Lipid nanoparticle formulations comprising lipidated cationic peptide compounds for nucleic acid delivery

Abstract

The present disclosure relates to lipid mixtures comprising lipidated cationic peptide compounds, such as tertiary amino lipidated and/or PEGylated cationic peptide compounds or lipitoids, for nucleic acid delivery. More specifically, the present disclosure relates to lipid nanoparticle formulations comprising lipidated cationic peptide compounds and other lipid components including structural lipid, phospholipid and shielding lipids. The present disclosure also relates to methods of preparing and using the lipid mixtures.

1 . A composition, comprising complexes of:
 one or more polyanionic compounds; and   lipid components, wherein the lipid components comprise:   (i) optionally one or more structural lipids;   (ii) one or more phospholipids;   (iii) one or more shielding lipids; and   (iv) one or more lipidated cationic peptide compounds, wherein the one or more lipidated cationic peptide compounds are selected from the group consisting of:
 (a) one or more tertiary amino lipidated and/or PEGylated cationic peptide compounds of formula (I) or salts thereof: 
   
       
         
           
           
               
               
           
         
       
       wherein: 
       m is an integer from 0 to 10; 
       n is an integer from 0 to 5; 
       s is an integer from 0 to 5; 
       t is an integer from 0 to 10;
 wherein at least one of m, n, s, and t is nonzero; 
 
       r is an integer from 1 to 20; 
       each o is independently an integer 0, 1, or 2; 
       each q is independently an integer 0, 1, or 2; 
       each p is independently an integer 1 or 2; 
       R 1  is —H, alkyl, alkylaryl, —COR 1a  or a lipid moiety,
 wherein R 1a  is —H, —OH, alkyl, aryl, alkylaryl, —O-alkyl, or —O-alkylaryl; 
 
       each R 2  is independently an ethylene glycol moiety of the formula
 —CH 2 CH 2 O(CH 2 CH 2 O) u CH 3 , and wherein each u is independently an integer from 2 to 200; 
 
       each R 3  is independently a lipid moiety; 
       each R 4  is independently a neutral spacer moiety or a lipid moiety; 
       each R 5  is independently a cationic moiety; 
       each R 6  is independently an ethylene glycol moiety of the formula
 —CH 2 CH 2 O(CH 2 CH 2 O) v CH 3 , and wherein each v is independently an integer from 2 to 200; 
 
       R 7  is —H, alkyl, acyl, —OH, —OR 7a , —NH 2 , —NHR 7a , or a lipid moiety, wherein R 7a  is alkyl, acyl, or a lipid moiety; and 
       each R a  and R b  is independently —H, C 1 -C 4 -alkyl, or a side chain moiety found on a naturally- or non-naturally-occurring amino acid;
   (b) one or more lipitoids of formula (II):   
 
       
         
           
           
               
               
           
         
       
       wherein: 
       x is an integer from 1 to 100; 
       each R 9  is independently a lipid moiety; and 
       each R 19  is independently a cationic or neutral spacer moiety; 
       and
   (c) any combination thereof.   
 
     
     
         2 . The composition of  claim 1 , comprising 40-80% w/w one or more lipidated cationic peptide compounds of the total weight of the lipid components. 
     
     
         3 . (canceled) 
     
     
         4 . The composition of  claim 1 , wherein the one or more lipidated cationic peptide compounds are one or more tertiary amino lipidated and/or PEGylated cationic peptide compounds of the formula (I) or salts thereof. 
     
     
         5 . The composition of  claim 1 , wherein at least one of n or s is nonzero. 
     
     
         6 . (canceled) 
     
     
         7 . The composition of  claim 1 , wherein each R 3  is independently C 4 -C 22 -alkyl or C 4 -C 22 -alkenyl, and wherein the C 4 -C 22 -alkenyl is optionally mono- or poly-unsaturated. 
     
     
         8 .- 24 . (canceled) 
     
     
         25 . The composition of  claim 1 , wherein the one or more lipidated cationic peptide compounds are one or more lipitoids of formula (II). 
     
     
         26 . The composition of  claim 25 , wherein the one or more lipitoids of formula (II) are Lipitoid 1. 
     
     
         27 . The composition of  claim 1 , comprising 0-40% w/w one or more structural lipids of the total weight of the lipid components. 
     
     
         28 . The composition of  claim 1 , comprising 0-25% w/w one or more structural lipids of the total weight of the lipid components. 
     
     
         29 . The composition of  claim 1 , wherein the one or more structural lipid are selected from the group consisting of cholesterol, fecosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatidine, ursolic acid, alpha-tocopherol, and mixtures thereof. 
     
