] In some embodiments, the composition comprises 0.5 mg or more of mRNA, 0.75 mg or more of mRNA, 1 mg or more of mRNA, 1.25 mg or more of mRNA, 1.5 mg or more of mRNA, or 1.75 mg or more of mRNA. Thus, in some embodiments, the composition comprises 0.5 mg or more of mRNA. In some embodiments, the composition comprises 0.75 mg or more of mRNA. In some embodiments, the composition comprises 1 mg or more of mRNA. In some embodiments, the composition comprises 1.25 mg or more of mRNA. In some embodiments, the composition comprises 1.5 mg or more of mRNA. In some embodiments, the composition comprises 1.75 mg or more of mRNA.

[0030] In some embodiments, the composition is formulated for a suppository of about 3 grams, about 2 grams, or about 1 gram. Thus, in some embodiments, the composition is formulated for a suppository of about 3 grams. In some embodiments, the composition is formulated for a suppository of about 2 grams. In some embodiments, the composition is formulated for a suppository of about 1 gram.

[0031] In some embodiments, the composition is formulated for 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 for a suppository having a volume of about 2.0 mL. In some embodiments, the composition is formulated for a suppository having a volume of about 3.5 mL. In some embodiments, the composition is formulated for a suppository having a volume of about 7.5 mL. In some embodiments, the composition is formulated for a suppository having a volume of about 10.0 mL.

[0032] In certain embodiments, the suppositories are refrigerated prior to administration.

[0033] In certain embodiments, the subject is first administered a permeation enhancer prior to administering the composition comprising the mRNA.

[0034] 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 before administering the composition comprising the mRNA. Thus, in some embodiments, the permeability enhancer is administered to the subject about 30 minutes before administering the composition comprising the mRNA. In some embodiments, the permeability enhancer is administered to the subject about 1 hour before administering the composition comprising the mRNA. In some embodiments, the permeability enhancer is administered to the subject about 2.5 hours before administering the composition comprising the mRNA. In some embodiments, the permeability enhancer is administered to the subject about 5.0 hours before administering the composition comprising the mRNA. In some embodiments, the permeability enhancer is administered to the subject about 12 hours before administering the composition comprising the mRNA.

[0035] In one aspect, the present invention provides a method for delivering messenger RNA (mRNA) to a subject for in vivo production of a protein or peptide in the subject, comprising administering to the subject via mucosal delivery a composition comprising mRNA encoding the protein or peptide and encapsulated in lipid nanoparticles, wherein administration of the composition results in detectable expression of the protein or peptide encoded by the mRNA in the subject at least about 24 hours, about 48 hours, about 72 hours, or about 96 hours after administration. Thus, in one embodiment, administration of the composition results in detectable expression of the protein or peptide encoded by the mRNA in the subject at least about 24 hours after administration. In one embodiment, administration of the composition results in detectable expression of the protein or peptide encoded by the mRNA in the subject at least about 48 hours after administration. In one embodiment, administration of the composition results in detectable expression of the protein or peptide encoded by the mRNA in the subject at least about 72 hours after administration. In one embodiment, administration of the composition results in detectable expression of the protein or peptide encoded by the mRNA in the subject at least about 96 hours after administration.

[0036] In some embodiments, the mRNA is detectable in the circulation, liver, kidney, colon, and / or rectum of a subject at least about 24 hours, about 48 hours, about 72 hours, or about 96 hours after administration. Thus, in some embodiments, the mRNA is detectable in the circulation of a subject at least about 24 hours after administration. In some embodiments, the mRNA is detectable in the circulation of a subject at least about 48 hours after administration. In some embodiments, the mRNA is detectable in the circulation of a subject at least about 72 hours after administration. In some embodiments, the mRNA is detectable in the circulation of a subject at least about 96 hours after administration. In some embodiments, the mRNA is detectable in the liver of a subject at least about 24 hours after administration. In some embodiments, the mRNA is detectable in the liver of a subject at least about 48 hours after administration. In some embodiments, the mRNA is detectable in the liver of a subject at least about 72 hours after administration. In some embodiments, the mRNA is detectable in the liver of a subject at least about 96 hours after administration. In some embodiments, the mRNA is detectable in the kidney of a subject at least about 24 hours after administration. In some embodiments, the mRNA is detectable in the subject's kidney at least about 48 hours after administration. In some embodiments, the mRNA is detectable in the subject's kidney at least about 72 hours after administration. In some embodiments, the mRNA is detectable in the subject's kidney at least about 96 hours after administration. In some embodiments, the mRNA is detectable in the subject's colon at least about 24 hours after administration. In some embodiments, the mRNA is detectable in the subject's colon at least about 48 hours after administration. In some embodiments, the mRNA is detectable in the subject's colon at least about 72 hours after administration. In some embodiments, the mRNA is detectable in the subject's colon at least about 96 hours after administration. In some embodiments, the mRNA is detectable in the subject's rectum at least about 24 hours after administration. In some embodiments, the mRNA is detectable in the subject's rectum at least about 48 hours after administration. In some embodiments, the mRNA is detectable in the subject's rectum at least about 72 hours after administration.In certain embodiments, the mRNA is detectable in the rectum of the subject at least about 96 hours after administration.

[0037] In some embodiments, mucosal delivery is rectal, vaginal, ocular, oral, or gastrointestinal. Thus, in some embodiments, mucosal delivery is rectal. In some embodiments, mucosal delivery is vaginal. In some embodiments, mucosal delivery is ocular. In some embodiments, mucosal delivery is oral. In some embodiments, mucosal delivery is gastrointestinal.

[0038] In some embodiments, oral delivery is buccal or sublingual. Thus, in some embodiments, oral delivery is buccal. In some embodiments, oral delivery is sublingual.

[0039] In some embodiments, the in vivo production of the protein or peptide is in the circulation, liver, kidney, colon, and / or rectum of a subject. Thus, in some embodiments, the in vivo production of the protein or peptide is in the circulation of a subject. In some embodiments, the in vivo production of the protein or peptide is in the liver of a subject. In some embodiments, the in vivo production of the protein or peptide is in the kidney of a subject. In some embodiments, the in vivo production of the protein or peptide is in the colon of a subject. In some embodiments, the in vivo production of the protein or peptide is in the rectum of a subject.

[0040] In one aspect, the present invention provides a suppository for rectal administration of mRNA, the suppository comprising mRNA encapsulated in lipid nanoparticles, the mRNA encoding a protein or peptide; and gelatin.

[0041] In some embodiments, the suppository contains 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 contains about 5% or more gelatin in water. For example, in some embodiments, the suppository contains about 5%, 6%, 7%, 8%, or 9% or more gelatin. In some embodiments, the suppository contains about 10% or more gelatin in water. For example, in some embodiments, the suppository contains about 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, or 19% or more gelatin in water. In some embodiments, the suppository contains about 20% or more gelatin in water. For example, in some embodiments, the suppository contains about 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, or 29% or more gelatin in water. In some embodiments, the suppository contains about 30% or more gelatin in water. For example, in some embodiments, the suppository contains 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 contains 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.

[0042] In certain embodiments, the suppository does not include a lipid-based suppository component.

[0043] In some embodiments, the lipid-based suppository component is cocoa butter, theobroma oil, a synthetic fat, or a synthetic base. Thus, in some embodiments, the lipid-based suppository component is cocoa butter. In some embodiments, the lipid-based suppository component is theobroma oil. In some embodiments, the lipid-based suppository component is a synthetic fat. In some embodiments, the lipid-based suppository component is a synthetic base. In some embodiments, the lipid-based suppository component is cocoa butter, theobroma oil, a synthetic fat, or a synthetic base, or any combination thereof.

[0044] In certain embodiments, the suppository comprises a permeation enhancer.

[0045] In some embodiments, the suppository comprises a permeation enhancer selected from bile salts, surfactants, fatty acids and derivatives, glycerides, chelating agents, salicylates, or polymers. 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.

[0046] In some embodiments, the suppository comprises a fatty acid and a derivative selected from sorbitan laurate, sodium caprate, sucrose, palmitate, lauroylcholine, sodium myristate, or palmitoylcarnitine. Thus, in some embodiments, the fatty acid and a derivative comprises sodium caprate. In some embodiments, the fatty acid and a derivative is sucrose. In some embodiments, the fatty acid and a derivative is palmitate. In some embodiments, the fatty acid and a derivative is lauroylcholine. In some embodiments, the fatty acid and a derivative is sodium myristate. In some embodiments, the fatty acid and a derivative is palmitoylcarnitine.

[0047] In certain embodiments, the suppository comprises a permeation enhancer in the form of a caprate.

[0048] In certain embodiments, the caprate-based permeation enhancer is sodium caprate.

[0049] In one embodiment, the permeation enhancer is Labrasol®.

[0050] In some embodiments, the suppository further comprises glycerin and / or PEG. Thus, in some embodiments, the suppository further comprises glycerin. In some embodiments, the suppository further comprises PEG.

[0051] In some embodiments, the suppository softens or melts at about 36-37° 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.

[0052] In certain 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.

[0053] The following figures are for illustrative purposes only and not for limitation. [Brief explanation of the drawings]

[0054] [Figure 1] 1 shows an exemplary suppository containing mRNA encapsulated in lipid nanoparticles, which can be delivered rectally. [Figure 2A] 1 shows exemplary imaging of mice 24 hours after rectal administration of saline as a negative control. [Figure 2B] 1 shows exemplary imaging of various tissues 24 hours after rectal administration of saline as a negative control, where no signal was detected when saline was administered intrarectally. [Figure 3A] 1 shows exemplary imaging of mice 24 hours after rectal administration of FFLuc mRNA-LNPs. [Figure 3B] Illustrates exemplary imaging of various tissues 24 hours after rectal administration of FFLuc mRNA-LNP. Luciferase activity signals were detected in mice. The colon showed strong luminescence. [Figure 4] Illustrated are exemplary images of mice 24 hours after rectal administration of FFLuc mRNA-LNPs at a dose of 0.2 mg (Group 1) or 0.05 mg (Group 2). Mice in Group 2 were pre-administered with sodium caprate prior to administration of mRNA-LNPs. [Figure 5] FIG. 10 is an exemplary graphical representation of luminescence detected for mice 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 with sodium caprate (Group 2). [Figure 6A] 1 shows exemplary imaging of a mouse 24 hours after rectal administration of a suppository containing FFLuc mRNA-LNPs. [Figure 6B] 1 shows exemplary imaging of various tissues in a rat 24 hours after rectal administration of a suppository. [Figure 7] FIG. 1 is an exemplary graphical representation of serum hEPO protein detected in rats x hours after administration of the composition. DETAILED DESCRIPTION OF THE INVENTION

[0055] definition In order that the present invention may be more readily understood, certain terms are first defined below. Further definitions of these and other terms are set forth throughout the specification.

[0056] Terms such as "more than," "at least," and "more than," e.g., "at least one," are used to mean, but are not 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, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 199, 199, 100, 101, 102, 103, 104, 105 It is understood to include 8, 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 any higher number than the stated value. Any higher number or fraction in between is also included.

[0057] Conversely, the term "less than or equal to" includes every value less than the recited value. For example, "100 or fewer 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, Includes 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 fractional number in between is also included.

[0058] Animal: As used herein, the term "animal" refers to any member of the animal kingdom. In certain embodiments, "animal" refers to humans at any stage of development. In certain embodiments, "animal" refers to non-human animals at any stage of development. In certain embodiments, the non-human animal is a mammal (e.g., a rodent, mouse, rat, rabbit, monkey, dog, cat, sheep, cow, primate, and / or pig). In certain embodiments, animals include, but are not limited to, mammals, birds, reptiles, amphibians, fish, insects, and / or worms. In certain embodiments, the animal may be a transgenic animal, a genetically modified animal, and / or a clone.

[0059] Approximately or about: As used herein, the term "approximately" or "about," when applied to one or more values ​​to which it refers, refers to a value similar to a stated reference value. In certain embodiments, the term "approximately" or "about" refers to a range of values ​​that fall within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater or less than) the stated reference value, unless otherwise stated or clear from the context (except when such number exceeds 100% of possible values).

[0060] Comprising: As used herein, the term "comprising" or variations such as "comprises" or "comprising" will be understood to imply the inclusion of the stated elements, integers or steps, or group of elements, integers or steps, but not the exclusion of any other elements, integers or steps, or group of elements, integers or steps.

[0061] Delivery: As used herein, the term "delivery" encompasses both local and systemic delivery. For example, delivery of mRNA encompasses a situation in which the mRNA is delivered to a target tissue and the encoded protein is expressed and retained within the target tissue (also referred to as "local distribution" or "local delivery"). Another exemplary situation includes a situation in which the mRNA is delivered to a target tissue and the encoded protein is expressed in the patient's circulatory system (e.g., serum), secreted, distributed throughout the body, and taken up by other tissues (also referred to as "systemic distribution" or "systemic delivery"). In another exemplary situation, the mRNA is delivered systemically and taken up by a wide variety of cells and tissues in vivo. In one exemplary situation, delivery is intravenous, intramuscular, or subcutaneous.

[0062] Dosing interval: As used herein, in the context of a method for treating a disease, the dosing interval is the frequency with which a therapeutic composition, e.g., an mRNA composition, is administered at an effective dose of mRNA to a subject (mammal) in need thereof such that one or more symptoms associated with the disease are alleviated; or one or more biomarkers associated with the disease are reduced over at least the duration of the dosing interval. Dosing frequency and dosing interval may be used interchangeably in this disclosure.

[0063] Efficacy: As used herein, the term "efficacy," or grammatical equivalents, refers to the improvement of a biologically relevant endpoint associated with delivery of mRNA encoding a relevant protein or peptide. In one embodiment, the biological endpoint is protection from an ammonium chloride challenge at a specific time point after administration.

[0064] Encapsulation: As used herein, the term "encapsulation," or grammatical equivalents thereof, refers to the process of entrapment of nucleic acid molecules within nanoparticles.

[0065] Expression: As used herein, "expression" of a nucleic acid sequence refers to the translation of mRNA into a polypeptide, the assembly of multiple polypeptides (e.g., heavy or light chains of an antibody) into an intact protein (e.g., an antibody), and / or the post-translational modification of the polypeptides or fully assembled protein (e.g., an antibody). In this application, the terms "expression" and "production," and their grammatical equivalents, are used interchangeably.

[0066] Effective dose: As used herein, an effective dose is a dose of mRNA in a pharmaceutical composition that, when administered to a subject in need thereof, here a mammalian subject, according to the methods of the present invention, is effective to produce the desired result in the subject, e.g., to alleviate symptoms associated with a disease.

[0067] Functional: As used herein, a "functional" biomolecule is a biomolecule in a form in which it exhibits a property and / or activity by which it is characterized.

[0068] Improve, increase, or alleviate: As used herein, the terms "improve," "increase," or "alleviate," or grammatical equivalents, refer to a value compared to a baseline measurement, such as a measurement in the same individual prior to the initiation of a treatment described herein, or a measurement in a control subject (or control subjects) without the treatment described herein. A "control subject" is a subject afflicted with the same form of disease as the subject being treated and is approximately the same age as the subject being treated.

[0069] In vitro: As used herein, the term "in vitro" refers to events that take place not within a multicellular organism but in an artificial environment, e.g., a test tube or reaction vessel, cell culture, etc.

[0070] In vivo: As used herein, the term "in vivo" refers to events that occur within multicellular organisms, such as humans and non-human animals. In the context of cell-based systems, the term can be used to refer to events that occur within living cells (e.g., as opposed to in vitro systems).

[0071] Liposome: As used herein, the term "liposome" refers to any lamellar, multilamellar, or solid nanoparticle vesicle. Typically, liposomes as used herein can be formed by mixing one or more lipids, or by mixing one or more lipids and a polymer. In some embodiments, liposomes suitable for the present invention contain a cationic lipid and optionally a non-cationic lipid, optionally a cholesterol-based lipid, and / or optionally a PEG-modified lipid.

[0072] 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 both modified and unmodified RNA. mRNA can contain one or more coding and non-coding regions. mRNA can be purified from natural sources, produced using recombinant expression systems, optionally purified, chemically synthesized, etc. Optionally, for example, in the case of chemically synthesized molecules, mRNA can contain nucleoside analogs, such as chemically modified bases or sugars, analogs with backbone modifications, etc. The mRNA sequence is presented in the 5' to 3' direction unless otherwise indicated.

[0073] N / P ratio: As used herein, the term " N / P ratio " refers to the molar ratio of the positively charged molecular unit in the cationic lipid in the lipid nanoparticle to the negatively charged molecular unit in the mRNA encapsulated in the lipid nanoparticle.Therefore, N / P ratio is typically calculated as the ratio of the mole number of amine group in the cationic lipid in the lipid nanoparticle to the mole number of phosphate group in the mRNA encapsulated in the lipid nanoparticle.

[0074] Nucleic Acid: As used herein, the term "nucleic acid" in its broadest sense refers to any compound and / or substance that is or can be incorporated into a polynucleotide chain. In certain embodiments, a nucleic acid is a compound and / or substance that is or can be incorporated into a polynucleotide chain via a phosphodiester bond. In certain embodiments, "nucleic acid" refers to individual nucleic acid residues (e.g., nucleotides and / or nucleosides). In certain embodiments, "nucleic acid" refers to a polynucleotide chain comprising individual nucleic acid residues. In certain embodiments, "nucleic acid" encompasses RNA as well as single- and / or double-stranded DNA and / or cDNA. Furthermore, the terms "nucleic acid," "DNA," "RNA," and / or similar terms include nucleic acid analogs, i.e., analogs having other than 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 invention. The term "nucleotide sequence encoding an amino acid sequence" includes all nucleotide sequences that are degenerate versions of each other and / or encode the same amino acid sequence. Nucleotide sequences encoding proteins and / or RNAs may contain introns. Nucleic acids can be purified from natural sources, produced using recombinant expression systems, optionally purified, chemically synthesized, etc. Optionally, for example, in the case of chemically synthesized molecules, nucleic acids can include nucleoside analogs, such as chemically modified bases or sugars, analogs having backbone modifications, etc. Nucleic acid sequences are presented in the 5' to 3' direction unless otherwise indicated.In certain embodiments, nucleic acids are selected from 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-methyladenosine, 5-methylcytidine, C-5 propynyl-cytidine, C-5 propynyl-uridine, 2-aminoadenosine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynyl-uridine, and C5-propynyl-uridine). The nucleic acid may be or contain: cytidine, 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); intercalation bases; modified sugars (e.g., 2'-fluororibose, ribose, 2'-deoxyribose, arabinose, and hexose); and / or modified phosphate groups (e.g., phosphorothioate and 5'-N-phosphoramidite linkages). In certain embodiments, the present invention is specifically directed to "unmodified nucleic acids," which refer to nucleic acids (e.g., polynucleotides and residues, including nucleotides and / or nucleosides) that have not been chemically modified to facilitate or achieve delivery. In certain embodiments, the nucleotides T and U are used interchangeably in describing sequences.

[0075] Patient: As used herein, the term "patient" or "subject" refers to any organism to which provided compositions can be administered, for example, for experimental, diagnostic, prophylactic, cosmetic, and / or therapeutic purposes. Typical patients include animals (e.g., mammals such as mice, rats, rabbits, non-human primates, and / or humans). In certain embodiments, the patient is a human. Human includes prenatal and postnatal forms.

[0076] Polypeptide: As used herein, a "polypeptide" generally refers to a series of at least two amino acids joined together by peptide bonds. In certain embodiments, a polypeptide may contain at least three to five amino acids, each of which is joined to each other by at least one peptide bond. Those skilled in the art will understand that polypeptides may contain "unnatural" amino acids or other substances, although these may be optionally incorporated into the polypeptide chain.

