MUCOSAL ADMINISTRATION METHODS AND FORMULATIONS

1 . A method for inducing a mucosal immune response, comprising

administering to a mucosal surface of a subject a composition comprising an mRNA encoding an antigen and a nanoparticle, wherein the nanoparticle comprises a lipid nanoparticle core comprising an ionizable lipid, a phospholipid, a structural lipid, and a PEG-lipid, and a cationic agent dispersed primarily on the outer surface of the core in an effective amount to induce a mucosal immune response.

2 . The method of claim 1 , wherein the mRNA is encapsulated within the core.

3 . The method of claim 1 , wherein the nanoparticle has a greater than neutral zeta potential at physiological pH.

4 . The method of claim 1 , wherein a weight ratio of the cationic agent to mRNA is about 1:1 to about 4:1, about 1.25:1 to about 3.75:1, about 1.25:1, about 2.5:1, or about 3.75:1.

5 . The method of claim 1 , wherein the antigen is an infectious disease antigen.

6 . The method of claim 1 , wherein the mucosal surface comprises a cell population selected from respiratory mucosal cells, oral mucosal cells, intestinal mucosal cells, vaginal mucosal cells, rectal mucosal cells, and buccal mucosal cells.

7 . A method for expressing a protein in mucosal tissue, comprising

administering to a mucosal surface of a subject a composition comprising an mRNA encoding a protein and a nanoparticle, wherein the nanoparticle comprises a lipid nanoparticle core comprising an ionizable lipid, a phospholipid, a structural lipid, and a PEG-lipid, and a cationic agent dispersed primarily on the outer surface of the core in an effective amount to induce expression of the protein in a mucosal tissue.

8 . The method of claim 7 , wherein the mRNA encodes a therapeutic protein.

9 .- 20 . (canceled)

21 . The method of claim 1 , wherein greater than about 80%, greater than 90%, greater than 95%, or greater than 95% of the cationic agent is on the surface on the nanoparticle.

22 . The method of claim 1 , wherein at least about 50%, at least about 75%, at least about 90%, or at least about 95% of the mRNA is encapsulated within the core.

23 - 30 . (canceled)

31 . The method of claim 1 , wherein the cationic agent is a cationic lipid and the cationic lipid is a sterol amine comprising a hydrophobic moiety and a hydrophilic moiety.

32 . The method of claim 31 , wherein the hydrophilic moiety comprises an amine group comprising one to four primary, secondary, or tertiary amines or mixtures thereof.

33 .- 35 . (canceled)

36 . The method of claim 32 , wherein the amine group has a pKa value of greater than about 8.

37 . (canceled)

38 . The method of claim 31 , wherein the sterol amine is a compound of Formula (A1):

A-L-B  (A1)

or a salt thereof, wherein:

A is an amine group, L is an optional linker, and B is a sterol.

39 . The method of claim 31 , wherein the sterol amine has Formula A2a:

or a salt thereof, wherein:

is a single or double bond;

R 1 is C 1-14 alkyl or C 1-14 alkenyl;

L a is absent, —O—, —S—S—, —OC(═O)—, —C(═O)N—, —OC(═O)N—, CH 2 —NH—C(O)—, —C(═O)O—, —OC(═O)—CH 2 —CH 2 —C(═O)N—, —S—S—CH 2 , —SS—CH 2 —CH 2 —C(═O)N—, or a group of formula (a):

Y 1 is C 1-10 alkyl, 3 to 8-membered heterocycloalkyl, 5 to 6-membered heteroaryl, —C 1-6 alkyl-(3 to 8-membered heterocycloalkyl), or —C 1-6 alkyl-(5 to 6-membered heteroaryl),

wherein the C 1-10 alkyl, 3 to 8-membered heterocycloalkyl, 5 to 6-membered heteroaryl, —C 1-6 alkyl-(3 to 8-membered heterocycloalkyl), and —C 1-6 alkyl-(5 to 6-membered heteroaryl) comprises one to five primary, secondary, or tertiary amines or combination thereof;

and wherein the C 1-10 alkyl, 3 to 8-membered heterocycloalkyl, 5 to 6-membered heteroaryl, —C 1-6 alkyl-(3 to 8-membered heterocycloalkyl), and —C 1-6 alkyl-(5 to 6-membered heteroaryl) are each optionally substituted with 1, 2, 3, or 4 substituents independently selected from C 1-6 alkyl, halo, —OH, —O(C 1-6 alkyl), —C 1-6 alkyl-OH, —NH 2 , —NH(C 1-6 alkyl), —N(C 1-6 alkyl) 2 , 3 to 8-membered heterocycloalkyl (optionally substituted with C 1-14 alkyl comprising one to five primary, secondary, or tertiary amines or combination thereof), 5 to 6-membered heteroaryl, —NH-(3 to 8-membered heterocycloalkyl), and —NH(5 to 6-membered heteroaryl); and

n is 1 or 2, and

optionally:

wherein is a double bond,

wherein is a single bond,

wherein L a is —OC(═O)—, —OC(═O)N—, or —OC(═O)—CH 2 —CH 2 —C(═O)N—,

wherein n is 1,

wherein n is 2,

wherein R 1 is C 1-14 alkyl,

wherein R 1 is C 1-14 alkenyl,

wherein R t is

and/or

wherein Y t is C 1-10 alkyl, 3 to 8-membered heterocycloalkyl, —C 1-6 alkyl-(3 to 8-membered heterocycloalkyl), or —C 1-6 alkyl-(5 to 6-membered heteroaryl), wherein the C 1-10 alkyl, 3 to 8-membered heterocycloalkyl, —C 1-6 alkyl-(3 to 8-membered heterocycloalkyl), and —C 1-6 alkyl-(5 to 6-membered heteroaryl) comprises one to five primary, secondary, or tertiary amines or combination thereof; and wherein the C 1-10 alkyl, C 1-6 alkyl-(3 to 8-membered heterocycloalkyl), and C 1-6 alkyl-(5 to 6-membered heteroaryl) are each optionally substituted with C 1-6 alkyl, —OH, —C 1-6 alkyl-OH, or —NH 2 .

40 . The method of claim 39 , wherein Y 1 is selected from:

(1);

(2)

(3)

(4)

(5)

(6)

(7)

(8)

(9)

(10)

(11)

(12)

(13)

(14)

(15)

(16)

(17)

(18)

(19)

(20)

(21)

(22)

(23)

(28) —N(CH 3 ) 2 ; (29)

(30)

(31)

and (32)

41 . The method of claim 31 , wherein the sterol amine has Formula A4

or a salt thereof, wherein:

Z 1 is —OH or C 3.6 alkyl;

L is absent, —O—, —S—S—, —OC(═O)—, —C(═O)N—, —OC(═O)N—, —CH 2 —NH—C(═O)—, —C(═O)O—, —OC(═O)—CH 2 —CH 2 —C(═O)N—, —S—S—CH 2 —, or —SS—CH 2 —CH 2 —C(O)N—;

Y 1 is C 1-10 alkyl, 3 to 8-membered heterocycloalkyl, 5 to 6-membered heteroaryl, —C 1-6 alkyl-(3 to 8 membered heterocycloalkyl), or —C 1-6 alkyl-(5 to 6 membered heteroaryl),

wherein the C 1-10 alkyl, 3 to 8-membered heterocycloalkyl, 5 to 6-membered heteroaryl, —C 1-6 alkyl-(3 to 8-membered heterocycloalkyl), and —C 1-6 alkyl-(5 to 6-membered heteroaryl) comprises one to five primary, secondary, or tertiary amines or combination thereof;

and wherein the C 1-10 alkyl, 3 to 8-membered heterocycloalkyl, 5 to 6 membered heteroaryl, —C 1-6 alkyl-(3 to 8-membered heterocycloalkyl), and —C 1-6 alkyl-(5 to 6-membered heteroaryl) are each optionally substituted with 1, 2, 3, or 4 substituents selected from C 1-6 alkyl, halo, —OH, —O(C 1-6 alkyl), —C 1-6 alkyl-OH, —NH 2 , —NH(C 1-6 alkyl), —N(C 1-6 alkyl) 2 , 3 to 8-membered heterocycloalkyl (optionally substituted with C 1-14 alkyl comprising one to five primary, secondary, or tertiary amines or combination thereof), 5 to 6-membered heteroaryl, —NH(3 to 8-membered heterocycloalkyl), and —NH(5 to 6-membered heteroaryl); and

n is 1 or 2, and

optionally:

wherein Z 1 is —OH,

wherein Z 1 is C 3-6 alkyl,

wherein L is —C(═O)N—, —CH 2 —NH—C(═O)—, or —C(═O)O—,

wherein Y 1 is C 1-10 alkyl comprising one to five primary, secondary, or tertiary amines or combination thereof,

wherein Y 1 is

wherein n is 1, and/or

wherein n is 2.

42 . The method of claim 31 , wherein the sterol amine is selected from: SA3, SA10, SA18, SA24, SA58, SA78, SA121, SA137, SA138, SA158, and SA183.

43 - 46 . (canceled)

47 . The method of claim 1 , wherein the ionizable lipid is a compound of Formula (I):

or a salt thereof, wherein:

R 1 is selected from the group consisting of C 5-30 alkyl, C 5-20 alkenyl, and —R″M′R′:

R 2 and R 3 are independently selected from the group consisting of C 1-14 alkyl and C 2-14 alkenyl;

R 4 is —(CH 2 ) n Q, wherein Q is —OR, wherein n is selected from 1, 2, 3, 4, and 5;

each R 5 is H;

each R 6 is H;

M and M′ are independently selected from —C(O)O— and —OC(O)—;

R 7 is H;

R is H;

R′ is selected from the group consisting of C 1-18 alkyl and C 2-18 alkenyl;

R″ is selected from the group consisting of C 3-14 alkyl and C 3-14 alkenyl; and

m is selected from 5, 6, 7, 8, 9, 10, 11, 12, and 13.

48 . (canceled)

49 . The method of claim 1 , wherein the phospholipid is 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC).

50 .- 52 . (canceled)