     
         30 . (canceled) 
     
     
         31 . The composition of  claim 1 , comprising 10-60% w/w one or more phospholipids of the total weight of the lipid components. 
     
     
         32 . The composition of  claim 1 , comprising 20-40% w/w one or more phospholipids of the total weight of the lipid components. 
     
     
         33 . The composition of  claim 1 , wherein the one or more phospholipids are selected from the group consisting of 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-diundecanoyl-sn-glycero-phosphocholine (DUPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-di-0-octadecenyl-sn-glycero-3-phosphocholine (18:0 Diether PC), 1-oleoyl-2-cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (C 16 Lyso PC), 1,2-dilinolenoyl-sn-glycero-3-phosphocholine, 1,2-diarachidonoyl-sn-glycero-3-phosphocholine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphocholine, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (ME 16.0 PE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinolenoyl-sn-glycero-3-phosphoethanolamine, 1,2-diarachidonoyl-sn-glycero-3-phosphoethanolamine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphoethanolamine, 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt (DOPG), sphingomyelin, and any mixtures thereof. 
     
     
         34 . (canceled) 
     
     
         35 . The composition of  claim 1 , comprising 1-5% w/w one or more shielding lipids of the total weight of the lipid components. 
     
     
         36 . The composition of  claim 1 , wherein the one or more shielding lipids are one or more PEG lipids selected from the group consisting of a PEG-modified phosphatidylethanolamine, a PEG-modified phosphatidic acid, a PEG-modified ceramide, a PEG-modified dialkylamine, a PEG-modified diacylglycerol, a PEG-modified dialkylglycerol, and mixtures thereof. 
     
     
         37 . (canceled) 
     
     
         38 . The composition of  claim 1 , wherein the one or more shielding lipids comprises one or more tertiary amino lipidated and/or PEGylated cationic peptide compounds of formula (I) or salts thereof: 
       
         
           
           
               
               
           
         
         wherein: 
         m is an integer from 0 to 10; 
         n is an integer from 0 to 5; 
         s is an integer from 0 to 5; 
         t is an integer from 0 to 10;
 wherein at least one of m and t is nonzero; 
 
         r is an integer from 1 to 20; 
         each o is independently an integer 0, 1, or 2; 
         each q is independently an integer 0, 1, or 2; 
         each p is independently an integer 1 or 2; 
         R 1  is —H, alkyl, alkylaryl, —COR 1a  or a lipid moiety, 
         wherein R 1a  is —H, —OH, alkyl, aryl, alkylaryl, —O-alkyl, or —O-alkylaryl; 
         each R 2  is independently an ethylene glycol moiety of the formula
 —CH 2 CH 2 O(CH 2 CH 2 O) u CH 3 , and wherein each u is independently an integer from 2 to 200; 
 
         each R 3  is independently a lipid moiety; 
         each R 4  is independently a neutral spacer moiety or a lipid moiety; 
         each R 5  is independently a cationic moiety; 
         each R 6  is independently an ethylene glycol moiety of the formula
 —CH 2 CH 2 O(CH 2 CH 2 O) v CH 3 , and wherein each v is independently an integer from 2 to 200; 
 
         R 7  is —H, alkyl, acyl, —OH, —OR 7a , —NH 2 , —NHR 7a , or a lipid moiety, wherein 
         R 7a  is alkyl, acyl, or a lipid moiety; and 
         each R a  and R b  are independently —H, C 1 -C 4 -alkyl, or a side chain moiety found on a naturally- or non-naturally-occurring amino acid. 
       
     
     
         39 .- 41 . (canceled) 
     
     
         42 . The composition of  claim 1 , wherein the lipid components comprise by total weight of the lipid components:
 a. 40-70% w/w one or more lipidated cationic peptide compounds;   b. 0-25% w/w cholesterol;   c. 20-40% w/w 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE); and   d. 1-5% w/w 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG2000).   
     
     
         43 . (canceled) 
     
     
         44 . The composition of  claim 1 , wherein the one or more polyanionic compounds comprise a nucleic acid. 
     
     
         45 . The composition of  claim 1 , wherein the one or more polyanionic compounds comprise a nucleic acid and wherein the nucleic acid is an mRNA encoding a polypeptide. 
     
     
         46 . (canceled) 
     
     
         47 . The composition of  claim 1 , wherein the mass ratio of the lipid components to the one or more polyanionic compounds is between 2:1 and 25:1. 
     
     
         48 .- 49 . (canceled) 
     
     
         50 . A method of delivering a polyanionic compound to a cell comprising contacting the cell with the composition of  claim 1 . 
     