[0077] Protein: As used herein, the term "protein" in "therapeutic protein" refers to a polypeptide (i.e., a series of at least two amino acids linked together by peptide bonds). Proteins may contain moieties other than amino acids (e.g., may be glycoproteins, proteoglycans, etc.) and / or may be otherwise processed or modified. Those of skill in the art will understand that a "protein" may be an entire polypeptide chain produced by a cell (with or without a signal sequence) or a characteristic portion thereof. Those of skill in the art will understand that a protein may comprise two or more polypeptide chains linked, for example, by one or more disulfide bonds or associated by other means. Polypeptides may contain l-amino acids, d-amino acids, or both, and may contain any of a variety of amino acid modifications or analogs known in the art. Useful modifications include, for example, terminal acetylation, amidation, methylation, etc. In certain embodiments, proteins may comprise natural amino acids, unnatural amino acids, synthetic amino acids, and combinations thereof. The term "peptide" is generally used to refer to a polypeptide having a length of less than about 100 amino acids, less than about 50 amino acids, less than 20 amino acids, or less than 10 amino acids. In certain embodiments, the protein is an antibody, an antibody fragment, a biologically active portion thereof, and / or a characteristic portion thereof.

[0078] Systemic distribution or delivery: As used herein, the terms "systemic distribution," "systemic delivery," or grammatical equivalents refer to a delivery or distribution mechanism or technique that affects the entire body or the entire organism. Typically, systemic distribution or delivery is achieved via the body's circulatory system, e.g., the bloodstream. Compare with the definition of "local distribution or delivery."

[0079] 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). Human includes prenatal and postnatal forms. In many embodiments, the subject is a human. A subject may be a patient, which refers to a person who visits a medical institution for diagnosis or treatment of a disease. The term "subject" is used interchangeably herein with "individual" or "patient." A subject may be suffering from or susceptible to a disease or disorder, or may not exhibit symptoms of a disease or disorder.

[0080] Substantially: As used herein, the term "substantially" refers to the qualitative state of exhibiting all or nearly all extent or degree of a characteristic or property of interest. Those skilled in the art of biology will understand that biological and chemical phenomena rarely, if ever, go to completion and / or proceed to completion, or achieve or avoid absolute results. Thus, the term "substantially" is used herein to capture the potential lack of completeness inherent in many biological and chemical phenomena.

[0081] Target tissue: As used herein, the term "target tissue" refers to any tissue affected by the disease being treated. In certain embodiments, the target tissue includes tissue that exhibits a pathology, symptom, or characteristic associated with the disease.

[0082] Therapeutically effective amount: As used herein, the term "therapeutically effective amount" of a therapeutic agent means an amount sufficient to treat, diagnose, prevent, and / or delay the onset of symptoms of a disease, disorder, and / or condition when administered to a subject suffering from or susceptible to the disease, disorder, and / or condition. It will be understood by those skilled in the art that a therapeutically effective amount is typically administered via a dosing regimen comprising at least one unit dose.

[0083] Treatment: As used herein, the terms "treat," "treatment," or "treating" refer to any method used to partially or completely alleviate, ameliorate, relieve, inhibit, prevent, delay the onset of, reduce the severity of, and / or reduce the incidence of one or more symptoms or characteristics of a particular disease, disorder, and / or condition. Treatment may be administered to subjects who do not show signs of the disease and / or who show only early signs of the disease in order to reduce the risk of developing pathology associated with the disease.

[0084] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this application belongs and as commonly used in the art to which this application belongs; such art is incorporated by reference in its entirety. In case of conflict, the present specification, including definitions, will control.

[0085] Detailed Description In particular, the present invention provides effective methods and compositions for delivering messenger RNA (mRNA) and / or its protein or polypeptide products to a subject via a mucosal route, for example, by rectal delivery. The present invention is based, in part, on the surprising discovery that mRNA and / or its protein or polypeptide products can be effectively delivered to a subject's circulation, liver, kidney, colon, and / or rectum by rectal delivery, despite many chemical and physical barriers.

[0086] Various aspects of the present invention are described in detail in the following sections. The use of sections is not intended to limit the invention. Each section may apply to any aspect of the present invention. In this application, the use of "or" means "and / or" unless otherwise stated.

[0087] Mucosal delivery of lipid-encapsulated mRNA The present invention provides various methods for delivering mRNA to target tissues. Delivery methods include administration of lipid-encapsulated mRNA across any mucosal tissue. For example, lipid-encapsulated mRNA can be delivered via rectal, vaginal, ocular, oral, and / or gastrointestinal routes.

[0088] In certain aspects, the present invention provides methods for delivery of messenger RNA (mRNA) to a subject, particularly for in vivo production of a protein or peptide in the subject, comprising administering to the subject by mucosal delivery a composition comprising mRNA encoding the protein or peptide and encapsulated in lipid nanoparticles, wherein administration of the composition results in detectable expression of the protein or peptide encoded by the mRNA in the subject at least about 24 hours, about 48 hours, about 72 hours, or about 96 hours after administration.

[0089] In one embodiment, mucosal delivery of lipid-encapsulated mRNA is via the rectum.

[0090] rectal delivery In one embodiment, the present invention provides a method for rectal delivery of lipid-encapsulated mRNA encoding a protein or peptide of interest.

[0091] Advantages of rectal delivery include ease of administration, allowing patients to remain at home. Rectal delivery does not require the specialized formulation of sterile drugs required in an inpatient setting and by intravenous administration. Therapeutic compositions can be administered rectally via suppositories, enemas, bulb syringes, and catheters. Rectal administration using a specialized rectal catheter can be performed by a clinician at home. Many oral forms of medication can be crushed and suspended in water for administration via a rectal catheter. Rectal administration is therefore particularly safe and convenient for infants and the elderly, and is useful for patients with needle aversion or gastrointestinal motility problems, such as dysphagia, ileus, or intestinal obstruction, which can hinder the progression of medication through the digestive tract. Furthermore, rectally administered drugs generally have a faster onset and higher bioavailability, and are less likely to cause nausea compared to oral drug administration. Drugs administered rectally avoid approximately two-thirds of first-pass metabolism, resulting in less drug transformation and higher drug concentrations in the patient's circulatory system. However, delivering mRNA via the rectal route is extremely challenging. The rectal region (i.e., the rectum and colon) contains a large amount of RNase, which can quickly degrade mRNA. In addition, the mucus layer in the rectum and / or colon can act as an absorption barrier. Furthermore, fecal impaction can hinder rectal delivery of drugs.

[0092] Despite these challenges, the method provided herein allows messenger RNA (mRNA) to be delivered via the rectal route.The present invention is based, in part, on the unexpected discovery that lipid-encapsulated mRNA can be effectively delivered to a subject's circulation, liver, kidney, colon, and / or rectum via rectal delivery, despite many barriers, such as RNase, mucus layer, and the presence of fecal impaction.Such a non-invasive delivery route unexpectedly provides an effective means for conveniently delivering lipid-encapsulated therapeutic compositions.

[0093] In particular, the present invention provides a method for delivering messenger RNA (mRNA) to a subject for in vivo production of a protein or peptide in the subject, comprising administering to the subject via rectal delivery a composition comprising mRNA encoding the protein or peptide and encapsulated in lipid nanoparticles, wherein administration of the composition results in detectable expression of the protein or peptide encoded by the mRNA in the subject at least about 24 hours, about 48 hours, about 72 hours, or about 96 hours after administration. This method allows for delivery of lipid-encapsulated mRNA via the rectal route, resulting in expression of the protein or peptide encoded by the mRNA in various tissues of the recipient subject. For example, this method allows for expression of the protein or peptide encoded by the mRNA in the liver, kidney, circulation, colon, or rectum of the subject.

[0094] The methods described herein are suitable for administering lipid-encapsulated mRNA in a home setting. In some embodiments, rectal delivery is by suppository, enema, catheter, or 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 types of specialized catheters can be used in the methods disclosed herein; for example, one such specialized catheter is a Macy catheter. In some embodiments, rectal delivery is by bulb syringe.

[0095] In some embodiments, the compositions of the present invention are delivered to various target tissues in a subject. Thus, the present invention can be used as a non-invasive means of delivering a desired protein or peptide and / or promoting the production of the protein or peptide encoded thereby in a target tissue. The methods and compositions described herein are useful for the management and treatment of many diseases caused by deficiencies of both secreted and non-secreted proteins and / or enzymes.

[0096] In some embodiments, rectal delivery of lipid-encapsulated mRNA results in the production 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, lipid-encapsulated mRNA results in the production of the desired protein or peptide encoded by the mRNA in the circulation. In some embodiments, lipid-encapsulated mRNA results in the production of the desired protein or peptide encoded by the mRNA in the liver. In some embodiments, lipid-encapsulated mRNA results in the production of the desired protein or peptide encoded by the mRNA in the kidney. In some embodiments, lipid-encapsulated mRNA results in the production of the desired protein or peptide encoded by the mRNA in the colon. In some embodiments, lipid-encapsulated mRNA results in the production of the desired protein or peptide encoded by the mRNA in the rectum. In some embodiments, lipid-encapsulated mRNA results in the production of the desired protein or peptide encoded by the mRNA in the heart. In some embodiments, lipid-encapsulated mRNA results in the production of the desired protein or peptide encoded by the mRNA in the spleen.

[0097] In some embodiments, rectal delivery of lipid-encapsulated mRNA results in the 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.

[0098] In some embodiments, the protein or peptide encoded by the mRNA is produced in the subject at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 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.

[0099] In certain embodiments, rectal delivery of lipid-encapsulated mRNA results in the detection of the desired protein or peptide encoded by the mRNA in the circulation, liver, kidney, colon, rectum, heart, and / or spleen. Thus, in certain embodiments, rectal delivery of lipid-encapsulated mRNA results in the detection of the desired protein or peptide encoded by the mRNA in the circulation. In certain embodiments, rectal delivery of lipid-encapsulated mRNA results in the detection of the desired protein or peptide encoded by the mRNA in the liver. In certain embodiments, rectal delivery of lipid-encapsulated mRNA results in the detection of the desired protein or peptide encoded by the mRNA in the kidney. In certain embodiments, rectal delivery of lipid-encapsulated mRNA results in the detection of the desired protein or peptide encoded by the mRNA in the colon. In certain embodiments, rectal delivery of lipid-encapsulated mRNA results in the detection of the desired protein or peptide encoded by the mRNA in the rectum. In certain embodiments, rectal delivery of lipid-encapsulated mRNA results in the detection of the desired protein or peptide encoded by the mRNA in the heart. In one embodiment, rectal delivery of lipid-encapsulated mRNA results in detection of the desired protein or peptide encoded by the mRNA in the spleen.

[0100] In some embodiments, the protein or peptide encoded by the mRNA is 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 is detectable in the subject at least about 6 hours after administration. In some embodiments, the protein or peptide encoded by the mRNA is detectable in the subject at least about 12 hours after administration. In some embodiments, the protein or peptide encoded by the mRNA is detectable in the subject at least about 18 hours after administration. In some embodiments, the protein or peptide encoded by the mRNA is detectable in the subject at least about 24 hours after administration. In some embodiments, the protein or peptide encoded by the mRNA is detectable in the subject at least about 36 hours after administration. In some embodiments, the protein or peptide encoded by the mRNA is detectable in the subject at least about 48 hours after administration. In some embodiments, the protein or peptide encoded by the mRNA is detectable in the subject at least about 60 hours after administration. In some embodiments, the protein or peptide encoded by the mRNA is detectable in the subject at least about 72 hours after administration. In some embodiments, the protein or peptide encoded by the mRNA is detectable in the subject at least about 96 hours after administration. In some embodiments, the protein or peptide encoded by the mRNA is detectable in the subject at least about 120 hours after administration. In some embodiments, the protein or peptide encoded by the mRNA is detectable in the subject at least about 144 hours after administration. In some embodiments, the protein or peptide encoded by the mRNA is detectable in the subject at least about 168 hours after administration.

[0101] In some embodiments, the protein or peptide encoded by the mRNA is detectable in the subject at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 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.

[0102] In some embodiments, the protein or peptide encoded by the mRNA is detectable in the circulation, liver, kidneys, colon, and / or rectum of a 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, kidneys, colon, and / or rectum of a 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, kidneys, colon, and / or rectum of a 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, kidneys, colon, and / or rectum of a 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, kidneys, colon, and / or rectum of a 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, kidneys, colon, and / or rectum of a 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, kidneys, colon, and / or rectum of a 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, kidneys, colon, and / or rectum of a 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, kidneys, colon, and / or rectum of a 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, kidneys, colon, and / or rectum of a 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, kidneys, colon, and / or rectum of a subject at least about 120 hours after administration.

[0103] In some embodiments, the protein or peptide encoded by the mRNA is detectable in the circulation, liver, kidneys, colon, and / or rectum of a subject at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 days after administration. In some embodiments, the protein or peptide encoded by the mRNA is detectable in the circulation, liver, kidneys, colon, and / or rectum of a subject at least about 1 day after administration. In some embodiments, the protein or peptide encoded by the mRNA is detectable in the circulation, liver, kidneys, colon, and / or rectum of a subject at least about 2 days after administration. In some embodiments, the protein or peptide encoded by the mRNA is detectable in the circulation, liver, kidneys, colon, and / or rectum of a subject at least about 3 days after administration. In some embodiments, the protein or peptide encoded by the mRNA is detectable in the circulation, liver, kidneys, colon, and / or rectum of a subject at least about 4 days after administration. In some embodiments, the protein or peptide encoded by the mRNA is detectable in the circulation, liver, kidneys, colon, and / or rectum of a subject at least about 5 days after administration. In some embodiments, the protein or peptide encoded by the mRNA is detectable in the circulation, liver, kidneys, colon, and / or rectum of a subject at least about 6 days after administration. In some embodiments, the protein or peptide encoded by the mRNA is detectable in the circulation, liver, kidneys, colon, and / or rectum of a subject at least about 7 days after administration. In some embodiments, the protein or peptide encoded by the mRNA is detectable in the circulation, liver, kidneys, colon, and / or rectum of a subject at least about 8 days after administration. In some embodiments, the protein or peptide encoded by the mRNA is detectable in the circulation, liver, kidneys, colon, and / or rectum of a subject at least about 9 days after administration. In some embodiments, the protein or peptide encoded by the mRNA is detectable in the circulation, liver, kidneys, colon, and / or rectum of a subject at least about 10 days after administration.

[0104] In certain embodiments, the lipid-encapsulated mRNA can translocate (e.g., travel intact by either active or passive means) after rectal delivery into the systemic blood supply, and then reach different cells or target tissues.

[0105] Thus, in one aspect, the present invention provides a method for delivering messenger RNA (mRNA) to a subject for in vivo production of a protein or peptide in the subject, comprising administering to the subject via mucosal delivery a composition comprising mRNA encoding the protein or peptide and encapsulated in lipid nanoparticles, wherein the mRNA is detectable in the subject's circulation, liver, kidneys, colon, and / or rectum at least about 24 hours, about 48 hours, about 72 hours, or about 96 hours after administration. In one embodiment, the in vivo production of the protein or peptide occurs in the subject's circulation, liver, kidneys, colon, and / or rectum. Thus, in one embodiment, the in vivo production of lipid-encapsulated mRNA delivered intrarectally occurs in the subject's circulation. In one embodiment, the in vivo production of lipid-encapsulated mRNA delivered intrarectally occurs in the subject's liver. In one embodiment, the in vivo production of lipid-encapsulated mRNA delivered intrarectally occurs in the subject's kidney. In certain embodiments, in vivo production of intrarectally delivered lipid-encapsulated mRNA occurs in the colon of the subject. In certain embodiments, in vivo production of intrarectally delivered lipid-encapsulated mRNA occurs in the rectum of the subject.

[0106] In some embodiments, the mRNA is detectable in the circulation, liver, kidney, colon, rectum, heart, and / or spleen of a subject. In some embodiments, the mRNA is detectable in the circulation of a subject. In some embodiments, the mRNA is detectable in the liver of a subject. In some embodiments, the mRNA is detectable in the kidney of a subject. In some embodiments, the mRNA is detectable in the colon of a subject. In some embodiments, the mRNA is detectable in the rectum of a subject. In some embodiments, the mRNA is detectable in the heart of a subject. In some embodiments, the mRNA is detectable in the spleen of a subject.

[0107] In some embodiments, the mRNA is 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, or about 120 hours after administration. In some embodiments, the mRNA is detectable in the subject at least about 6 hours after administration. In some embodiments, the mRNA is detectable in the subject at least about 12 hours after administration. In some embodiments, the mRNA is detectable in the subject at least about 18 hours after administration. In some embodiments, the mRNA is detectable in the subject at least about 24 hours after administration. In some embodiments, the mRNA is detectable in the subject at least about 36 hours after administration. In some embodiments, the mRNA is detectable in the subject at least about 48 hours after administration. In some embodiments, the mRNA is detectable in the subject at least about 60 hours after administration. In some embodiments, the mRNA is detectable in the subject at least about 72 hours after administration. In some embodiments, the mRNA is detectable in the subject at least about 96 hours after administration. In certain embodiments, the mRNA is detectable in the subject at least about 120 hours after administration.

[0108] In some embodiments, mRNA is detectable in a subject at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 days after administration. In some embodiments, mRNA is detectable in a subject at least about 1 day after administration. In some embodiments, mRNA is detectable in a subject at least about 2 days after administration. In some embodiments, mRNA is detectable in a subject at least about 3 days after administration. In some embodiments, mRNA is detectable in a subject at least about 4 days after administration. In some embodiments, mRNA is detectable in a subject at least about 5 days after administration. In some embodiments, mRNA is detectable in a subject at least about 6 days after administration. In some embodiments, mRNA is detectable in a subject at least about 7 days after administration. In some embodiments, mRNA is detectable in a subject at least about 8 days after administration. In some embodiments, mRNA is detectable in a subject at least about 9 days after administration. In some embodiments, mRNA is detectable in a subject at least about 10 days after administration.

[0109] Composition of the formulation The present invention provides effective compositions for delivering messenger RNA (mRNA), particularly via rectal delivery. The compositions described herein are suitable for delivering mRNA through mucosal tissues such as the rectum.

[0110] In some embodiments, the composition comprises an 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 permeation enhancer.

[0111] suppositories Compositions for rectal or vaginal (e.g., vaginal) administration are typically suppositories that can be prepared by mixing the composition with a suitable non-irritating excipient such as cocoa butter, polymers, hydrogels, glycerin, gelatin, polyethylene glycol, or a suppository wax that is solid at ambient temperature but liquid at body temperature and therefore melts in the rectum or vaginal cavity and releases the active ingredient. The type of material used depends on the type of suppository, the type of drug, and the conditions under which the suppository will be stored.

[0112] The present invention provides a suppository for effective delivery of mRNA encapsulated in lipid nanoparticles, particularly via mucosal routes such as rectal, vaginal, ocular, oral, and / or gastrointestinal routes. In some embodiments, the lipid-encapsulated mRNA is delivered via the rectal or vaginal route. The suppositories described herein, which include lipid nanoparticles and mRNA, are solid at room temperature and melt when administered rectally or vaginally. The melting of the suppository after being placed in the rectum or vagina allows for effective release of the lipid nanoparticles loaded with mRNA.

[0113] In certain embodiments, the suppositories are refrigerated prior to administration.

[0114] In some embodiments, the suppository softens or melts at about 30-42°C. In some embodiments, the suppository softens or melts at about 32-40°C. In some embodiments, the suppository softens or melts at about 34-38°C. In some embodiments, the suppository softens or melts at about 36-37°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.

[0115] In some embodiments, the suppository softens or melts within 30 minutes of being administered to a subject. In some embodiments, the suppository softens or melts within 20 minutes of being administered to a subject. In some embodiments, the suppository softens or melts within 15 minutes of being administered to a subject. In some embodiments, the suppository softens or melts within 10 minutes of being administered to a subject. In some embodiments, the suppository softens or melts within 5 minutes of being administered to a subject. In some embodiments, the suppository softens or melts within 3 minutes of being administered to a subject. In some embodiments, the suppository softens or melts within 1 minute of being administered to a subject.

[0116] As a non-limiting example, formulations for rectal and / or vaginal administration can be prepared by mixing the drug with a suitable non-irritating excipient that is solid at room temperature but liquid at rectal temperature and therefore melts in the rectum and / or vagina to release the drug. Such materials include cocoa butter and polyethylene glycol.

[0117] Pharmaceutical compositions for rectal or vaginal administration may contain at least one inactive ingredient. Any or all of the inactive ingredients used may be approved by the US Food and Drug Administration (FDA). A non-exhaustive list of inactive ingredients for use in pharmaceutical compositions for rectal or vaginal administration includes methylcellulose, hydroxypropyl methylcellulose, hydroxymethylcellulose, poloxamer, polyvinyl alcohol, polyvinylpyrrolidone, polyacrylamide, polyethylene oxide, modified starch, adipic acid, denatured alcohol, allantoin, anhydrous lactose, apricot kernel oil PEG-6 ester, barium sulfate, beeswax, bentonite, benzoic acid, benzyl alcohol, butylated hydroxyanisole ... Hydroxytoluene, calcium lactate, carbomer 934, carbomer 934p, microcrystalline cellulose, ceteth-20, cetostearyl alcohol, cetyl alcohol, cetyl esters wax, cetyl palmitate, cholesterol, cholesterol, citric acid, citric acid monohydrate, hydrogenated coconut / palm kernel glycerides, crospovidone, edetate disodium, ethylcellulose, ethylene-vinyl acetate copolymer (28% vinyl acetate), ethylene-vinyl acetate copolymer (9% vinyl acetate), fatty alcohols, FD&C Yellow No. 5, gelatin, dl-glutamic acid, glycerin, glyceryl isostearate, glyceryl monostearate, glyceryl stearate, guar gum, high-density polyethylene, hydrogel polymer, hydrogenated palm oil, hypromellose 2208 (15,000 MPa).S), Hypromellose, Isopropyl Myristate, Lactic Acid, DL-Lactic Acid, Lactose, Lactose Monohydrate, Lactose Hydrate, Lanolin, Anhydrous Lanolin, Lecithin, Soy Lecithin, Light Mineral Oil, Magnesium Aluminum Silicate, Magnesium Aluminum Silicate Hydrate, Magnesium Stearate, Methyl Stearate, Methylparaben, Microcrystalline Wax, Mineral Oil, Nitric Acid, Octyldodecanol, Peanut Oil, PEG-4 Stearate 6-32 / glycol stearate, peg-100 stearate, peg-120 glyceryl stearate, peg-2 stearate, peg-5 oleate, pegoxol 7 stearate, white petrolatum, phenylmercuric acetate, phospholipon 90g, phosphoric acid, piperazine hexahydrate, poly(dimethylsiloxane / methylvinylsiloxane / methylhydrogensiloxane) dimethylvinyl or dimethylhydroxy or trimethyl end-stopped, polycarbophil, polyester, polyethylene 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 Ingredients include: d, propylene glycol, propylene glycol monopalmitostearate, propylparaben, quaternium-15 cis form, silicon dioxide, colloidal silicon dioxide, silicone, sodium bicarbonate, sodium citrate, sodium hydroxide, sodium lauryl sulfate, sodium metabisulfite, disodium phosphate anhydrous, monosodium phosphate anhydrous, sorbic acid, sorbitan monostearate, sorbitol, sorbitol solution, spermaceti, stannous 2-ethylhexanoate, starch, Starch 1500, corn starch, stearamidoethyl diethylamine, stearic acid, stearyl alcohol, dl-tartaric acid, tert-butylhydroquinone, tetrapropyl orthosilicate, trolamine, urea, hydrogenated vegetable oil, Wecobee FS, white ceresin wax, and white wax.

[0118] In some embodiments, gelatin aqueous solution is used in the formulation to keep lipid nanoparticles intact.Gelatin aqueous solution is mucoadhesive and can help suppository contact with mucous membrane.Therefore, the presence of gelatin can help mRNA-loaded lipid nanoparticles move into the systemic circulation.In addition, gelatin solution is gel at room temperature, which prevents mRNA-loaded nanoparticles from dripping out of the rectum.Gelatin also melts gradually at physiological temperature, allowing mRNA-loaded lipid nanoparticles to contact with mucous membrane.

[0119] In some embodiments, the suppository contains 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, or 90% or more gelatin in water. In some embodiments, the suppository contains about 1% or more gelatin in water. In some embodiments, the suppository contains about 3% or more gelatin in water. In some embodiments, the suppository contains about 5% or more gelatin in water. In some embodiments, the suppository contains about 10% or more gelatin in water. In some embodiments, the suppository contains about 15% or more gelatin in water. In some embodiments, the suppository contains about 20% or more gelatin in water. In some embodiments, the suppository contains about 30% or more gelatin in water. In some embodiments, the suppository contains about 40% or more gelatin in water. In some embodiments, the suppository contains about 50% or more gelatin in water. In some embodiments, the suppository contains about 60% or more gelatin in water. In some embodiments, the suppository contains about 70% or more gelatin in water. In some embodiments, the suppository contains about 80% or more gelatin in water. In some embodiments, the suppository contains about 90% or more gelatin in water.

[0120] In certain embodiments, the suppository does not adversely affect the integrity of the lipid nanoparticles.

[0121] In some embodiments, the composition does not include a lipid-based suppository component. In some embodiments, the composition includes a lipid-based suppository component. In some embodiments, the lipid-based suppository component is cocoa butter, theobroma oil, a synthetic fat, or a synthetic base. In some embodiments, the lipid-based suppository component is cocoa butter, theobroma oil, a synthetic fat, or a synthetic base. In some embodiments, the lipid-based suppository component is cocoa butter. In some embodiments, the lipid-based suppository component is theobroma oil. In some embodiments, the lipid-based suppository component is a synthetic fat. In some embodiments, the lipid-based suppository component is a synthetic base.

[0122] In some embodiments, the composition comprises an aqueous suppository component. In some embodiments, the aqueous suppository component is selected from glycerin, gelatin, or polyethylene glycol (PEG), or a combination thereof. In some embodiments, the aqueous suppository component is glycerin. In some embodiments, the aqueous suppository component is gelatin. In some embodiments, the aqueous suppository component is polyethylene glycol (PEG). In some embodiments, the only aqueous suppository component is gelatin.

[0123] In some embodiments, the suppository comprises glycerin and / or PEG. In some embodiments, the suppository comprises glycerin. In some embodiments, the suppository comprises PEG. In some embodiments, the suppository comprises less than about 10% glycerin. In some embodiments, the suppository comprises less than about 8% glycerin. In some embodiments, the suppository comprises less than about 6% glycerin. In some embodiments, the suppository comprises less than about 4% glycerin. In some embodiments, the suppository comprises less than about 2% glycerin. In some embodiments, the suppository comprises less than about 1% glycerin. In some embodiments, the suppository comprises less than about 0.1% glycerin. 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 certain embodiments, the suppository contains less than about 0.1% PEG.

[0124] In some embodiments, the suppository further comprises glycerol. In some embodiments, the amount of glycerol present in the suppository does not destroy the lipid nanoparticles. In some embodiments, the suppository does not contain glycerol. In some embodiments, the suppository comprises less than about 30% glycerol. In some embodiments, the suppository comprises less than about 20% glycerol. In some embodiments, the suppository comprises less than about 15% glycerol. 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 0.5% glycerol. In some embodiments, the suppository comprises less than about 0.1% glycerol.

[0125] The present invention provides, inter alia, suppositories for rectal administration of mRNA. In one embodiment, the suppository comprises mRNA encapsulated in lipid nanoparticles, the mRNA encoding a protein or peptide, and gelatin.

[0126] The suppositories described herein are formulated to hold various concentrations of mRNA and are sized to allow for convenient, non-invasive administration via rectal or vaginal delivery. Such non-invasive delivery routes unexpectedly provide an effective means for conveniently delivering therapeutic compositions.

[0127] In some embodiments, the composition comprises 0.25 mg / mL or more of mRNA, 0.5 mg / mL or more of mRNA, 0.75 mg / mL or more of mRNA, or 1 mg / mL or more of mRNA. In some embodiments, the composition comprises 0.1 mg / mL or more of mRNA. In some embodiments, the composition comprises 0.25 mg / mL or more of mRNA. In some embodiments, the composition comprises 0.5 mg / mL or more of mRNA. In some embodiments, the composition comprises 0.75 mg / mL or more of mRNA. In some embodiments, the composition comprises 1 mg / mL or more of mRNA. In some embodiments, the composition comprises 2 mg / mL or more of mRNA. In some embodiments, the composition comprises 2.5 mg / mL or more of mRNA. In some embodiments, the composition comprises 5 mg / mL or more of mRNA.

[0128] In some embodiments, the composition comprises 0.5 mg or more of mRNA, 0.75 mg or more of mRNA, 1 mg or more of mRNA, 1.25 mg or more of mRNA, 1.5 mg or more of mRNA, or 1.75 mg or more of mRNA. In some embodiments, the composition comprises 0.1 mg or more of mRNA. In some embodiments, the composition comprises 0.25 mg or more of mRNA. In some embodiments, the composition comprises 0.5 mg or more of mRNA. In some embodiments, the composition comprises 0.75 mg or more of mRNA. In some embodiments, the composition comprises 1 mg or more of mRNA. In some embodiments, the composition comprises 1.25 mg or more of mRNA. In some embodiments, the composition comprises 1.5 mg or more of mRNA. In some embodiments, the composition comprises 1.75 mg or more of mRNA. In some embodiments, the composition comprises 2 mg or more of mRNA. In some embodiments, the composition comprises 2.5 mg or more of mRNA. In some embodiments, the composition comprises 5 mg or more of mRNA.

[0129] In some embodiments, the composition is formulated for about 3 gram, about 2 gram, or about 1 gram suppository. In some embodiments, the composition is formulated for about 20 gram suppository. In some embodiments, the composition is formulated for about 10 gram suppository. In some embodiments, the composition is formulated for about 5 gram suppository. In some embodiments, the composition is formulated for about 3 gram suppository. In some embodiments, the composition is formulated for about 2 gram suppository. In some embodiments, the composition is formulated for about 1 gram suppository. In some embodiments, the composition is formulated for about 0.5 gram suppository.

[0130] In some embodiments, the composition is formulated for 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 for a suppository having a volume of about 1.0 mL. In some embodiments, the composition is formulated for a suppository having a volume of about 2.0 mL. In some embodiments, the composition is formulated for a suppository having a volume of about 2.5 mL. In some embodiments, the composition is formulated for a suppository having a volume of about 3.0 mL. In some embodiments, the composition is formulated for a suppository having a volume of about 3.5 mL. In some embodiments, the composition is formulated for a suppository having a volume of about 4.0 mL. In some embodiments, the composition is formulated for a suppository having a volume of about 5.0 mL. In some embodiments, the composition is formulated for a suppository having a volume of about 7.5 mL. In some embodiments, the composition is formulated for a suppository having a volume of about 10.0 mL. In some embodiments, the composition is formulated for a suppository having a volume of about 12.5 mL. In some embodiments, the composition is formulated for a suppository having a volume of about 15.0 mL. In some embodiments, the composition is formulated for a suppository having a volume of about 17.5 mL. In some embodiments, the composition is formulated for a suppository having a volume of about 20.0 mL.

[0131] Permeation enhancers Penetration or permeability enhancers have been used to improve the bioavailability of drugs with poor absorption across mucosal routes (e.g., rectal, vaginal, ocular, oral, or gastrointestinal). In some embodiments for rectal or vaginal administration, the suppository further comprises a permeability enhancer. In some embodiments, the suppository does not comprise a permeability enhancer. In some embodiments, the permeability enhancer does not adversely affect the integrity of the lipid nanoparticles.

[0132] In certain embodiments, the permeation enhancer is selected from bile salts, surfactants, fatty acids and derivatives, glycerides, chelating agents, salicylates, or polymers. In certain embodiments, the permeation enhancer is a bile salt. In certain embodiments, the permeation enhancer is a fatty acid and its derivatives. In certain embodiments, the permeation enhancer is a glyceride. In certain embodiments, the permeation enhancer is a chelating agent. In certain embodiments, the permeation enhancer is a salicylate. In certain embodiments, the permeation enhancer is a polymer.

[0133] In some embodiments, the fatty acids and derivatives are selected from sorbitan laurate, sodium caprate, sucrose, palmitate, lauroylcholine, sodium myristate, or palmitoylcarnitine. In some embodiments, the fatty acids and derivatives include sorbitan laurate. In some embodiments, the fatty acids and derivatives include sodium caprate. In some embodiments, the fatty acids and derivatives are sucrose. In some embodiments, the fatty acids and derivatives include palmitate. In some embodiments, the fatty acids and derivatives include lauroylcholine. In some embodiments, the fatty acids and derivatives include sodium myristate. In some embodiments, the fatty acids and derivatives include palmitoylcarnitine.

[0134] In certain embodiments, the permeation enhancer is in the form of caprate. In certain embodiments, the caprate-based permeation enhancer is sodium caprate.

[0135] In some embodiments, the permeation enhancer includes cholate. In some embodiments, the permeation enhancer is citric acid. In some embodiments, the permeation enhancer is ethylenediaminetetraacetic acid (EDTA). In some embodiments, the permeation enhancer is oleic acid. In some embodiments, the permeation enhancer is caprate. In some embodiments, the permeation enhancer is a surfactant. In some embodiments, the permeation enhancer is sodium dodecyl sulfate (SDS). In some embodiments, the permeation enhancer is Cremophor®. In some embodiments, the permeation enhancer is Tween® 80, and in some embodiments, the permeation enhancer is Labrasol®. In some embodiments, the permeation enhancer is a self-microemulsifying drug delivery system (SMEDDS). In some embodiments, the permeation enhancer is a natural bioenhancer. In some embodiments, the permeation enhancer is allicin. In some embodiments, the permeation enhancer is piperine. In some embodiments, the permeation enhancer is curcumin. In some embodiments, the permeation enhancer is quercetin.

[0136] Several different formulations of lipid-encapsulated mRNA compositions have been devised to facilitate delivery to a subject, including administering a permeability enhancer before administering the composition containing mRNA.Administering a permeability enhancer promotes the movement of mRNA-loaded lipid nanoparticles from the colon into the systemic circulation.

[0137] In some embodiments, a subject is first administered a permeability enhancer before administering a composition comprising mRNA. 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 before administering a composition comprising mRNA. In some embodiments, the permeability enhancer is administered to the subject about 1 minute before administering a composition comprising mRNA. In some embodiments, the permeability enhancer is administered to the subject about 3 minutes before administering a composition comprising mRNA. In some embodiments, the permeability enhancer is administered to the subject about 5 minutes before administering a composition comprising mRNA. In some embodiments, the permeability enhancer is administered to the subject about 10 minutes before administering a composition comprising mRNA. In some embodiments, the permeability enhancer is administered to the subject about 15 minutes before administering a composition comprising mRNA. In some embodiments, the permeability enhancer is administered to the subject about 20 minutes before administering a composition comprising mRNA. In some embodiments, the permeability enhancer is administered to the subject about 25 minutes before administering a composition comprising mRNA. In some embodiments, the permeation enhancer is administered to the subject about 30 minutes before administering the composition comprising mRNA. In some embodiments, the permeation enhancer is administered to the subject about 45 minutes before administering the composition comprising mRNA. In some embodiments, the permeation enhancer is administered to the subject about 1 hour before administering the composition comprising mRNA. In some embodiments, the permeation enhancer is administered to the subject about 1.5 hours before administering the composition comprising mRNA. In some embodiments, the permeation enhancer is administered to the subject about 2 hours before administering the composition comprising mRNA. In some embodiments, the permeation enhancer is administered to the subject about 2.5 hours before administering the composition comprising mRNA. In some embodiments, the permeation enhancer is administered to the subject about 5 hours before administering the composition comprising mRNA. In some embodiments, the permeation enhancer is administered to the subject about 12 hours before administering the composition comprising mRNA. In some embodiments, the permeation enhancer is administered to the subject about 18 hours before administering the composition comprising mRNA. In one embodiment, the permeation enhancer is administered to the subject about 24 hours before administering the composition comprising the mRNA.

[0138] In some embodiments, the permeability enhancer is administered to the subject simultaneously with the composition comprising mRNA. In some embodiments, the subject is administered the permeability enhancer after administering the composition comprising mRNA. In some embodiments, the subject is administered the permeability enhancer about 5 minutes after administering the composition comprising mRNA. In some embodiments, the subject is administered the permeability enhancer about 10 minutes after administering the composition comprising mRNA. In some embodiments, the subject is administered the permeability enhancer about 15 minutes after administering the composition comprising mRNA. In some embodiments, the subject is administered the permeability enhancer about 20 minutes after administering the composition comprising mRNA. In some embodiments, the subject is administered the permeability enhancer about 30 minutes after administering the composition comprising mRNA. In some embodiments, the subject is administered the permeability enhancer about 45 minutes after administering the composition comprising mRNA. In some embodiments, the subject is administered the permeability enhancer about 1 hour after administering the composition comprising mRNA. In some embodiments, the subject is administered the permeability enhancer about 2 hours after administering the composition comprising mRNA. In some embodiments, the subject is administered the permeability enhancer about 2.5 hours after administering the composition comprising mRNA. In some embodiments, the subject is administered the permeability enhancer about 5 hours after administering the composition comprising mRNA. In some embodiments, the subject is administered the permeability enhancer about 12 hours after administering the composition comprising mRNA. In some embodiments, the subject is administered the permeability enhancer about 18 hours after administering the composition comprising mRNA. In some embodiments, the subject is administered the permeability enhancer about 24 hours after administering the composition comprising mRNA.

[0139] mRNA synthesis The mRNA of the present invention can be synthesized according to any of a variety of known methods. Various methods are described in U.S. Patent Application Publication No. 2018 / 0258423 and can be used to practice the present invention, all of which are incorporated herein by reference. For example, the mRNA of the present invention can be synthesized by in vitro transcription (IVT). Briefly, IVT is typically performed using a linear or circular DNA template containing a promoter, a pool of ribonucleotide triphosphates, a buffer system that may contain DTT and magnesium ions, and an appropriate RNA polymerase (e.g., T3, T7, or SP6 RNA polymerase), DNAse I, pyrophosphatase, and / or an RNAse inhibitor. The exact conditions will vary depending on the specific application.

[0140] In some embodiments, the preferred mRNA sequence is an mRNA sequence that encodes a protein or peptide. In some embodiments, the preferred mRNA sequence is codon-optimized for efficient expression in human cells. In some embodiments, the preferred mRNA sequence is a natural or wild-type sequence. In some embodiments, the preferred mRNA sequence encodes a protein or peptide containing one or more mutations in the amino acid sequence.

[0141] The present invention can be used to deliver mRNAs of various lengths, hi certain embodiments, the present invention 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 more in length. In certain embodiments, the present invention can be used to deliver in vitro synthesized mRNA in the length range of about 1 to 20 kb, about 1 to 15 kb, about 1 to 10 kb, about 5 to 20 kb, about 5 to 15 kb, about 5 to 12 kb, about 5 to 10 kb, about 8 to 20 kb, or about 8 to 50 kb.

[0142] In one embodiment, a DNA template is transcribed in vitro to prepare mRNA of the present invention. A suitable DNA template typically has a promoter, e.g., a T3, T7, or SP6 promoter for in vitro transcription, followed by the desired nucleotide sequence for the desired mRNA and a termination signal.