     
         51 .- 52 . (canceled) 
     
     
         53 . The method of  claim 50 , wherein the polyanionic compound comprises a nucleic acid which is the mRNA encoding the polypeptide and the cell expresses the polypeptide after being contacted with the complex. 
     
     
         54 . (canceled) 
     
     
         55 . A method of preparing the composition according to  claim 1 , comprising:
 combining one or more lipidated cationic peptide compounds, one or more phospholipids, one or more shielding lipids, and optionally one or more structural lipids with one or more polyanionic compounds.   
     
     
         56 . (canceled) 
     
     
         57 . The composition of  claim 1 , wherein the one or more lipidated cationic peptide compounds are one or more tertiary amino lipidated and/or PEGylated cationic peptide compounds of formula (I) or salts thereof, wherein:
 (a) m is 0; n is 0; s is an integer from 1 to 5; t is 0; r is an integer from 1 to 5; o is 0; q is 2; and p is 1, and formula (I) has a structure:   
       
         
           
           
               
               
           
         
          or 
         (b) m is 0; n is 0; s is an integer from 1 to 5; t is 0; r is an integer from 2 to 10; o is 0; q is 0; and p is 1, and formula (I) has a structure: 
       
       
         
           
           
               
               
           
         
          or 
         (c) m is 0; n is 0; s is 3 or 4; t is 0; r is 1; o is 0; q is 2; and p is 2, and formula (I) has a structure: 
       
       
         
           
           
               
               
           
         
          or 
         (d) m is 0; n is 0; s is 3 or 4; t is 0; r is 3; o is 0; q is 2; and p is 1, and formula (I) has a structure: 
       
       
         
           
           
               
               
           
         
         (e) or combinations thereof. 
       
     
     
         58 . The composition of  claim 57 , wherein
 R 1  is H or alkyl;   each R 3  independently is C 1 -C 24 alkyl;   each R 4  is independently a neutral spacer moiety that is a C 1 -C 4 -alkyl substituted by cycloalkyl, heterocyclylalkyl, alkylaryl, arylalkyl, alkylheteroaryl, heteroarylalkyl, alkoxy, alkoxyalkyl, or hydroxyalkyl, wherein each cycloalkyl, heterocyclylalkyl, alkylaryl, arylalkyl, alkylheteroaryl, heteroarylalkyl, alkoxy, alkoxyalkyl, or hydroxyalkyl is optionally substituted with one or more substituents —OH, halo, or alkoxy;   each R 5  is independently selected from the group consisting of aminoalkyl, alkylaminoalkyl, aminoalkylaminoakyl, guanidinoalkyl, N-heterocyclylalkyl, and N-heteroaryl, hydroxyalkyl, hydroxyether, alkoxyalkyl, and hydroxylheteroalkyl;   R 7  is NH 2 ; and   each of R a  and R b  is H.   
     
     
         59 . The composition of  claim 57 , wherein
 each R 3  independently is   
       
         
           
           
               
               
           
         
         each R 4  independently is selected from the group consisting of 
       
       
         
           
           
               
               
           
         
          and 
         each R 5  is independently selected from the group consisting of 
       
       
         
           
           
               
               
           
         
       
     
     
         60 . The composition of  claim 38 , wherein m is 1; n is 0; s is 3 or 4; t is 0; r is 1 or 2; o is 0; q is 2; and p is 1, and having a structure: 
       
         
           
           
               
               
           
         
       
     
     
         61 . The composition of  claim 60 , wherein:
 R 1  is H;   R 2  is an ethylene glycol moiety of the formula —CH 2 CH 2 O(CH 2 CH 2 O) u CH 3 , and wherein each u is independently an integer from 2 to 50;   each R 3  independently is C 8 -C 24 alkyl;   each R 4  is independently a neutral spacer moiety that is a C 1 -C 4 -alkyl substituted by cycloalkyl, heterocyclylalkyl, alkylaryl, arylalkyl, alkylheteroaryl, heteroarylalkyl, alkoxy, alkoxyalkyl, or hydroxyalkyl, wherein each cycloalkyl, heterocyclylalkyl, alkylaryl, arylalkyl, alkylheteroaryl, heteroarylalkyl, alkoxy, alkoxyalkyl, or hydroxyalkyl is optionally substituted with one or more substituents —OH, halo, or alkoxy;   each R 5  is independently selected from the group consisting of aminoalkyl, alkylaminoalkyl, aminoalkylaminoakyl, guanidinoalkyl, N-heterocyclylalkyl, and N-heteroaryl, hydroxyalkyl, hydroxyether, alkoxyalkyl, and hydroxylheteroalkyl;   R 7  is NH 2 ; and   each of R a  and R b  is H.