[0143] nucleotide A variety of natural or modified nucleosides can be used to produce the mRNA of the present invention. In certain embodiments, the mRNA is prepared using natural nucleosides (or unmodified nucleotides; e.g., adenosine, guanosine, cytidine, uridine); nucleoside analogs (e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine, 3-methyladenosine, 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, the base is or contains: 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, O(6)-methylguanine, pseudouridine (e.g., N-1-methyl-pseudouridine), 2-thiouridine, and 2-thiocytidine; chemically modified bases; biologically modified bases (e.g., methylated bases); intercalation bases; modified sugars (e.g., 2'-fluororibose, ribose, 2'-deoxyribose, arabinose, and hexose); and / or modified phosphate groups (e.g., phosphorothioate and 5'-N-phosphoramidite linkages).

[0144] In certain embodiments, suitable mRNAs may contain backbone, sugar, and / or base modifications. For example, modified nucleotides include, but are not limited to, modified purines (adenine (A), guanine (G)) or pyrimidines (thymine (T), cytosine (C), uracil (U)), as well as modified nucleotide analogs or derivatives of purines and pyrimidines, such as 1-methyl-adenine, 2-methyl-adenine, 2-methylthio-N-6-isopentenyl-adenine, N6-methyl-adenine, N6-isopentenyl-adenine, N6-isopenten ... 1-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), dihydro-uracil, 2-thio-uracil, 4-thio-uracil, 5-carboxymethyl uracil-5-oxyacetic acid methyl ester, uracil-5-oxyacetic acid (v), 1-methyl-pseudouracil, queosine, β-D-mannosyl-queosine, wybutosine, as well as phosphoramidates, phosphorothioates, peptide nucleotides, methylphosphonates, 7-deazaguanosine, 5-methylcytosine, and inosine.The preparation of such analogs is known to those skilled in the art from, for example, U.S. Pat. Nos. 4,373,071, 4,401,796, 4,415,732, 4,458,066, 4,500,707, 4,668,777, 4,973,679, 5,047,524, 5,132,418, 5,153,319, 5,262,530, and 5,700,642, the disclosures of which are incorporated by reference in their entireties.

[0145] In some embodiments, the mRNA comprises one or more non-standard nucleotide residues. Non-standard nucleotide residues may include, for example, 5-methyl-cytidine ("5mC"), pseudouridine ("ΨU"), and / or 2-thio-uridine ("2sU"). For a description of such residues and their incorporation into mRNA, see, for example, U.S. Pat. No. 8,278,036 or WO 2011 / 012316. The mRNA may be RNA, defined as RNA in which 25% of U residues are 2-thio-uridine and 25% of C residues are 5-methylcytidine. Teachings of the use of RNA are disclosed in U.S. Pat. App. Pub. No. 2012 / 0195936 and WO 2011 / 012316, both of which are incorporated herein by reference in their entireties. The presence of non-standard nucleotide residues may render an 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 may contain one or more non-standard nucleotide residues selected from isocytosine, pseudoisocytosine, 5-bromouracil, 5-propynyluracil, 6-aminopurine, 2-aminopurine, inosine, diaminopurine, and 2-chloro-6-aminopurine cytosine, as well as combinations of these and other nucleobase modifications. Some embodiments may further include additional modifications to the furanose ring or nucleobase. Additional modifications may include, for example, sugar modifications or substitutions (e.g., one or more of 2'-O-alkyl modifications, locked nucleic acids (LNAs)). In some embodiments, the RNA may be complexed or hybridized with additional polynucleotides and / or peptide polynucleotides (PNAs). In some embodiments where the sugar modification is a 2'-O-alkyl modification, such modifications can include, but are not limited to, 2'-deoxy-2'-fluoro, 2'-O-methyl, 2'-O-methoxyethyl, and 2'-deoxy modifications. In some embodiments, any of these modifications can be present in 0-100% of the nucleotides—e.g., individually or in combination, 0%, 1%, 10%, 25%, 50%, 75%, 85%, 90%, 95%, or greater than 100% of the constituent nucleotides.

[0146] In some embodiments, mRNA can comprise RNA backbone modification.Typically, backbone modification is the modification that the backbone phosphate of the nucleotide contained in RNA is chemically modified.Exemplary backbone modification typically includes, but is not limited to, from the group consisting of methyl phosphonate, methyl phosphoramidate, phosphoramidate, phosphorothionate (for example, cytidine 5'-O-(1-thiophosphate)), boranophosphate, positively charged guanidinium group, etc., which means that phosphodiester bond is replaced with other anionic, cationic or neutral group.

[0147] In certain embodiments, the mRNA may contain sugar modifications. Exemplary sugar modifications are chemical modifications of the sugars of the nucleotides it contains, including, but not limited to, 2'-deoxy-2'-fluoro-oligoribonucleotides (2'-fluoro-2'-deoxycytidine 5'-triphosphate, 2'-fluoro-2'-deoxyuridine 5'-triphosphate), 2'-deoxy-2'-deamine-oligoribonucleotides (2'-amino-2'-deoxycytidine 5'-triphosphate, 2'-amino-2'-deoxyuridine 5'-triphosphate), 2'-O-alkyloligoribonucleotides (2'-fluoro-2'-deoxyuridine 5'-triphosphate, 2'-fluoro-2'-deoxyuridine 5'-triphosphate), 2'-O-alkyl-2'-deoxyuridine 5'-triphosphate, ... The oligoribonucleotides contain sugar modifications selected from the group consisting of oligoribonucleotides, 2'-deoxy-2'-C-alkyl oligoribonucleotides (2'-O-methylcytidine 5'-triphosphate, 2'-methyluridine 5'-triphosphate), 2'-C-alkyl oligoribonucleotides, and their isomers (2'-aracytidine 5'-triphosphate, 2'-aruridine 5'-triphosphate), or azidotriphosphates (2'-azido-2'-deoxycytidine 5'-triphosphate, 2'-azido-2'-deoxyuridine 5'-triphosphate).

[0148] Post-composite processing Typically, a 5' cap and / or a 3' tail may be added post-synthetically. The presence of a cap is important in providing resistance to nucleases found in most eukaryotic cells. The presence of a "tail" serves to protect the mRNA from exonuclease degradation.

[0149] A 5' cap is typically added as follows: first, an RNA terminal phosphatase removes one of the terminal phosphate groups from the 5' nucleotide, leaving two terminal phosphates; next, guanosine triphosphate (GTP) is added to the terminal phosphate via a guanylyltransferase, creating a 5'5'5 triphosphate linkage; and then the 7-nitrogen of guanine is methylated by a methyltransferase. Examples of cap structures include, but are not limited to, m7G(5')ppp(5'(A, G(5')ppp(5')A, and G(5')ppp(5')G. Additional cap structures are described in U.S. Patent Application Publication Nos. 2016 / 0032356 and 2018 / 0125989, which are incorporated herein by reference.

[0150] Typically, the tail structure comprises a poly(A) and / or poly(C) tail. The poly-A or poly-C tail at the 3' end of the mRNA typically comprises 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, respectively. tide, 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 adenosine or cytosine nucleotides.In certain embodiments, the poly A or poly C tail comprises about 10 to 800 adenosine or cytosine nucleotides, respectively (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 The poly(A) tail structure may be 200-600 adenosine or cytosine nucleotides, about 250-600 adenosine or cytosine nucleotides, about 300-600 adenosine or cytosine nucleotides, about 350-600 adenosine or cytosine nucleotides, about 400-600 adenosine or cytosine nucleotides, about 450-600 adenosine or cytosine nucleotides, about 500-600 adenosine or cytosine nucleotides, about 10-150 adenosine or cytosine nucleotides, about 10-100 adenosine or cytosine nucleotides, about 20-70 adenosine or cytosine nucleotides, or about 20-60 adenosine or cytosine nucleotides. In certain embodiments, the tail structure comprises a combination of poly(A) and poly(C) tails of various lengths as described herein. In some embodiments, the tail structure comprises at least 50%, 55%, 65%, 70%, 75%, 80%, 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, or 99% adenosine nucleotides, hi some embodiments, the tail structure comprises at least 50%, 55%, 65%, 70%, 75%, 80%, 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, or 99% cytosine nucleotides.

[0151] As described herein, the addition of a 5' cap and / or a 3' tail facilitates the detection of abortive transcripts that occur during in vitro synthesis because, without capping and / or tailing, the size of these prematurely terminated mRNA transcripts would be too small to be detected. Thus, in certain embodiments, a 5' cap and / or a 3' tail is added to a synthetic mRNA before the purity of the mRNA (e.g., the level of abortive transcripts present in the mRNA) is tested. In certain embodiments, a 5' cap and / or a 3' tail is added to a synthetic mRNA before the mRNA is purified as described herein. In other embodiments, a 5' cap and / or a 3' tail is added to a synthetic mRNA after the mRNA has been purified as described herein.

[0152] The mRNA synthesized according to the present invention can be used without further purification. In particular, the mRNA synthesized according to the present invention can be used without a step to remove shortmers. In certain embodiments, the mRNA synthesized according to the present invention can be further purified. Various methods can be used to purify the mRNA synthesized according to the present invention. For example, purification of the mRNA can be carried out using centrifugation, filtration, and / or chromatography methods. In certain embodiments, the synthesized mRNA is purified by ethanol precipitation or filtration or chromatography, or gel purification, or any other suitable means. In certain embodiments, the mRNA is purified by HPLC. In certain embodiments, the mRNA is extracted in a standard phenol:chloroform:isoamyl alcohol solution, as is well known to those skilled in the art. In certain embodiments, the mRNA is purified using tangential flow filtration. Suitable purification methods include those described in U.S. Patent Application Publication No. 2016 / 0040154, U.S. Patent Application Publication No. 2015 / 0376220, U.S. Patent Application Publication No. 2018 / 0251755, U.S. Patent Application Publication No. 2018 / 0251754, U.S. Provisional Patent Application No. 62 / 757,612 filed November 8, 2018, and U.S. Provisional Patent Application No. 62 / 891,781 filed August 26, 2019, all of which are incorporated by reference herein and may be used in practicing the present invention.

[0153] In some embodiments, the mRNA is purified before capping and tailing. In some embodiments, the mRNA is purified after capping and tailing. In some embodiments, the mRNA is purified before and after capping and tailing.

[0154] In one embodiment, the mRNA is purified by centrifugation either before, after, or both before and after capping and tailing.

[0155] In one embodiment, the mRNA is purified by filtration either before, after, or both before and after capping and tailing.

[0156] In one embodiment, the mRNA is purified by tangential flow filtration (TFF) either before, after, or both before and after capping and tailing.

[0157] In one embodiment, the mRNA is purified by chromatography either before, after, or both before and after capping and tailing.

[0158] Characterization of purified mRNA The mRNA compositions described herein are substantially free of contaminants, including short truncated RNA species, long truncated RNA species, double-stranded RNA (dsRNA), residual plasmid DNA, residual in vitro transcriptase, residual solvents, and / or residual salts.

[0159] The mRNA compositions described herein have a purity of about 60% to about 100%. Thus, in certain embodiments, the purified mRNA has a purity of about 60%. In certain embodiments, the purified mRNA has a purity of about 65%. In certain embodiments, the purified mRNA has a purity of about 70%. In certain embodiments, the purified mRNA has a purity of about 75%. In certain embodiments, the purified mRNA has a purity of about 80%. In certain embodiments, the purified mRNA has a purity of about 85%. In certain embodiments, the purified mRNA has a purity of about 90%. In certain embodiments, the purified mRNA has a purity of about 91%. In certain embodiments, the purified mRNA has a purity of about 92%. In certain embodiments, the purified mRNA has a purity of about 93%. In certain embodiments, the purified mRNA has a purity of about 94%. In certain embodiments, the purified mRNA has a purity of about 95%. In certain 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%.

[0160] In certain 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% of impurities other than full-length mRNA. Impurities include IVT contaminants such as proteins, enzymes, DNA templates, free nucleotides, residual solvents, residual salts, double-stranded RNA (dsRNA), prematurely terminated RNA sequences ("shortmers" or "short truncated RNA species"), and / or long truncated RNA species. In certain embodiments, the purified mRNA is substantially free of processing enzymes.

[0161] In certain embodiments, the residual plasmid DNA in the purified mRNA of the present invention is 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. Thus, the residual plasmid DNA in the purified mRNA is less than about 1 pg / mg. In certain embodiments, the residual plasmid DNA in the purified mRNA is less than about 2 pg / mg. In certain embodiments, the residual plasmid DNA in the purified mRNA is less than about 3 pg / mg. In certain embodiments, the residual plasmid DNA in the purified mRNA is less than about 4 pg / mg. In certain embodiments, the residual plasmid DNA in the purified mRNA is less than about 5 pg / mg. In certain embodiments, the residual plasmid DNA in the purified mRNA is less than about 6 pg / mg. In some embodiments, the residual plasmid DNA in the purified mRNA is less than about 7 pg / mg. In some embodiments, the residual plasmid DNA in the purified mRNA is less than about 8 pg / mg. In some embodiments, the residual plasmid DNA in the purified mRNA is less than about 9 pg / mg. In some embodiments, the residual plasmid DNA in the purified mRNA is less than about 10 pg / mg. In some embodiments, the residual plasmid DNA in the purified mRNA is less than about 11 pg / mg. In some embodiments, the residual plasmid DNA in the purified mRNA is less than about 12 pg / mg.

[0162] In some embodiments, methods of the present invention remove more than about 90%, 95%, 96%, 97%, 98%, 99%, or substantially all prematurely interrupted RNA sequences (also known as "shortmers"). In some embodiments, the mRNA composition is substantially free of prematurely interrupted RNA sequences. In some embodiments, the mRNA composition contains less than about 5% (e.g., less than about 4%, 3%, 2%, or 1%) prematurely interrupted 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%) prematurely interrupted RNA sequences. In some embodiments, the mRNA composition lacks detectable prematurely terminated RNA sequences as determined, for example, by high-performance liquid chromatography (HPLC) (e.g., a shoulder or a discrete peak), ethidium bromide, Coomassie staining, capillary electrophoresis, or glyoxal gel electrophoresis (e.g., the presence of a discrete lower band). As used herein, the terms "shortmer," "short truncated RNA species," "prematurely terminated RNA sequence," or "long truncated RNA species" refer to any transcript that is less than full length. In some embodiments, "shortmers," "short truncated RNA species," or "prematurely terminated RNA sequences" are less than 100, 90, 80, 70, 60, 50, 40, 30, 20, or 10 nucleotides in length. In some embodiments, shortmers are detected or quantified after the addition of a 5'-cap and / or a 3'-polyA tail. In some embodiments, the prematurely interrupted RNA transcript contains fewer than 15 bases (e.g., fewer than 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, or 3 bases). In some embodiments, the prematurely interrupted RNA transcript contains about 8-15, 8-14, 8-13, 8-12, 8-11, or 8-10 bases.

[0163] In certain embodiments, purified mRNA of the present invention is substantially free of enzymatic reagents used in in vitro synthesis, including, but not limited to, T7 RNA polymerase, DNAse I, pyrophosphatase, and / or RNAse inhibitors. In certain embodiments, purified mRNA of the present invention contains less than about 5% (e.g., less than about 4%, 3%, 2%, or 1%) of the enzymatic reagents used in in vitro synthesis. In certain embodiments, 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 the enzymatic reagents used in in vitro synthesis. In certain embodiments, purified mRNA contains undetectable enzymatic reagents used in in vitro synthesis, as determined, for example, by silver staining, gel electrophoresis, high-performance liquid chromatography (HPLC), ultra-performance liquid chromatography (UPLC), and / or capillary electrophoresis, ethidium bromide and / or Coomassie staining.

[0164] In various embodiments, the purified mRNA of the present invention maintains a high degree of integrity. As used herein, the term "mRNA integrity" generally refers to the quality of mRNA after purification. mRNA integrity can be determined using methods well known in the art, for example, by RNA agarose gel electrophoresis. In certain embodiments, mRNA integrity can be determined by the band pattern of RNA agarose gel electrophoresis. In certain embodiments, the purified mRNA of the present invention exhibits few or no bands compared to the reference bands of RNA agarose gel electrophoresis. In certain embodiments, the purified mRNA of the present invention has an integrity of greater than about 95% (e.g., greater than about 96%, 97%, 98%, 99%, or more). In certain embodiments, the purified mRNA of the present invention has an integrity of greater than 98%. In certain embodiments, the purified mRNA of the present invention has an integrity of greater than 99%. In certain embodiments, the purified mRNA of the present invention has an integrity of about 100%.

[0165] In some embodiments, purified mRNA is evaluated for one or more of the following characteristics: appearance, identity, quantity, concentration, presence of impurities, microbiological evaluation, pH level, and activity. In some embodiments, acceptable appearance includes a clear, colorless solution substantially free of visible particulate matter. In some embodiments, mRNA identity is evaluated by sequencing methods. In some embodiments, concentration is evaluated by a suitable method, such as UV spectrophotometry. In some embodiments, a suitable concentration is approximately 90%-110% nominal (0.9-1.1 mg / mL).

[0166] In some embodiments, assessing mRNA purity includes assessing mRNA integrity, residual plasmid DNA, and residual solvents. In some embodiments, an acceptable level of mRNA integrity is assessed by agarose gel electrophoresis. The gel is analyzed to determine whether the band pattern and apparent nucleotide length are consistent with analytical reference standards. Additional methods for assessing RNA integrity include, for example, assessing purified mRNA using capillary gel electrophoresis (CGE). In some embodiments, acceptable purity of purified mRNA, as determined by CGE, is a purified mRNA composition having no more than about 55% long-chain truncated / degraded species. In some embodiments, residual plasmid DNA is assessed by methods known in the art, for example, by using qPCR. In some embodiments, an acceptable level of residual plasmid DNA is 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). In some embodiments, acceptable residual solvent levels are 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, or less. Thus, in some embodiments, acceptable residual solvent levels are 10,000 ppm or less. In some embodiments, acceptable residual solvent levels are 9,000 ppm or less. In some embodiments, the acceptable residual solvent level is 8,000 ppm or less. In some embodiments, the acceptable residual solvent level is 7,000 ppm or less. In some embodiments, the acceptable residual solvent level is 6,000 ppm or less. In some embodiments, the acceptable residual solvent level is 5,000 ppm or less. In some embodiments, the acceptable residual solvent level is 4,000 ppm or less. In some embodiments, the acceptable residual solvent level is 3,000 ppm or less. In some embodiments, the acceptable residual solvent level is 2,000 ppm or less.In certain embodiments, the acceptable residual solvent level is 1,000 ppm or less.

[0167] In some embodiments, microbiological testing is performed on the purified mRNA, including, for example, evaluation 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 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 no more than 1 CFU / 100 mL.

[0168] In certain embodiments, the pH of the purified mRNA is evaluated. In certain embodiments, the acceptable pH of the purified mRNA is 5 to 8. Thus, in certain embodiments, the purified mRNA has a pH of about 5. In certain embodiments, the purified mRNA has a pH of about 6. In certain embodiments, the purified mRNA has a pH of about 7. In certain embodiments, the purified mRNA has a pH of about 7. In certain embodiments, the purified mRNA has a pH of about 8.

[0169] In one embodiment, the translation fidelity of the purified mRNA is assessed. Translation fidelity can be assessed by a variety of methods, including, for example, transfection and Western blot analysis. Acceptable characteristics of purified mRNA include a band pattern in a Western blot that migrates at a similar molecular weight to a reference standard.

[0170] In certain embodiments, the purified mRNA is assessed for conductivity. In certain embodiments, acceptable characteristics of the purified mRNA include a conductivity of about 50% to 150% of the reference standard.

[0171] The purified mRNA is also evaluated for cap percentage and poly-A tail length. In one embodiment, acceptable cap percentages include Cap1, area%:NLT90. In one embodiment, acceptable poly-A tail lengths are approximately 100 to 1500 nucleotides (e.g., 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, and 1000, 1100, 1200, 1300, 1400, or 1500 nucleotides).

[0172] In certain embodiments, the purified mRNA is also evaluated for residual PEG. In certain embodiments, the purified mRNA has less than 10 ng PEG / mg of purified mRNA to 1000 ng PEG / mg of mRNA. Thus, in certain embodiments, the purified mRNA has less than about 10 ng PEG / mg of purified mRNA. In certain embodiments, the purified mRNA has less than about 100 ng PEG / mg of purified mRNA. In certain embodiments, the purified mRNA has less than about 250 ng PEG / mg of purified mRNA. In certain embodiments, the purified mRNA has less than about 500 ng PEG / mg of purified mRNA. In certain embodiments, the purified mRNA has less than about 750 ng PEG / mg of purified mRNA. In certain embodiments, the purified mRNA has less than about 1000 ng PEG / mg of purified mRNA.

[0173] Various methods for detecting and quantifying mRNA purity are known in the art. For example, such methods include blotting, capillary electrophoresis, chromatography, fluorescence, gel electrophoresis, HPLC, silver staining, spectroscopy, ultraviolet (UV), or UPLC, or a combination thereof. In some embodiments, mRNA is first denatured with glyoxal dye before gel electrophoresis ("glyoxal gel electrophoresis"). In some embodiments, synthetic mRNA is characterized before capping or tailing. In some embodiments, synthetic mRNA is characterized after capping and tailing.

[0174] Delivery Vehicle According to the present invention, mRNA or MCNA encoding a protein or peptide described herein (e.g., full-length, fragment, or portion of a protein or peptide) can be delivered as naked RNA (unpackaged) or via a delivery vehicle. As used herein, the terms "delivery vehicle," "transfer vehicle," "nanoparticle," or grammatical equivalents are used interchangeably.

[0175] The delivery vehicle can be formulated in a pharmacological composition in combination with one or more additional nucleic acids, carriers, targeting ligands, or stabilizing reagents, or in which it is mixed with a suitable excipient. Techniques for drug formulation and administration can be found in "Remington's Pharmaceutical Sciences," Mack Publishing Co., Easton, Pa., latest edition. A particular delivery vehicle is selected based on its ability to facilitate transfection of the nucleic acid into target cells.

[0176] In some embodiments, mRNA or MCNA encoding at least one protein or peptide can be delivered via a single delivery vehicle.In some embodiments, mRNA or MCNA encoding at least one protein or peptide can be delivered via one or more delivery vehicles of different compositions.In some embodiments, one or more mRNAs and / or MCNAs are encapsulated in the same lipid nanoparticle.In some embodiments, one or more mRNAs are encapsulated in separate lipid nanoparticles.

[0177] 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, exosomes of both natural and synthetic origin, natural, synthetic and semi-synthetic lamellar bodies, nanoparticulates, calcium phosphate-silicate nanoparticulates, calcium phosphate nanoparticulates, silicon dioxide nanoparticulates, nanocrystalline microparticles, semiconductor nanoparticulates, poly(D-arginine), sol-gels, nanodendrimers, starch-based delivery systems, micelles, emulsions, niosomes, multi-domain block polymers (vinyl polymers, polypropylacrylic acid polymers, dynamic polyconjugates), dry powder formulations, plasmids, viruses, calcium phosphate nucleotides, aptamers, peptides and other vector tags. The use of bio-nanocapsules and other viral capsid protein assemblies as suitable transfer vehicles is also envisaged (Hum. Gene Ther. 2008 September;19(9):887-95).

[0178] Liposomal Delivery Vehicles In some embodiments, a suitable delivery vehicle is a liposome delivery vehicle, such as a lipid nanoparticle. As used herein, a liposome delivery vehicle, such as a lipid nanoparticle, is generally characterized as a microscopic vesicle having an internal aqueous space separated from the external medium by one or more bilayer membranes. The bilayer membrane of a liposome is typically formed by amphiphilic molecules, such as synthetic or naturally occurring lipids, containing spatially separated hydrophilic and hydrophobic domains (Lasic, Trends Biotechnol., 16:307-321, 1998). The bilayer membrane of a liposome can also be formed by amphiphilic polymers and surfactants (e.g., polymerosomes, niosomes, etc.). In the context of the present invention, a liposome delivery vehicle typically serves 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 liposomes comprise 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 liposomes comprise three or fewer different lipid components. In some embodiments, one different lipid component is a sterol-based cationic lipid.

[0179] cationic lipids As used herein, the phrase "cationic lipid" refers to any of several lipid species that have a net positive charge at a selected pH, such as physiological pH.

[0180] Cationic lipids suitable for use in the compositions and methods of the invention include those 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 invention comprise: [ka] The cationic lipids having the compound structure (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate and pharmaceutically acceptable salts thereof.

[0181] Other suitable cationic lipids for use in the compositions and methods of the invention include ionizable cationic lipids such as those described in International Patent Publication WO 2013 / 149140, which is incorporated herein by reference. In some embodiments, the compositions and methods of the invention comprise ionizable cationic lipids of the following formula: [ka] or a pharmaceutically acceptable salt thereof, wherein R1 and R2 are each independently hydrogen, optionally substituted variably saturated or unsaturated C1-C 20 Alkyl and optionally substituted variably saturated or unsaturated C-C 20 acyl; wherein L1 and L2 are 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; where m and o are each independently selected from the group consisting of 0 and any positive integer (e.g., when m is 3); and n is 0 or any positive integer (e.g., when n is 1). In certain embodiments, the compositions and methods of the present invention include: [ka] and pharmaceutically acceptable salts thereof. In certain embodiments, the compositions and methods of the present invention comprise a cationic lipid (15Z,18Z)-N,N-dimethyl-6-((9Z,12Z)-octadeca-9,12-dien-1-yl)tetracosa-15,18-dien-1-amine ("HGT5000") having the compound structure: [ka] and pharmaceutically acceptable salts thereof. In certain embodiments, the compositions and methods of the present invention comprise a cationic lipid (15Z,18Z)-N,N-dimethyl-6-((9Z,12Z)-octadeca-9,12-dien-1-yl)tetracosa-4,15,18-trien-1-amine ("HGT5001") having the compound structure: [ka] and (15Z,18Z)-N,N-dimethyl-6-((9Z,12Z)-octadeca-9,12-dien-1-yl)tetracosa-5,15,18-trien-1-amine (“HGT5002”), and pharmaceutically acceptable salts thereof.

[0182] Other suitable cationic lipids for use in the compositions and methods of the invention include those cationic lipids described as amino alcohol lipid oils in International Patent Publication WO 2010 / 053572, which is incorporated herein by reference. In certain embodiments, the compositions and methods of the invention comprise: [ka] and pharmaceutically acceptable salts thereof.

[0183] Other suitable cationic lipids for use in the compositions and methods of the invention include those 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 invention comprise: [ka] and pharmaceutically acceptable salts thereof.

[0184] Other suitable cationic lipids for use in the compositions and methods of the invention include those 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 invention comprise: [ka] and pharmaceutically acceptable salts thereof.

[0185] Other suitable cationic lipids for use in the compositions and methods of the present invention include cationic lipids having the formula 14,25-ditridecyl 15,18,21,24-tetraaza-octatriacontane, and pharmaceutically acceptable salts thereof.

[0186] Other suitable cationic lipids for use in the compositions and methods of the invention include those 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 invention comprise cationic lipids having the following formula: [ka] or a pharmaceutically acceptable salt thereof, wherein R L Each occurrence of is independently an optionally substituted C6 to C 40 In certain embodiments, the compositions and methods of the present invention include: [ka] and pharmaceutically acceptable salts thereof. In certain embodiments, the compositions and methods of the present invention comprise cationic lipids having the compound structure: [ka] and pharmaceutically acceptable salts thereof. In certain embodiments, the compositions and methods of the present invention comprise cationic lipids having the compound structure: [ka] and pharmaceutically acceptable salts thereof. In certain embodiments, the compositions and methods of the present invention comprise cationic lipids having the compound structure: [ka] and pharmaceutically acceptable salts thereof.

[0187] Other suitable cationic lipids for use in the compositions and methods of the invention include those 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 invention comprise cationic lipids of the following formula: [ka] 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; and each R A are 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; each R Bare 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 invention provide a cationic lipid, "Target 23," having the following compound structure: [ka] and pharmaceutically acceptable salts thereof.

[0188] Other suitable cationic lipids for use in the compositions and methods of the invention include those cationic lipids described in International Patent Publication WO 2016 / 004202, which is incorporated herein by reference. In some embodiments, the compositions and methods of the invention provide cationic lipids having the compound structure: [ka] or a pharmaceutically acceptable salt thereof. In some embodiments, the compositions and methods of the present invention comprise a cationic lipid having the compound structure: [ka] or a pharmaceutically acceptable salt thereof. In some embodiments, the compositions and methods of the present invention comprise a cationic lipid having the compound structure: [ka] or a pharmaceutically acceptable salt thereof.

[0189] Other suitable cationic lipids for use in the compositions and methods of the present invention include those cationic lipids as described in U.S. Provisional Patent Application No. 62 / 758,179, which is incorporated herein by reference. In some embodiments, the compositions and methods of the present invention comprise cationic lipids of the following formula: [ka] or a pharmaceutically acceptable salt thereof, wherein each R 1 and R 2 is independently H or a C1-C6 aliphatic; each m is independently an integer having a value from 1 to 4; each A is independently a covalent bond or arylene; and each L 1 is independently an ester, thioester, disulfide, or anhydride group; each L 2 But independently, C2~C 10 is aliphatic; each X 1 is independently H or OH; each R 3 But independently, C6~C 20 In some embodiments, the compositions and methods of the present invention provide a cycloaliphatic alkyl group having the following formula: [ka] or a pharmaceutically acceptable salt thereof. In one embodiment, the compositions and methods of the present invention comprise a cationic lipid of the following formula: [ka] or a pharmaceutically acceptable salt thereof. In one embodiment, the compositions and methods of the present invention comprise a cationic lipid of the following formula: [ka] or a pharmaceutically acceptable salt thereof.

[0190] Other suitable cationic lipids for use in the compositions and methods of the present invention include those described in J. McClellan, MCKing, 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 of the compositions and methods of the present invention are: [ka] and pharmaceutically acceptable salts thereof.

[0191] Other suitable cationic lipids for use in the compositions and methods of the invention include those 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 invention provide cationic lipids having the compound structure: [ka] and pharmaceutically acceptable salts thereof. In some embodiments, the compositions and methods of the present invention comprise a cationic lipid having the compound structure: [ka] and pharmaceutically acceptable salts thereof. In some embodiments, the compositions and methods of the present invention comprise a cationic lipid having the compound structure: [ka] and pharmaceutically acceptable salts thereof. In some embodiments, the compositions and methods of the present invention comprise a cationic lipid having the compound structure: [ka] and pharmaceutically acceptable salts thereof. In some embodiments, the compositions and methods of the present invention comprise a cationic lipid having the compound structure: [ka] and pharmaceutically acceptable salts thereof. In some embodiments, the compositions and methods of the present invention comprise a cationic lipid having the compound structure: [ka] and pharmaceutically acceptable salts thereof. In some embodiments, the compositions and methods of the present invention comprise a cationic lipid having the compound structure: [ka] and pharmaceutically acceptable salts thereof. In some embodiments, the compositions and methods of the present invention comprise a cationic lipid having the compound structure: [ka] and pharmaceutically acceptable salts thereof. In some embodiments, the compositions and methods of the present invention comprise a cationic lipid having the compound structure: [ka] and pharmaceutically acceptable salts thereof. In some embodiments, the compositions and methods of the present invention comprise a cationic lipid having the compound structure: [ka] and pharmaceutically acceptable salts thereof. In some embodiments, the compositions and methods of the present invention comprise a cationic lipid having the compound structure: [ka] and pharmaceutically acceptable salts thereof. In some embodiments, the compositions and methods of the present invention comprise a cationic lipid having the compound structure: [ka] and pharmaceutically acceptable salts thereof. In some embodiments, the compositions and methods of the present invention comprise a cationic lipid having the compound structure: [ka] and pharmaceutically acceptable salts thereof.

[0192] Other suitable cationic lipids for use in the compositions and methods of the invention include those cationic lipids described in International Patent Publication WO 2017 / 004143, which is incorporated herein by reference. In some embodiments, the compositions and methods of the invention comprise cationic lipids having the compound structure: [ka] and pharmaceutically acceptable salts thereof. In some embodiments, the compositions and methods of the present invention comprise a cationic lipid having the compound structure: [ka] and pharmaceutically acceptable salts thereof. In some embodiments, the compositions and methods of the present invention comprise a cationic lipid having the compound structure: [ka] and pharmaceutically acceptable salts thereof. In some embodiments, the compositions and methods of the present invention comprise a cationic lipid having the compound structure: [ka] and pharmaceutically acceptable salts thereof. In some embodiments, the compositions and methods of the present invention comprise a cationic lipid having the compound structure: [ka] and pharmaceutically acceptable salts thereof. In some embodiments, the compositions and methods of the present invention comprise a cationic lipid having the compound structure: [ka] and pharmaceutically acceptable salts thereof. In some embodiments, the compositions and methods of the present invention comprise a cationic lipid having the compound structure: [ka] and pharmaceutically acceptable salts thereof. In some embodiments, the compositions and methods of the present invention comprise a cationic lipid having the compound structure: [ka] and pharmaceutically acceptable salts thereof. In some embodiments, the compositions and methods of the present invention comprise a cationic lipid having the compound structure: [ka] and pharmaceutically acceptable salts thereof. In some embodiments, the compositions and methods of the present invention comprise a cationic lipid having the compound structure: [ka] and pharmaceutically acceptable salts thereof. In some embodiments, the compositions and methods of the present invention comprise a cationic lipid having the compound structure: [ka] and pharmaceutically acceptable salts thereof. In some embodiments, the compositions and methods of the present invention comprise a cationic lipid having the compound structure: [ka] and pharmaceutically acceptable salts thereof. In some embodiments, the compositions and methods of the present invention comprise a cationic lipid having the compound structure: [ka] and pharmaceutically acceptable salts thereof. In some embodiments, the compositions and methods of the present invention comprise a cationic lipid having the compound structure: [ka] and pharmaceutically acceptable salts thereof. In some embodiments, the compositions and methods of the present invention comprise a cationic lipid having the compound structure: [ka] and pharmaceutically acceptable salts thereof. In some embodiments, the compositions and methods of the present invention comprise a cationic lipid having the compound structure: [ka] and pharmaceutically acceptable salts thereof. In some embodiments, the compositions and methods of the present invention comprise a cationic lipid having the compound structure: [ka] and pharmaceutically acceptable salts thereof.

[0193] Other suitable cationic lipids for use in the compositions and methods of the invention include those 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 invention comprise cationic lipids of the following formula: [ka] or a pharmaceutically acceptable salt thereof, wherein L 1 or L 2 One of the groups is -O(C=O)-, -(C=O)O-, -C(=O)-, -O-, -S(O) x , -SS-, -C(=O)S-, -SC(=O)-, -NR a C(=O)-, -C(=O)NR a -, NR a C(=O)NR a -, -OC(=O)NR a - or -NR a C(=O)O-;L 1 or L 2 The other is -O(C=O)-, -(C=O)O-, -C(=O)-, -O-, -S(O) x , -SS-, -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 to C 12 Alkylene or C1-C12 Alkenylene; G 3 However, C1~C 24 Alkylene, C1-C 24 alkenylene, C3-C8 cycloalkylene, C3-C8 cycloalkenylene; R a But H or C1~C 12 alkyl; R 1 and R 2 are independently C6~C 24 Alkyl or C6-C 24 alkenyl; R 3 But, H, OR 5 , CN, -C(=O)OR 4 , -OC(=O)R 4 or -NR 5 C(=O)R 4 and;R 4 However, C1~C 12 alkyl; R 5 is H or C1-C6 alkyl; and x is 0, 1 or 2.

[0194] Other suitable cationic lipids for use in the compositions and methods of the invention include those cationic lipids described in International Patent Publication WO 2017 / 117528, which is incorporated herein by reference. In some embodiments, the compositions and methods of the invention provide cationic lipids having the compound structure: [ka] and pharmaceutically acceptable salts thereof. In some embodiments, the compositions and methods of the present invention comprise a cationic lipid having the compound structure: [ka] and pharmaceutically acceptable salts thereof. In some embodiments, the compositions and methods of the present invention comprise a cationic lipid having the compound structure: [ka] and pharmaceutically acceptable salts thereof.

[0195] Other suitable cationic lipids for use in the compositions and methods of the invention include those cationic lipids as described in International Patent Publication WO 2017 / 049245, which is incorporated herein by reference. In some embodiments, the cationic lipid of the compositions and methods of the invention has the following formula: [ka] and pharmaceutically acceptable salts thereof. For any one of these four formulas, R4 is independently -(CH2) n Q and -(CH2) n Q is selected from -OR, -OH, -O(CH2) n and n is selected from the group consisting of N(R), -OC(O)R, -CX, -CN, -N(R)C(O)R, -N(H)C(O)R, -N(R)S(O)R, -N(H)S(O)R, -N(R)C(O)N(R), -N(H)C(O)N(R), -N(H)C(O)N(H)(R), -N(R)C(S)N(R), -N(H)C(S)N(R), -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 invention comprise: [ka] and pharmaceutically acceptable salts thereof. In certain embodiments, the compositions and methods of the present invention comprise cationic lipids having the compound structure: [ka] and pharmaceutically acceptable salts thereof. In certain embodiments, the compositions and methods of the present invention comprise cationic lipids having the compound structure: [ka] and pharmaceutically acceptable salts thereof. In certain embodiments, the compositions and methods of the present invention comprise cationic lipids having the compound structure: [ka] and pharmaceutically acceptable salts thereof.

[0196] Other suitable cationic lipids for use in the compositions and methods of the invention include those cationic lipids as described in International Patent Publications WO 2017 / 173054 and WO 2015 / 095340, each of which is incorporated herein by reference. In certain embodiments, the compositions and methods of the invention comprise: [ka] and pharmaceutically acceptable salts thereof. In certain embodiments, the compositions and methods of the present invention comprise cationic lipids having the compound structure: [ka] and pharmaceutically acceptable salts thereof. In certain embodiments, the compositions and methods of the present invention comprise cationic lipids having the compound structure: [ka] and pharmaceutically acceptable salts thereof. In certain embodiments, the compositions and methods of the present invention comprise cationic lipids having the compound structure: [ka] and pharmaceutically acceptable salts thereof.

[0197] Other suitable cationic lipids for use in the compositions and methods of the invention include cleavable cationic lipids such as those described in International Patent Publication WO 2012 / 170889, which is incorporated herein by reference. In some embodiments, the compositions and methods of the invention comprise cleavable cationic lipids of the formula: [ka] wherein R1 is selected from the group consisting of imidazole, guanidinium, amino, imine, enamine, optionally substituted alkylamino (e.g., alkylamino such as dimethylamino), and pyridyl; and R2 is selected from the group consisting of the following two formulae: [ka] wherein R3 and R4 are each independently an optionally substituted variably saturated or unsaturated C6-C 20 Alkyl and optionally substituted variably saturated or unsaturated C6-C 20 acyl; where n is 0 or any positive integer (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more). In certain embodiments, the compositions and methods of the present invention provide a cationic lipid, "HGT4001," having the following compound structure: [ka] and pharmaceutically acceptable salts thereof. In certain embodiments, the compositions and methods of the present invention include a cationic lipid, "HGT4002," having the following compound structure: [ka] and pharmaceutically acceptable salts thereof. In certain embodiments, the compositions and methods of the present invention include a cationic lipid, "HGT4003," having the following compound structure: [ka] and pharmaceutically acceptable salts thereof. In certain embodiments, the compositions and methods of the present invention include a cationic lipid, "HGT4004," having the following compound structure: [ka] and pharmaceutically acceptable salts thereof. In certain embodiments, the compositions and methods of the present invention include a cationic lipid "HGT4005" having the following compound structure: [ka] and pharmaceutically acceptable salts thereof.

[0198] Other suitable cationic lipids for use in the compositions and methods of the invention include cleavable cationic lipids such as those described in International Application No. PCT / US2019 / 032522, which is incorporated herein by reference. In certain embodiments, the compositions and methods of the invention comprise a cationic lipid having any of the general formulas described in International Application No. PCT / US2019 / 032522 or any of structures (1a)-(21a), (1b)-(21b), and (22)-(237). In certain embodiments, the compositions and methods of the invention comprise a cationic lipid having formula (I') [ka] and a cationic lipid having a structure represented by the formula: R X But independently, -H, -L 1 -R 1 , or -L 5A -L 5B -B'; L 1 , L 2 , and L 3 each independently represents a covalent bond, —C(O)—, —C(O)O—, —C(O)S—, or —C(O)NR L - and; Each L 4A and L 5A are independently —C(O)—, —C(O)O—, or —C(O)NR L - and; Each L 4B and L 5B However, independently, C1~C 20 Alkylene; C2-C 20 Alkenylene; or C2-C 20 is alkynylene; Each B and B' is NR 4 R 5 or a 5- to 10-membered nitrogen-containing heteroaryl; Each R 1 , R 2 , and R 3 But independently, C6~C 30 Alkyl, C6-C 30 Alkenyl, or C6-C 30 is alkynyl; Each R 4 and R 5 are independently hydrogen, C1 to C 10 Alkyl; C2-C 10 Alkenyl; or C2-C 10 is alkynyl; Each R L are independently hydrogen, C1 to C 20 Alkyl, C2-C 20 Alkenyl or C2-C 20 It is alkynyl.

[0199] In certain embodiments, the compositions and methods of the present invention comprise: [ka] The cationic lipid is compound (139) of International Application No. PCT / US2019 / 032522, which has the compound structure:

[0200] In certain embodiments, the compositions and methods of the present invention include the cationic lipid, N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (“DOTMA”). (Feigner et al. (Proc. Nat'l Acad. Sci. 84, 7413 (1987); U.S. Pat. No. 4,897,355, incorporated herein by reference). Other cationic lipids suitable for the compositions and methods of the present invention include, for example, 5-carboxyspermylglycine dioctadecylamide ("DOGS"); 2,3-dioleyloxy-N-[2(spermine-carboxamido)ethyl]-N,N-dimethyl-1-propanaminium ("DOSPA") (Behr et al. Proc. Nat'l Acad. Sci. 86, 6982 (1989); U.S. Pat. No. 5,171,678; U.S. Pat. No. 5,334,761); 1,2-dioleoyl-3-dimethylammonium-propane ("DODAP"); 1,2-dioleoyl-3-trimethylammonium-propane ("DOTAP").

[0201] Further exemplary cationic lipids suitable for the compositions and methods of the present invention also include 1,2-distearyloxy-N,N-dimethyl-3-aminopropane ("DSDMA"); 1,2-dioleyloxy-N,N-dimethyl-3-aminopropane ("DODMA"); 1,2-dilinoleyloxy-N,N-dimethyl-3-aminopropane ("DLinDMA"); 1,2-dilinolenyloxy-N,N-dimethyl-3-aminopropane ("DLenDMA"); N-dioleyl-N,N-dimethylammoni N,N-distearyl-N,N-dimethylammonium bromide ("DDAB"); N-(l,2-dimyristyloxyprop-3-yl)-N,N-dimethyl-N-hydroxyethylammonium bromide ("DMRIE"); 3-dimethylamino-2-(cholest-5-en-3-β-oxybutan-4-oxy)-l-(cis,cis-9,12-octadecadienoxy)propane ("CLinDMA"); 2-[5'-(cholest-5-en-3-β-oxy] N,N-dimethyl-3,4-dioleyloxybenzylamine ("DMOBA"); 1,2-N,N'-dioleylcarbamyl-3-dimethylaminepropane ("DOcarbDAP"); 2,3-dilinoleoyloxy-N,N-dimethylpropylamine ("DLinDAP"); 1,2-N,N'-dilinoleylcarbamyl-3-dimethylamine 1,2-Dilinoleoylcarbamyl-3-dimethylaminepropane ("DLinCDAP"); 2,2-Dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane ("DLin-K-DMA"); 2-((8-[(3P)-cholest-5-en-3-yloxy]octyl)oxy)-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-1-amine ("Octyl-CLinDMA");(2R)-2-((8-[(3β)-cholest-5-en-3-yloxy]octyl)oxy)-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-1-amine ("Octyl-CLinDMA(2R)"); (2S)-2-((8-[(3P)-cholest-5-en-3-yloxy]octyl)oxy)-N,fsl-dimethyl(dimethyh)3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-1-amine Examples include Pan-1-amine ("Octyl-CLinDMA(2S)"); 2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane ("DLin-K-XTC2-DMA"); and 2-(2,2-di((9Z,12Z)-octadeca-9,12-dien-1-yl)-1,3-dioxolan-4-yl)-N,N-dimethylethanamine ("DLin-KC2-DMA") (see WO 2010 / 042877; Semple et al., Nature Biotech. 28:172-176 (2010), which are incorporated by reference herein). (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 certain embodiments, one or more of the cationic lipids comprises at least one imidazole, dialkylamino, or guanidinium moiety;

[0202] In certain embodiments, the one or more cationic lipids suitable for the compositions and methods of the present invention include 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane ("XTC"); (3aR,5s,6aS)-N,N-dimethyl-2,2-di((9Z,12Z)-octadeca-9,12-dienyl)tetrahydro-3aH-cyclopenta[d][1,3]dioxol-5-amine ("ALNY-100") and / or 4,7,13-tris(3-oxo-3-(undecylamino)propyl)-N1,N16-diundecyl-4,7,10,13-tetraazahexadecane-1,16-diamide ("NC98-5").

[0203] In some embodiments, the compositions of the present invention comprise one or more cationic lipids that constitute at least about 5%, 10%, 20%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, or 70% of the total lipid content in the composition, e.g., as measured by weight of lipid nanoparticles. In some embodiments, the compositions of the present invention comprise one or more cationic lipids that constitute at least about 5%, 10%, 20%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, or 70% of the total lipid content in the composition, e.g., as measured as mol% of lipid nanoparticles. In certain embodiments, the compositions of the present invention comprise one or more cationic lipids that constitute 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 in the composition, e.g., measured by weight of the lipid nanoparticles. In certain embodiments, the compositions of the present invention comprise one or more cationic lipids that constitute 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 in the composition, e.g., measured as mol% of the lipid nanoparticles.

[0204] Non-cationic / Helper Lipids In some embodiments, the liposome contains one or more non-cationic ("helper") lipids. As used herein, the term "non-cationic lipid" refers to any neutral, zwitterionic, or anionic lipid. As used herein, the term "anionic lipid" refers to any of several lipid species that have 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 (DPPG), and oleoylphosphatidylethanolamine (DOPE). and mixtures thereof.

[0205] In certain embodiments, the non-cationic lipid is a neutral lipid, ie, a lipid that has no net electrical charge in the conditions in which the composition is formulated and / or administered.

[0206] In certain embodiments, such non-cationic lipids can be used alone, but are preferably used in combination with other lipids, such as cationic lipids.

[0207] In some embodiments, the noncationic lipids may 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 lipids present in the composition. In some embodiments, the total noncationic lipids may 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 lipids present in the composition. In some embodiments, the percentage of non-cationic lipids in the liposomes may 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 total non-cationic lipid percentage in the liposomes may 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 total non-cationic lipid percentage in the liposomes may be less than about 5 mol%, less than about 10 mol%, less than about 20 mol%, less than about 30 mol%, or less than about 40 mol%. In some embodiments, the total non-cationic lipid percentage in the liposomes may be less than about 5 mol%, less than about 10 mol%, less than about 20 mol%, less than about 30 mol%, or less than about 40 mol%.

[0208] In some embodiments, the noncationic lipids may 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 lipids present in the composition. In some embodiments, the total noncationic lipids may 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 lipids present in the composition. In some embodiments, the percentage of non-cationic lipids in the liposomes may be greater than about 5% by weight, greater than about 10% by weight, greater than about 20% by weight, greater than about 30% by weight, or greater than about 40% by weight. In some embodiments, the total non-cationic lipid percentage in the liposomes may be greater than about 5% by weight, greater than about 10% by weight, greater than about 20% by weight, greater than about 30% by weight, or greater than about 40% by weight. In some embodiments, the total non-cationic lipid percentage in the liposomes may be less than about 5% by weight, less than about 10% by weight, less than about 20% by weight, less than about 30% by weight, or less than about 40% by weight. In some embodiments, the total non-cationic lipid percentage in the liposomes may be less than about 5% by weight, less than about 10% by weight, less than about 20% by weight, less than about 30% by weight, or less than about 40% by weight.

[0209] Cholesterol-based lipids In some embodiments, the liposome comprises one or more cholesterol-based lipids. For example, suitable cholesterol-based cationic lipids include DC-Choi (N,N-dimethyl-N-ethylcarboxamidocholesterol), 1,4-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. Pat. No. 5,744,335), or imidazole cholesterol ester (ICE) having the following structure: [ka] Examples include:

[0210] In an embodiment, the cholesterol-based lipid is cholesterol.

[0211] In some embodiments, the cholesterol-based lipid may comprise 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 may 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 may be about 5 mol% or less, about 10 mol% or less, about 20 mol% or less, about 30 mol% or less, or about 40 mol% or less.

[0212] In some embodiments, the cholesterol-based lipid may 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 may 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 may be about 5 wt% or less, about 10 wt% or less, about 20 wt% or less, about 30 wt% or less, or about 40 wt% or less.

[0213] PEG modified lipid In certain embodiments, the liposome comprises one or more PEGylated lipids.

[0214] For example, the use of derivatized lipids, such as polyethylene glycol (PEG)-modified phospholipids and 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, including transfer vehicles (e.g., lipid nanoparticles), is also contemplated by the present invention.

[0215] Contemplated PEG-modified lipids include, but are not limited to, C6-C 20 The PEG-modified or PEGylated lipid comprises a polyethylene glycol chain of up to 5 kDa in length covalently attached to a lipid having an alkyl chain of up to 5 kDa in length. In one embodiment, the PEG-modified or PEGylated lipid is PEGylated cholesterol or PEG-2K. The addition of such components can prevent complex aggregation, increase circulation lifetime, and may also provide a means for increasing delivery of lipid-nucleic acid compositions to target tissues (Klibanov et al. (1990) FEBS Letters, 268(1):235-237), or they can be selected to rapidly exchange from the formulation in vivo (see U.S. Pat. No. 5,885,613). Particularly useful exchangeable lipids have shorter acyl chains (e.g., C 14 or C 18 ) is a PEG-ceramide.

[0216] The PEG-modified phospholipids and derivatized lipids of the present invention may 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 lipids present in the liposome transfer vehicle. In some embodiments, one or more PEG-modified lipids comprise about 4% of the total lipids by molar ratio. In some embodiments, one or more PEG-modified lipids comprise about 5% of the total lipids by molar ratio. In some embodiments, one or more PEG-modified lipids comprise about 6% of the total lipids by molar ratio.

[0217] Amphiphilic Block Copolymers In certain embodiments, a suitable delivery vehicle contains an amphiphilic block copolymer (eg, a poloxamer).

[0218] A variety of amphiphilic block copolymers can be used in practicing the present invention. In certain embodiments, the amphiphilic block copolymers are also referred to as surfactants or nonionic surfactants.

[0219] In certain embodiments, amphiphilic polymers suitable for the present invention are selected from poloxamers (Pluronic®), poloxamines (Tetronic®), polyoxyethylene glycol sorbitan alkyl esters (polysorbates), and polyvinylpyrrolidone (PVP).

[0220] Poloxamer In some embodiments, a suitable amphiphilic polymer is a poloxamer. For example, a suitable poloxamer has the following structure: [ka] wherein a is an integer of 10 to 150, and b is an integer of 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.

[0221] In one embodiment, poloxamers suitable for the present invention have from about 10 to about 150 ethylene oxide units. In one embodiment, poloxamers have from about 10 to about 100 ethylene oxide units.

[0222] 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.

[0223] In some embodiments, suitable poloxamers have an average molecular weight of about 4,000 g / mol to about 20,000 g / mol. In some embodiments, suitable poloxamers have an average molecular weight of about 1,000 g / mol to about 50,000 g / mol. In some embodiments, suitable poloxamers have an average molecular weight of about 1,000 g / mol. In some embodiments, suitable poloxamers have an average molecular weight of about 2,000 g / mol. In some embodiments, suitable poloxamers have an average molecular weight of about 3,000 g / mol. In some embodiments, suitable poloxamers have an average molecular weight of about 4,000 g / mol. In some embodiments, suitable poloxamers have an average molecular weight of about 5,000 g / mol. In some embodiments, suitable poloxamers have an average molecular weight of about 6,000 g / mol. In some embodiments, suitable poloxamers have an average molecular weight of about 7,000 g / mol. In some embodiments, suitable poloxamers have an average molecular weight of about 8,000 g / mol. In some embodiments, suitable poloxamers have an average molecular weight of about 9,000 g / mol. In some embodiments, suitable poloxamers have an average molecular weight of about 10,000 g / mol. In some embodiments, suitable poloxamers have an average molecular weight of about 20,000 g / mol. In some embodiments, suitable poloxamers have an average molecular weight of about 25,000 g / mol. In some embodiments, suitable poloxamers have an average molecular weight of about 30,000 g / mol. In some embodiments, suitable poloxamers have an average molecular weight of about 40,000 g / mol. In some embodiments, suitable poloxamers have an average molecular weight of about 50,000 g / mol.

[0224] Other amphiphilic polymers In certain embodiments, the amphiphilic polymer is a poloxamine, such as tetronic 304 or tetronic 904.

[0225] In certain embodiments, the amphiphilic polymer is polyvinylpyrrolidone (PVP), such as PVP having a molecular weight of 3 kDa, 10 kDa, or 29 kDa.

[0226] In some embodiments, the amphiphilic polymer is polyethylene glycol ether (Brij), polysorbate, sorbitan, and derivatives thereof. In some embodiments, the amphiphilic polymer is a polysorbate, such as PS 20.

[0227] In certain embodiments, the amphiphilic polymer is a polyethylene glycol ether (Brij), a poloxamer, a polysorbate, a sorbitan, or a derivative thereof.

[0228] In some embodiments, the amphiphilic polymer is a polyethylene glycol ether. In some embodiments, a suitable polyethylene glycol ether has the formula (S-1): [ka] or a salt or isomer thereof, wherein: t is an integer from 1 to 100; R 1BRIJ But independently, C 10~40 Alkyl, C 10~40 Alkenyl, or C 10~40 alkynyl; optionally, R 5PEG one or more methylene groups are independently selected from C 3~10 Carbocyclylene, 4-10 membered heterocyclylene, C 6~10 Arylene, 4- to 10-membered heteroarylene, -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-substituted; R N Each case is independently hydrogen, C 1~6 alkyl, or nitrogen protecting groups.

[0229] In one embodiment, R 1BRIJ where C is alkyl. For example, polyethylene glycol ethers are represented by the formula (S-1a): [ka] or a salt or isomer thereof, wherein s is an integer of 1 to 100.

[0230] In one embodiment, R 1BRIJ where C is alkenyl. For example, a suitable polyethylene glycol ether is represented by the formula (S-1b): [ka] or a salt or isomer thereof, wherein s is an integer of 1 to 100.

[0231] 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 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% less than its CMC. In some embodiments, the amphiphilic polymer (e.g., poloxamer) is present in the mixture in an amount about 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, or 0.1% less than its CMC. In certain embodiments, the amphiphilic polymer (e.g., poloxamer) is present in the mixture in an amount that is about 55%, 60%, 65%, 70%, 75%, 80%, 90%, or 95% less than its CMC.

[0232] In some embodiments, less than about 0.1%, 0.09%, 0.08%, 0.07%, 0.06%, 0.05%, 0.04%, 0.03%, 0.02%, or 0.01% of the original amount of amphiphilic polymer (e.g., poloxamer) present in the formulation remains after removal. In some embodiments, a residual amount of amphiphilic polymer (e.g., poloxamer) remains in the formulation after removal. As used herein, residual amount refers to the amount remaining after substantially all of the substance (e.g., amphiphilic polymer described herein, such as poloxamer) in the composition has been removed. The residual amount may be detectable qualitatively or quantitatively using known techniques. The residual amount may not be detectable using known techniques.

[0233] In some embodiments, a suitable delivery vehicle comprises less than 5% amphiphilic block copolymer (e.g., poloxamer). In some embodiments, a suitable delivery vehicle comprises less than 3% amphiphilic block copolymer (e.g., poloxamer). In some embodiments, a suitable delivery vehicle comprises less than 2.5% amphiphilic block copolymer (e.g., poloxamer). In some embodiments, a suitable delivery vehicle comprises less than 2% amphiphilic block copolymer (e.g., poloxamer). In some embodiments, a suitable delivery vehicle comprises less than 1.5% amphiphilic block copolymer (e.g., poloxamer). In some embodiments, a suitable delivery vehicle comprises less than 1% amphiphilic block copolymer (e.g., poloxamer). In some embodiments, a suitable delivery vehicle comprises less than 0.5% (e.g., less than 0.4%, 0.3%, 0.2%, or 0.1%) amphiphilic block copolymer (e.g., poloxamer). In some embodiments, a suitable delivery vehicle comprises less than 0.09%, 0.08%, 0.07%, 0.06%, 0.05%, 0.04%, 0.03%, 0.02%, or 0.01% of an amphiphilic block copolymer (e.g., poloxamer). In some embodiments, a suitable delivery vehicle comprises less than 0.01% of an amphiphilic block copolymer (e.g., poloxamer). In some embodiments, a suitable delivery vehicle contains a residual amount of an amphiphilic polymer (e.g., poloxamer). As used herein, residual amount refers to the amount remaining after substantially all of the substance (an amphiphilic polymer described herein, such as a poloxamer) in the composition has been removed. The residual amount may be detectable qualitatively or quantitatively using known techniques. The residual amount may not be detectable using known techniques.

[0234] polymer In some embodiments, suitable delivery vehicles are formulated with polymers as carriers, either alone or in combination with other carriers, including various lipids, as described herein. Thus, in some embodiments, the term "liposome delivery vehicle," as used herein, also encompasses nanoparticles containing polymers. Suitable polymers may include, for example, polyacrylate, polyalkylcyanoacrylate, polylactide, polylactide-polyglycolide copolymer, polycaprolactone, dextran, albumin, gelatin, alginate, collagen, chitosan, cyclodextrin, protamine, PEGylated protamine, PLL, PEGylated PLL, and polyethyleneimine (PEI). When PEI is present, it may be branched PEI with a molecular weight ranging from 10 to 40 kDa, e.g., 25 kDa branched PEI (Sigma #408727).

[0235] According to various embodiments, the selection of cationic lipids, non-cationic lipids, PEG-modified lipids, cholesterol-based lipids, and / or amphiphilic block copolymers, including lipid nanoparticles, and the relative molar ratio of these components (lipids) to each other are based on the characteristics of the selected lipids, the nature of the intended target cells, and the characteristics of the nucleic acid to be delivered. Further considerations include, for example, the saturation of alkyl chains, as well as the size, charge, pH, pKa, fusogenicity, and toxicity of the selected lipids. Therefore, the molar ratio can be adjusted accordingly.

[0236] Ratio of different lipid components Suitable liposomes for the present invention may contain one or more of the cationic lipids, non-cationic lipids, cholesterol lipids, PEG-modified lipids, amphiphilic block copolymers, and / or polymers described herein in various ratios. In some embodiments, the lipid nanoparticles contain five or fewer different nanoparticle components. In some embodiments, the lipid nanoparticles contain four or fewer different nanoparticle components. In some embodiments, the lipid nanoparticles contain three or fewer different nanoparticle components. As non-limiting examples, suitable liposome formulations may contain a combination 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.

[0237] In various embodiments, the cationic lipid (e.g., cKK-E12, C12-200, ICE, and / or HGT4003) constitutes 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 by molar ratio. 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% or more of the liposome by molar ratio.

[0238] In some embodiments, the ratio of cationic lipids to non-cationic lipids to cholesterol-based lipids to PEG-modified lipids may be about 30-60:25-35:20-30:1-15, respectively. In some embodiments, the ratio of cationic lipids to non-cationic lipids to cholesterol-based lipids to PEG-modified lipids is about 40:30:20:10, respectively. In some embodiments, the ratio of cationic lipids to non-cationic lipids to cholesterol-based lipids to PEG-modified lipids is about 40:30:25:5, respectively. In some embodiments, the ratio of cationic lipids to non-cationic lipids to cholesterol-based lipids to PEG-modified lipids is about 40:32:25:3, respectively. In some embodiments, the ratio of cationic lipids to non-cationic lipids to cholesterol-based lipids to PEG-modified lipids is about 50:25:20:5.

[0239] In embodiments in which the lipid nanoparticle comprises three or fewer different components of lipids, the ratio of the total lipid content (i.e., the ratio of lipid component (1):lipid component (2):lipid component (3)) can be expressed as x:y:z, where: (y+z)=100-x is.

[0240] In one embodiment, each of "x," "y," and "z" represents the molar percentage of three different components of the lipid, and the ratios are molar ratios.

[0241] In one embodiment, "x," "y," and "z" each represent the weight percentage of three different components of the lipid, and the ratios are by weight.

[0242] In certain embodiments, the lipid component (1), represented by the variable "x," is a sterol-based cationic lipid.

[0243] In certain embodiments, the lipid component (2), represented by the variable "y", is a helper lipid.

[0244] In one embodiment, the lipid component (3), represented by the variable "z," is a PEG lipid.

[0245] In certain embodiments, the variable "x", which represents the molar percentage of lipid component (1) (e.g., 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%.

[0246] In some embodiments, the variable "x," representing the molar percentage of lipid component (1) (e.g., sterol-based cationic lipid), is 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% or less. In some embodiments, the variable "x" is about 65%, about 60%, about 55%, about 50%, about 40%, or less.

[0247] In some embodiments, the variable "x", representing the molar percentage 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 some 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%.

[0248] In certain embodiments, the variable "x," representing the weight percentage of lipid component (1) (e.g., 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%.

[0249] In some embodiments, the variable "x," representing the weight percentage of lipid component (1) (e.g., sterol-based cationic lipid), is 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% or less. In some embodiments, the variable "x" is about 65%, about 60%, about 55%, about 50%, about 40%, or less.

[0250] In some embodiments, the variable "x", representing the weight percentage 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 some 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%.

[0251] In some embodiments, the variable "z", which represents the molar percentage of lipid component (3) (e.g., PEG lipid), is less than or equal to about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, or 25%. In some embodiments, the variable "z", which represents the molar percentage of lipid component (3) (e.g., PEG lipid), is less than or equal to about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10%. In embodiments, the variable "z," representing the molar percentage of lipid component (3) (e.g., PEG lipid), is between about 1% and about 10%, between about 2% and about 10%, between about 3% and about 10%, between about 4% and about 10%, between about 1% and about 7.5%, between about 2.5% and about 10%, between about 2.5% and about 7.5%, between about 2.5% and about 5%, between about 5% and about 7.5%, or between about 5% and about 10%.

[0252] In some embodiments, the variable "z", which represents the weight percentage of lipid component (3) (e.g., PEG lipid), is less than or equal to about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, or 25%. In some embodiments, the variable "z", which represents the weight percentage of lipid component (3) (e.g., PEG lipid), is less than or equal to about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10%. In embodiments, the variable "z," representing the weight percentage of lipid component (3) (e.g., PEG lipid), is between about 1% and about 10%, between about 2% and about 10%, between about 3% and about 10%, between about 4% and about 10%, between about 1% and about 7.5%, between about 2.5% and about 10%, between about 2.5% and about 7.5%, between about 2.5% and about 5%, between about 5% and about 7.5%, or between about 5% and about 10%.

[0253] For compositions having three and only three different lipid components, the variables "x," "y," and "z" can be in any combination as long as the sum of the three variables equals 100% of the total lipid content.

[0254] Formation of liposomes encapsulating mRNA The liposome transfer vehicle for use in the composition of the present invention can be prepared by various techniques currently known in the art.For example, multilamellar vesicles (MLVs) can be prepared according to conventional techniques, such as by dissolving lipids in a suitable solvent, then evaporating the solvent to leave a thin film inside the vessel, or by spray-drying to deposit the selected lipids on the inner wall of a suitable container or vessel.Then, an aqueous phase can be added to the vessel with a vortex action, thereby forming MLVs.Unilamellar vesicles (ULVs) can then be formed by homogenizing, sonicating, or extruding the multilamellar vesicles.Furthermore, unilamellar vesicles can be formed by detergent removal techniques.

[0255] Various methods can be used to practice the present invention, as described in U.S. Patent Application Publication No. 2011 / 0244026, U.S. Patent Application Publication No. 2016 / 0038432, U.S. Patent Application Publication No. 2018 / 0153822, U.S. Patent Application Publication No. 2018 / 0125989, and U.S. Provisional Patent Application No. 62 / 877,597, filed July 23, 2019, all of which are incorporated herein by reference. As used herein, Process A refers to the conventional method of encapsulating mRNA by mixing the mRNA with a mixture of lipids without first preforming the lipids into lipid nanoparticles, as described in U.S. Patent Application Publication No. 2016 / 0038432. As used herein, Process B refers to a method of encapsulating messenger RNA (mRNA) by mixing preformed lipid nanoparticles with the mRNA, as described in U.S. Patent Application Publication No. 2018 / 0153822.

[0256] Briefly, a method for preparing lipid liposomes loaded with mRNA or MCNA includes heating (i.e., applying heat from a heat source to) one or more solutions to (or maintaining at) a temperature above ambient temperature; another solution is a solution containing preformed lipid nanoparticles, a solution containing mRNA, and a mixed solution containing lipid nanoparticle-encapsulated mRNA. In some embodiments, the method includes heating one or both of the mRNA solution and the preformed lipid nanoparticle solution prior to the mixing step. In some embodiments, the method includes heating one or more of the solution containing preformed lipid nanoparticles, the solution containing mRNA, and the solution containing lipid nanoparticle-encapsulated mRNA during the mixing step. In some embodiments, the method includes heating the lipid nanoparticle-encapsulated mRNA after the mixing step. In some embodiments, the temperature to which one or more of the solutions is heated (or one or more of the solutions is maintained) is about 30°C, 37°C, 40°C, 45°C, 50°C, 55°C, 60°C, 65°C, or 70°C or higher. In certain embodiments, the temperature to which one or more of the solutions are heated ranges from about 25-70° C., about 30-70° C., about 35-70° C., about 40-70° C., about 45-70° C., about 50-70° C., or about 60-70° C. In certain embodiments, the temperature above ambient temperature to which one or more of the solutions are heated is about 65° C.

[0257] Various methods can be used to prepare mRNA solutions suitable for the present invention. In some embodiments, mRNA can be directly dissolved in a buffer solution as described herein. In some embodiments, an mRNA solution can be generated by mixing an mRNA stock solution with a buffer solution before mixing with a lipid solution for encapsulation. In some embodiments, an mRNA solution can be generated by mixing an mRNA stock solution with a buffer solution immediately before mixing with a lipid solution for encapsulation. In some embodiments, a suitable mRNA stock solution can contain mRNA in water at a concentration of about 0.2 mg / ml, 0.4 mg / ml, 0.5 mg / ml, 0.6 mg / ml, 0.8 mg / ml, 1.0 mg / ml, 1.2 mg / ml, 1.4 mg / ml, 1.5 mg / ml, 1.6 mg / ml, 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 or more.

[0258] In certain embodiments, the mRNA stock solution is mixed with the buffer using a pump. Exemplary pumps include, but are not limited to, gear pumps, peristaltic pumps, and centrifugal pumps.

[0259] Typically, the buffer is mixed at a rate higher than that of the mRNA stock solution. For example, the buffer may be mixed at a rate at least 1x, 2x, 3x, 4x, 5x, 6x, 7x, 8x, 9x, 10x, 15x, or 20x higher than that of the mRNA stock solution. In certain embodiments, the buffer is mixed at a flow rate in the range of 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 certain embodiments, the buffer 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.

[0260] In one embodiment, the mRNA stock solution is mixed at a flow rate in the range of about 10 to 600 ml / min (e.g., about 5 to 50 ml / min, about 10 to 30 ml / min, about 30 to 60 ml / min, about 60 to 120 ml / min, about 120 to 240 ml / min, about 240 to 360 ml / min, about 360 to 480 ml / min, or about 480 to 600 ml / min). In certain 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.

[0261] According to the present invention, the lipid solution contains a mixture of lipids suitable for forming lipid nanoparticles for encapsulating mRNA. In one embodiment, a suitable lipid solution is ethanol-based. For example, a suitable lipid solution may contain a mixture of desired lipids dissolved in pure ethanol (i.e., 100% ethanol). In another embodiment, a suitable lipid solution is isopropyl alcohol-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, isopropyl alcohol, and dimethyl sulfoxide.

[0262] Suitable lipid solutions may contain a mixture of desired lipids at various concentrations, for example, 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 more. In certain embodiments, a suitable lipid solution may 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 certain embodiments, a suitable lipid solution may contain a mixture of desired lipids at a total concentration of up to about 100 mg / ml, 90 mg / ml, 80 mg / ml, 70 mg / ml, 60 mg / ml, 50 mg / ml, 40 mg / ml, 30 mg / ml, 20 mg / ml, or 10 mg / ml.

[0263] 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 cationic lipids, helper lipids (e.g., non-cationic lipids and / or cholesterol lipids), amphiphilic block copolymers (e.g., poloxamers), and / or PEGylated lipids. In some embodiments, a suitable lipid solution contains a mixture of desired lipids, including one or more cationic lipids, one or more helper lipids (e.g., non-cationic lipids and / or cholesterol lipids), and one or more PEGylated lipids.

[0264] In certain embodiments, the provided compositions comprise liposomes, wherein mRNA is associated with both surfaces of the liposome and encapsulated within the same liposome. For example, during the preparation of the compositions of the present invention, cationic liposomes can be associated with mRNA or MCNA through electrostatic interactions.

[0265] In some embodiments, the compositions and methods of the present invention include mRNA encapsulated in liposomes. In some embodiments, one or more mRNA species may be encapsulated in the same liposome. In some embodiments, one or more mRNA species may be encapsulated in different liposomes. In some embodiments, the mRNA is encapsulated in one or more liposomes, which 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 may have different compositions of sterol-based cationic lipids, neutral lipids, PEG-modified lipids, and / or combinations thereof. In some embodiments, one or more liposomes may have different molar ratios of cholesterol-based cationic lipids, neutral lipids, and PEG-modified lipids used to generate the liposomes.

[0266] 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, incorporated herein by reference. The nucleic acid incorporated into a liposome may be located entirely or partially within the liposome's bilayer membrane, in the interior space of the liposome, or associated with the outer surface of the liposome membrane. The incorporation of a nucleic acid into a liposome is also referred to herein as "encapsulation," in which the nucleic acid is completely contained within the interior space of the liposome. The purpose of incorporating mRNA into a transfer vehicle such as a liposome is often to protect the nucleic acid from an environment that may contain enzymes or chemicals that degrade the nucleic acid and / or systems or receptors that cause the nucleic acid to be rapidly excreted. Thus, in certain embodiments, a suitable delivery vehicle can promote the stability of the mRNA contained therein and / or facilitate the delivery of a therapeutic agent (e.g., mRNA or MCNA) to a target cell or tissue.

[0267] Suitable liposomes according to the present invention can be made in a variety of sizes. In some embodiments, the provided liposomes can be made smaller than known liposomes. In some embodiments, the reduced size of the liposomes is associated with more efficient delivery of a therapeutic agent (e.g., mRNA or MCNA). The selection of an appropriate liposome size can take into account the site of the target cell or tissue and, to some extent, the intended use for which the liposomes are made.

[0268] In some embodiments, the appropriate size of liposomes is selected to promote the systemic distribution of the antibody encoded by mRNA.In some embodiments, it may be desirable to restrict the transfection of mRNA to specific cells or tissues.For example, to target hepatocytes, liposomes can be sized so that their dimensions are smaller than the perforations of the endothelial layer covering the hepatic sinusoids in the liver; in this case, liposomes can easily penetrate these endothelial perforations to reach the target hepatocytes.

[0269] Alternatively or additionally, liposomes can be sized such that the liposome dimensions are of sufficient diameter to limit or significantly avoid distribution to particular cells or tissues.

[0270] Various alternative methods known in the art are available for sizing liposome populations. One such sizing method is described in U.S. Pat. No. 4,737,323, incorporated herein by reference. Sonicating a liposome suspension, either by bath or probe sonication, gradually reduces the size to small ULVs less than about 0.05 microns in diameter. Homogenization is another method that relies on shear energy to disrupt large liposomes into smaller ones. In a typical homogenization procedure, MLVs are recirculated through a standard emulsion homogenizer until selected liposome sizes, typically about 0.1 to 0.5 microns, are observed. Liposome size can be determined by quasi-electric light scattering (QELS), as described in Bloomfield, Ann. Rev. Biophys. Bioeng., 10:421-450 (1981), incorporated herein by reference. The average liposome diameter can be reduced by sonication of the formed liposomes. Intermittent sonication cycles can be alternated with QELS assessment to induce efficient liposome synthesis.

[0271] Provided nanoparticles encapsulating mRNA In certain embodiments, the 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, have a size of about 150 nm (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 have a size of about 150 nm (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).

[0272] In some embodiments, the lipid nanoparticles have an average size of less than 150 nm. In some embodiments, the lipid nanoparticles have an average size of less than 120 nm. In some embodiments, the lipid nanoparticles have an average size of less than 100 nm. In some embodiments, the lipid nanoparticles have an average size of less than 90 nm. In some embodiments, the lipid nanoparticles have an average size of less than 80 nm. In some embodiments, the lipid nanoparticles have an average size of less than 70 nm. In some embodiments, the lipid nanoparticles have an average size of less than 60 nm. In some embodiments, the lipid nanoparticles have an average size of less than 50 nm. In some embodiments, the lipid nanoparticles have an average size of less than 30 nm. In some embodiments, the lipid nanoparticles have an average size of less than 20 nm.

[0273] In certain embodiments, the dispersity, or molecular size heterogeneity measure (PDI), of the nanoparticles in the compositions provided herein is less than about 0.5. In certain embodiments, the lipid nanoparticles have a PDI of less than about 0.5. In certain embodiments, the lipid nanoparticles have a PDI of less than about 0.4. In certain embodiments, the lipid nanoparticles have a PDI of less than about 0.3. In certain embodiments, the lipid nanoparticles have a PDI of less than about 0.28. In certain embodiments, the lipid nanoparticles have a PDI of less than about 0.25. In certain embodiments, the lipid nanoparticles have a PDI of less than about 0.23. In certain embodiments, the lipid nanoparticles have a PDI of less than about 0.20. In certain embodiments, the lipid nanoparticles have a PDI of less than about 0.18. In certain embodiments, the lipid nanoparticles have a PDI of less than about 0.16. In certain embodiments, the lipid nanoparticles have a PDI of less than about 0.14. In certain embodiments, the lipid nanoparticles have a PDI of less than about 0.12. In certain embodiments, the lipid nanoparticles have a PDI of less than about 0.10. In certain embodiments, the lipid nanoparticles have a PDI of less than about 0.08.

[0274] In some embodiments, greater than about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of the purified lipid nanoparticles in a composition provided herein encapsulate mRNA within each individual particle. In some embodiments, substantially all of the purified lipid nanoparticles in a composition encapsulate mRNA within each individual particle. In some embodiments, the lipid nanoparticles have an encapsulation efficiency of 50% to 99%. In some embodiments, the lipid nanoparticles have an encapsulation efficiency of greater than about 60%. In some embodiments, the lipid nanoparticles have an encapsulation efficiency of greater than about 65%. In some embodiments, the lipid nanoparticles have an encapsulation efficiency of greater than about 70%. In some embodiments, the lipid nanoparticles have an encapsulation efficiency of greater than about 75%. In some embodiments, the lipid nanoparticles have an encapsulation efficiency of greater than about 80%. In some embodiments, the lipid nanoparticles have an encapsulation efficiency of greater than about 85%. In some embodiments, the lipid nanoparticles have an encapsulation efficiency of greater than about 90%. In some embodiments, the lipid nanoparticles have an encapsulation efficiency of greater than about 92%. In some embodiments, the lipid nanoparticles have an encapsulation efficiency of greater than about 95%. In some embodiments, the lipid nanoparticles have an encapsulation efficiency of greater than about 98%. In some embodiments, the lipid nanoparticles have an encapsulation efficiency of greater than about 99%.

[0275] In some embodiments, the lipid nanoparticles have an N / P ratio of 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 nanoparticles to negatively charged molecular units in the mRNA encapsulated within the lipid nanoparticles. Thus, the N / P ratio is typically calculated as the ratio of the number of moles of amine groups in the cationic lipids in the lipid nanoparticles to the number of moles of phosphate groups in the mRNA encapsulated within the lipid nanoparticles. In some embodiments, the lipid nanoparticles have an N / P ratio greater than 1. In some embodiments, the lipid nanoparticles have an N / P ratio of about 1. In some embodiments, the lipid nanoparticles have an N / P ratio of about 2. In some embodiments, the lipid nanoparticles have an N / P ratio of about 3. In some embodiments, the lipid nanoparticles have an N / P ratio of about 4. In some embodiments, the lipid nanoparticles have an N / P ratio of about 5. In some embodiments, the lipid nanoparticles have an N / P ratio of about 6. In some embodiments, the lipid nanoparticles have an N / P ratio of about 7. In certain embodiments, the lipid nanoparticles have an N / P ratio of about 8.

[0276] In some embodiments, compositions of the present invention 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, compositions contain between about 0.1 mg and 1000 mg of encapsulated mRNA. In some embodiments, compositions contain at least about 0.5 mg of encapsulated mRNA. In some embodiments, compositions contain at least about 0.8 mg of encapsulated mRNA. In some embodiments, compositions contain at least about 1 mg of encapsulated mRNA. In some embodiments, compositions contain at least about 5 mg of encapsulated mRNA. In some embodiments, compositions contain at least about 8 mg of encapsulated mRNA. In some embodiments, compositions contain at least about 10 mg of encapsulated mRNA. In some embodiments, compositions contain at least about 50 mg of encapsulated mRNA. In some embodiments, compositions contain at least about 100 mg of encapsulated mRNA. In some embodiments, compositions contain at least about 500 mg of encapsulated mRNA. In some embodiments, compositions contain at least about 1000 mg of encapsulated mRNA.

[0277] Therapeutic Uses of the Composition To facilitate expression of mRNA in vivo, delivery vehicles such as liposomes can be combined with one or more additional nucleic acids, carriers, targeting ligands, or stabilizing reagents, or formulated into pharmacological compositions in which they are mixed with suitable excipients. Techniques for drug formulation and administration can be found in "Remington's Pharmaceutical Sciences," Mack Publishing Co., Easton, Pa., latest edition.

[0278] In some embodiments, the composition comprises 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.

[0279] The provided mRNA-loaded nanoparticles and compositions containing them can be administered and dosed in accordance with current medical practice, taking into account the subject's clinical condition, the site and method of administration, the administration schedule, the subject's age, sex, and weight, and other factors relevant to a clinician of ordinary skill in the art. For purposes herein, an "effective amount" can be determined by experimental clinical studies, pharmacological, clinical, and other relevant considerations known to those skilled in the art. In certain embodiments, the amount administered is effective to achieve at least some stabilization, improvement, or elimination of symptoms, and other indicators selected by those skilled in the art as appropriate measures of disease progression, regression, or improvement. For example, a suitable amount and administration regimen is one that causes at least transient protein (e.g., enzyme) production.

[0280] The present invention provides a method for delivering mRNA for in vivo protein production, comprising administering the mRNA to a subject in need thereof. In some embodiments, the mRNA is administered by a route selected from the group consisting of intravenous, subcutaneous, oral, subcutaneous, intraocular, intratracheal instillation, pulmonary (e.g., nebulization), intramuscular, intrathecal, or intraarticular delivery.

[0281] Suitable routes of administration include, for example, oral, rectal, vaginal, transmucosal, pulmonary, including intratracheal or inhalation, or intestinal administration; intradermal, transdermal (topical), intramuscular, subcutaneous, intramedullary injection, and parenteral delivery, including intrathecal, direct intraventricular, 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, administration results in delivery of the mRNA to muscle cells. In some embodiments, administration results in delivery of the mRNA to hepatocytes (i.e., liver cells). In certain embodiments, intramuscular administration results in delivery of the mRNA to muscle cells.

[0282] Further teachings of pulmonary delivery and nebulization are described in U.S. Patent Application Publication Nos. 2018 / 0125989 and 2018 / 0333457, each of which is incorporated by reference in its entirety.

[0283] Alternatively or additionally, the mRNA-loaded nanoparticles and compositions of the present invention, preferably in sustained-release formulations, can be administered locally rather than systemically, for example, by injection of the pharmaceutical composition directly into the targeted tissue. Local delivery can be affected in various ways depending on the targeted tissue. For example, an aerosol containing the present compositions can be inhaled (for nasal, tracheal, or bronchial delivery); the present compositions can be injected, for example, at the site of an injury, disease symptom, or pain; the compositions can be provided in a lozenge for oral, tracheal, or esophageal application; they can be provided in liquid, tablet, or capsule form for gastric or intestinal administration, in suppository form for rectal or vaginal application, or even delivered to the eye by the use of a cream, drops, or injection. Formulations containing the provided compositions complexed with therapeutic molecules or ligands can even be administered surgically, for example, with a polymer or other structure or substance that can allow the composition to diffuse from the implantation site to surrounding cells. Alternatively, they can be applied surgically without the use of a polymer or carrier.

[0284] The provided methods of the present invention contemplate single administration as well as multiple administrations of a therapeutically effective amount of a therapeutic agent (e.g., mRNA) described herein. The therapeutic agent may be administered at regular intervals depending on the nature, severity, and extent of the subject's condition. In certain embodiments, a therapeutically effective amount of a therapeutic agent (e.g., mRNA) of the present invention may be administered intrathecally at regular intervals (e.g., once a year, once every six months, once every five months, once every three months, every other month (once every two months), monthly (once every month), every other week (once every two weeks), twice a month, once every 30 days, once every 28 days, once every 14 days, once every 10 days, once every 7 days, weekly, twice a week, daily, or continuously).

[0285] In certain embodiments, the provided liposomes and / or compositions are formulated to be suitable for sustained release of the mRNA contained therein. Such sustained-release compositions can be conveniently administered to a subject at extended intervals. For example, in one embodiment, the compositions of the present invention are administered to a subject twice daily, daily, or every other day. In a preferred embodiment, the compositions of the present invention 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 2 weeks, once every 3 weeks, or more preferably, once every 4 weeks, once monthly, twice monthly, once every 6 weeks, once every 8 weeks, once every other month, once every 3 months, once every 4 months, once every 6 months, once every 8 months, once every 9 months, or annually. 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 are also contemplated. Preferably, the sustained release means used is combined with modifications made to the mRNA to enhance stability.

[0286] As used herein, the term "therapeutically effective amount" is determined primarily based on the total amount of the therapeutic agent contained in the pharmaceutical composition of the present invention. Generally, a therapeutically effective amount is sufficient to achieve a significant benefit to the subject (e.g., treat, regulate, cure, prevent, and / or ameliorate a disease or disorder). For example, a therapeutically effective amount may be an amount sufficient to achieve a desired therapeutic and / or preventive effect. Generally, the amount of a therapeutic agent (e.g., mRNA) administered to a subject in need thereof will depend on the subject's characteristics. Such characteristics include the subject's condition, disease severity, overall health, age, sex, and weight. Those skilled in the art can easily determine the appropriate dosage depending on these and other relevant factors. Furthermore, both objective and subjective assays can optionally be used to ascertain optimal dosage ranges.

[0287] A therapeutically effective amount is generally administered in a dosage regimen that may include multiple unit doses. For any particular therapeutic protein, the therapeutically effective amount (and / or appropriate unit dose within an effective dosage regimen) may vary, for example, depending on the route of administration and on the combination with other pharmaceutical agents. In addition, the specific therapeutically effective amount (and / or unit dose) for any particular patient may depend on a variety of factors, including the disorder being treated and the severity of the disorder; the activity of the specific pharmaceutical agent used; the specific composition used; the patient's age, weight, overall health, sex, and dietary habits; the time of administration, route of administration, and / or excretion or metabolic rate of the specific protein used; the duration of treatment; and similar factors well known in the medical field.

[0288] In one embodiment, the therapeutically effective dose is about 0.005 mg / kg body weight to 500 mg / kg body weight, for example, about 0.005 mg / kg body weight to 400 mg / kg body weight, about 0.005 mg / kg body weight to 300 mg / kg body weight, about 0.005 mg / kg body weight to 200 mg / kg body weight, about 0.005 mg / kg body weight to 100 mg / kg body weight, about 0.005 mg / kg body weight to 90 mg / kg body weight, about 0.005 mg / kg body weight to 80 mg / kg body weight, about 0.005 mg / kg body weight to 10 ... The ranges are about 0.005mg / kg body weight to 70mg / kg body weight, about 0.005mg / kg body weight to 60mg / kg body weight, about 0.005mg / kg body weight to 50mg / kg body weight, about 0.005mg / kg body weight to 40mg / kg body weight, about 0.005mg / kg body weight to 30mg / kg body weight, about 0.005mg / kg body weight to 25mg / kg body weight, about 0.005mg / kg body weight to 20mg / kg body weight, about 0.005mg / kg body weight to 15mg / kg body weight, and about 0.005mg / kg body weight to 10mg / kg body weight.

[0289] In certain 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, or greater than about 500 mg / kg body weight. In certain embodiments, the therapeutically effective dose is 1.0 mg / kg. In certain embodiments, a therapeutically effective dose of 1.0 mg / kg is administered intramuscularly or intravenously.

[0290] Lyophilized pharmaceutical compositions comprising one or more of the liposomes disclosed herein, and related methods for using such compositions, as disclosed, for example, in U.S. Provisional Patent Application No. 61 / 494,882, filed June 8, 2011, the entire teachings of which are incorporated herein by reference, are also contemplated herein. For example, lyophilized pharmaceutical compositions of the present invention can be reconstituted prior to administration or can be reconstituted in vivo. For example, lyophilized pharmaceutical compositions can be formulated into an appropriate dosage form (e.g., an intradermal dosage form such as a disk, rod, or membrane) and administered such that the dosage form is rehydrated in vivo over time by the individual's body fluids.

[0291] The provided liposomes and compositions can be administered to any desired tissue. In certain 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 certain embodiments, the delivered mRNA is expressed in a tissue different from the tissue to which the liposomes and / or compositions are administered. Exemplary tissues to which the delivered mRNA may be delivered and / or expressed include, but are not limited to, the liver, kidney, heart, spleen, serum, brain, skeletal muscle, lymph nodes, skin, and / or cerebrospinal fluid.

[0292] In some embodiments, administration of a provided composition increases mRNA expression levels in a biological sample from a subject compared to baseline expression levels before treatment. Typically, the baseline level is measured immediately before treatment. Biological samples include, for example, whole blood, serum, plasma, urine, and tissue samples (e.g., muscle, liver, skin fibroblasts). In some embodiments, administration of a provided composition increases mRNA expression levels by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% compared to baseline levels immediately before treatment. In some embodiments, administration of a provided composition increases mRNA expression levels compared to mRNA expression levels in an untreated subject.

[0293] According to various embodiments, the timing of expression of the delivered mRNA can be tailored to suit specific medical needs. In certain embodiments, expression of the protein encoded by the delivered mRNA is detectable 1, 2, 3, 6, 12, 24, 48, 72, and / or 96 hours after administration of provided liposomes and / or compositions. In certain embodiments, expression of the protein encoded by the delivered mRNA is detectable 1 week, 2 weeks, and / or 1 month after administration.

[0294] The invention also provides for the delivery of compositions having mRNA molecules encoding peptides or polypeptides of interest for use in treating a subject, e.g., a human subject, or cells of a human subject, or cells to be treated and delivered to a human subject. [Example]

[0295] While certain compounds, compositions and methods of the present invention have been specifically described according to certain embodiments, the following examples are merely illustrative of the compounds of the present invention and are not intended to limit the invention.

[0296] Example 1. Formulation of mRNA-LNP Compositions for Rectal Delivery This example demonstrates an exemplary method for making a composition comprising mRNA encapsulated within lipid nanoparticles (LNPs) suitable for rectal delivery.

[0297] Using Process B, messenger RNA was encapsulated in lipid nanoparticles containing ML-2:DOPE:cholesterol:DMG-PEG (40:30:25:5). As used herein, Process B refers to the process of encapsulating mRNA by mixing preformed lipid nanoparticles with mRNA, as described in U.S. Patent Application Publication No. 2018 / 0153822, the entire contents of which are incorporated herein by reference. Suppositories were prepared with gelatin at a 10% w / v concentration in LNPs. Briefly, gelatin was directly dissolved in LNPs at 65°C within 5 minutes and poured into disposable molds. The molds were then frozen at -80°C. The suppositories containing mRNA-encapsulated LNPs remained intact. An exemplary suppository containing mRNA-LNPs is shown in Figure 1.

[0298] Labrasol (permeation enhancer) solution was prepared by dissolving 153 mg of Labrasol in 1 mL of water.

[0299] Varying amounts of gelatin can be used to control the melting rate of the suppository, thereby controlling the release time of the mRNA encapsulated in the LNP after administration to a subject. Additionally, viscosity-adjusting excipients can be added to control the release time.

[0300] Example 2. Rectal delivery of FFLuc mRNA-LNPs to mice This example demonstrates successful rectal delivery of mRNA in lipid nanoparticles.

[0301] Mice were intrarectally administered either firefly luciferase (FFLuc) mRNA encapsulated in lipid nanoparticles (0.2 mg per animal) or saline. Whole-body and individual tissues were imaged 24 hours after rectal administration, as shown in Figures 2 and 3.

[0302] As shown in Figures 2A and 2B, mice administered saline intrarectally did not exhibit luminescence, as expected. Mice administered FFLuc mRNA-LNP intrarectally exhibited luminescence, particularly in the rectum (Figure 3A). As shown in Figure 3B, clones exhibited strong luminescence within the tissues.

[0303] This example demonstrates that mRNA-LNP can be successfully delivered into the rectum for in vivo expression of protein. Expression was detected in the rectum and colon.

[0304] Example 3. Rectal delivery of FFLuc mRNA-LNPs with permeability enhancers This example demonstrates the successful delivery of mRNA in lipid nanoparticles with sodium caprate, a permeability enhancer. Rectal administration of mRNA-LNPs with sodium caprate significantly increased in vivo expression of the protein.

[0305] As described in Example 2, one group of mice was administered 0.2 mg of FFLuc mRNA-LNP (Group 1). A second group of mice was pre-administered with sodium caprate (200 mg / ml solution—50 μl 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.

[0306] As shown in Figure 4, mice administered 0.05 mg of FFLuc mRNA-LNPs intrarectally with sodium caprate exhibited a significantly higher signal than mice administered 0.2 mg of FFLuc mRNA-LNPs. Figure 5 shows that there was a nearly two-fold increase in luminescence for Group 2, even though the mRNA-LNP dose was 25% of the dose in Group 1.

[0307] This example demonstrates that permeability enhancers can increase the expression of proteins delivered by mRNA-LNPs.

[0308] Example 4. Rectal delivery of FFLuc mRNA-LNPs in suppositories to mice and rats This example demonstrates the successful rectal delivery of mRNA-LNPs in a suppository formulation. Rectal administration of mRNA-LNPs in a suppository significantly increased in vivo protein expression, and protein expression was detected in various tissues.

[0309] Mice or rats were intrarectally administered 30 μl of Labrasol solution (153 mg / ml). 30 minutes later, a suppository formulation of FFLuc mRNA-LNP was intrarectally administered. Whole-body and individual tissues were imaged 24 hours after rectal administration.

[0310] Figure 6A shows that mice administered FFLuc mRNA-LNPs in a suppository intrarectally exhibited significantly higher luminescence than mice administered mRNA-LNPs without a suppository intrarectally. Furthermore, strong luminescence signals were observed in different tissues (e.g., rectum, colon, liver, kidney, etc.). As illustrated in Figure 6B, two out of four rats exhibited luminescence in the liver, colon, and rectum. Less variability was observed in mice compared to rats.

[0311] This example demonstrates that a significant increase in protein expression is observed when mRNA-LNPs are delivered in the form of suppositories. This example also confirms that suppositories can help mRNA-LNPs overcome RNase and mucus barriers and control the release of mRNA-LNPs within a subject's body after delivery. Furthermore, in vivo protein expression was detected in various tissues, including the kidney. This is important because targeted drug delivery into the mouse kidney is known to be difficult and can require laparotomy. Furthermore, FFL expression in the liver suggests uptake of LNPs into the systemic circulation.

[0312] Example 5. Rectal delivery of EPO mRNA-LNPs in suppositories to rats This example demonstrates the successful rectal delivery of mRNA-LNPs in suppositories for secreted proteins.

[0313] Rats were intrarectally administered 30 μl of Labrasol solution (153 mg / ml). 30 minutes later, a suppository formulation of hEPO mRNA-LNP was intrarectally administered. 24 hours after administration, serum hEPO levels were measured. As shown in Figure 7, rats rectally administered with hEPO mRNA-LNP in a suppository exhibited detectable serum hEPO levels. These levels are much higher than normal physiological levels of EPO.

[0314] This example demonstrates that rectal delivery of mRNA-LNPs in the form of suppositories can successfully provide the expressed protein in the systemic circulation.

[0315] Equivalents and Scope Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. The scope of the present invention is not intended to be limited to the above Description, but is instead set forth in the following claims.

Claims

1. 1. A combination comprising a permeation enhancer and another composition comprising messenger RNA (mRNA) encoding a protein or peptide for use in treating a disease in a subject in need thereof and encapsulated within lipid nanoparticles, wherein the composition is formulated for administration to the subject by rectal delivery, wherein administration of the composition results in expression in the subject of the protein or peptide encoded by the mRNA, and the protein or peptide encoded by the mRNA is detectable in the subject at least about 24 hours, about 48 hours, about 72 hours, or about 96 hours after administration, and the permeation enhancer comprises a form of Labrasol®, caprate, sorbitan laurate, palmitate, lauroylcholine, sodium myristate, or palmitoylcarnitine; and The combination, wherein the permeation enhancer is administered to the subject prior to administering the composition for treating a disease in the subject.

2. The combination of claim 1 , wherein said rectal delivery of said composition is by suppository.

3. 2. The combination of claim 1, wherein 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, about 48 hours, about 72 hours, or about 96 hours after administration of the composition.

4. The lipid nanoparticles are i) one or more cationic lipids, one or more non-cationic lipids, and one or more PEG-modified lipids; and / or ii) Cholesterol 2. The combination of claim 1, comprising:

5. 10. The combination of claim 1, wherein the rectal delivery is by suppository, enema, catheter, or bulb syringe.

6. The combination of claim 2, wherein the composition does not include a lipid-based suppository component.

7. 7. The combination of claim 6, wherein the lipid-based suppository component is cocoa butter, theobroma oil, a synthetic fat, or a synthetic base.

8. 3. The combination of claim 2, wherein the composition further comprises an aqueous suppository component selected from glycerin, gelatin, or polyethylene glycol (PEG), or a combination thereof.

9. 9. The combination of claim 8, wherein the aqueous suppository component comprises gelatin.

10. A combination according to any one of claims 1 to 9, wherein the permeation enhancer comprises sodium caprate.

11. The combination of any one of claims 1 to 9, wherein the permeation enhancer comprises Labrasol®.

12. 10. The combination of claim 9, wherein the only aqueous suppository component is gelatin.

13. 13. The combination of claim 12, wherein 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.

14. 10. The combination of any one of claims 1 to 9, wherein the composition comprises 0.25 mg / mL or more of mRNA, 0.5 mg / mL or more of mRNA, 0.75 mg / mL or more of mRNA, 1 mg / mL or more of mRNA, 1.25 mg / mL or more of mRNA, 1.5 mg / mL or more of mRNA, or 1.75 mg / mL or more of mRNA.

15. 15. The combination of claim 14, wherein the composition is formulated for a suppository having a volume of about 3 grams, about 2 grams, or about 1 gram, and / or about 2.0 mL, about 3.5 mL, about 7.5 mL, or about 10.0 mL.

16. 16. The combination of claim 15, wherein the suppository is refrigerated prior to administration.

17. The combination of any one of claims 1 to 9, wherein the permeation enhancer promotes passage of the lipid nanoparticles from the colon into the circulatory system of a subject.

18. 10. The combination of any one of claims 1 to 9, wherein the permeation enhancer is administered to the subject about 30 minutes, about 1 hour, about 2.5 hours, about 5 hours, or about 12 hours before administering the composition.

19. A combination comprising a permeation enhancer and another suppository for rectal administration of mRNA to a subject, the permeation enhancer comprises sodium caprate or Labrasol®; wherein the permeation enhancer is administered to the subject prior to administering the suppository during use; The suppository is a. the lipid nanoparticles comprise one or more cationic lipids and either one or more non-cationic lipids, one or more cholesterol-based lipids, or one or more PEG-modified lipids; b. mRNA encapsulated within lipid nanoparticles; c. Gelatin and The combination comprising:

20. i) 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; and / or ii) the suppository does not include a lipid-based suppository component; 20. The combination of claim 19.

21. 21. The combination of claim 20, wherein the lipid-based suppository component is cocoa butter, theobroma oil, a synthetic fat, or a synthetic base.

22. 20. The combination of claim 19, wherein the lipid nanoparticles comprise dioleoylphosphatidylethanolamine (DOPE), cholesterol, and 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol (DMG-PEG).

23. 20. The combination of claim 19, wherein the one or more cationic lipids constitute at least about 5%, 10%, 20%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, or 70% by weight of the total lipid content in the lipid nanoparticle.

24. The combination of claim 19, wherein the suppository further comprises glycerin and / or PEG.

25. The combination according to any one of claims 19 to 24, wherein the suppository softens or melts at about 36 to 37°C.

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