Polynucleotides encoding porphobilinogen deaminase for the treatment of acute intermittent porphyria

Abstract

The invention relates to mRNA therapy for the treatment of Acute Intermittent Porphyria (AIP). mRNAs for use in the invention, when administered in vivo, encode human porphobilinogen deaminase (PBGD), isoforms thereof, functional fragments thereof, and fusion proteins comprising PBGD. mRNAs of the invention are preferably encapsulated in lipid nanoparticles (LNPs) to affect efficient delivery to cells and/or tissues in subjects, when administered thereto. mRNA therapies of the invention increase and/or restore deficient levels of PBGD expression and/or activity in subjects. mRNA therapies of the invention further decrease levels of toxic metabolites associated with deficient PBGD activity in subjects, namely porphobilinogen and aminolevulinate (PBG and ALA).

1 .- 28 . (canceled) 
     
     
         29 . A pharmaceutical composition comprising a lipid nanoparticle, wherein the lipid nanoparticle comprises a compound having the Formula (I) 
       
         
           
           
               
               
           
         
         or a salt or stereoisomer thereof, wherein
 R 1  is selected from the group consisting of C 5-30  alkyl, C 5-20  alkenyl, —R*YR″, —YR″, and —R″M′R′; 
 R 2  and R 3  are independently selected from the group consisting of H, C 1-14  alkyl, C 2-14  alkenyl, —R*YR″, —YR″, and —R*OR″, or R 2  and R 3 , together with the atom to which they are attached, form a heterocycle or carbocycle; 
 R 4  is selected from the group consisting of a C 3-6  carbocycle, —(CH 2 ) n Q, —(CH 2 ) n CHQR, —CHQR, —CQ(R) 2 , and unsubstituted C 1-6  alkyl, where Q is selected from a carbocycle, heterocycle, —OR, —O(CH 2 ) n N(R) 2 , —C(O)OR, —OC(O)R, —CX 3 , —CX 2 H, —CXH 2 , —CN, —N(R) 2 , —C(O)N(R) 2 , —N(R)C(O)R, —N(R)S(O) 2 R, —N(R)C(O)N(R) 2 , —N(R)C(S)N(R) 2 , —N(R)R 8 , —O(CH 2 ) n OR, —N(R)C(═NR 9 )N(R) 2 , —N(R)C(═CHR 9 )N(R) 2 , —OC(O)N(R) 2 , —N(R)C(O)OR, —N(OR)C(O)R, —N(OR)S(O) 2 R, —N(OR)C(O)OR, —N(OR)C(O)N(R) 2 , —N(OR)C(S)N(R) 2 , —N(OR)C(═NR 9 )N(R) 2 , —N(OR)C(═CHR 9 )N(R) 2 , —C(═NR 9 )N(R) 2 , —C(═NR 9 )R, —C(O)N(R)OR, and —C(R)N(R) 2 C(O)OR, and each n is independently selected from 1, 2, 3, 4, and 5; 
 each R 5  is independently selected from the group consisting of C 1-3  alkyl, C 2-3  alkenyl, and H; 
 each R 6  is independently selected from the group consisting of C 1-3  alkyl, C 2-3  alkenyl, and H; 
 M and M′ are independently selected from —C(O)O—, —OC(O)—, —C(O)N(R′)—, —N(R′)C(O)—, —C(O)—, —C(S)—, —C(S)S—, —SC(S)—, —CH(OH)—, —P(O)(OR′)O—, —S(O) 2 —, —S—S—, an aryl group, and a heteroaryl group; 
 R 7  is selected from the group consisting of C 1-3  alkyl, C 2-3  alkenyl, and H; 
 R 8  is selected from the group consisting of C 3-6  carbocycle and heterocycle; 
 R 9  is selected from the group consisting of H, CN, NO 2 , C 1-6  alkyl, —OR, —S(O) 2 R, —S(O) 2 N(R) 2 , C 2-6  alkenyl, C 3-6  carbocycle and heterocycle; 
 each R is independently selected from the group consisting of C 1-3  alkyl, C 2-3  alkenyl, and H; 
 each R′ is independently selected from the group consisting of C 1-18  alkyl, C 2-18  alkenyl, —R*YR″, —YR″, and H; 
 each R″ is independently selected from the group consisting of C 3-14  alkyl and C 3-14  alkenyl; 
 each R* is independently selected from the group consisting of C 1-12  alkyl and C 2-12  alkenyl; 
 each Y is independently a C 3-6  carbocycle; 
 each X is independently selected from the group consisting of F, Cl, Br, and I; and m is selected from 5, 6, 7, 8, 9, 10, 11, 12, and 13; and 
 provided that when R 4  is —(CH 2 ) n Q, —(CH 2 ) n CHQR, —CHQR, or —CQ(R) 2 , then (i) Q is not —N(R) 2  when n is 1, 2, 3, 4 or 5, or (ii) Q is not 5, 6, or 7-membered heterocycloalkyl when n is 1 or 2, 
 wherein the lipid nanoparticle comprises an mRNA that comprises an open reading frame (ORF) encoding an porphobilinogen deaminase (PBGD) polypeptide, wherein the composition is suitable for administration to a human subject in need of treatment for acute intermittent porphyria (AIP). 
 
       
     
     
         30 . The pharmaceutical composition of  claim 29 , wherein the lipid nanoparticle comprises the compound is of Formula (IA): 
       
         
           
           
               
               
           
         
         or a salt or stereoisomer thereof, wherein 
         l is selected from 1, 2, 3, 4, and 5; 
         m is selected from 5, 6, 7, 8, and 9; 
         M 1  is a bond or M′; 
         R 4  is unsubstituted C 1-3  alkyl, or —(CH 2 ) n Q, in which Q is OH, —NHC(S)N(R) 2 , —NHC(O)N(R) 2 , —N(R)C(O)R, —N(R)S(O) 2 R, —N(R)R 8 , —NHC(═NR 9 )N(R) 2 , —NHC(═CHR 9 )N(R) 2 , —OC(O)N(R) 2 , —N(R)C(O)OR, heteroaryl or heterocycloalkyl; 
         M and M′ are independently selected from —C(O)O—, —OC(O)—, —C(O)N(R′)—, —P(O)(OR′)O—, —S—S— an aryl group, and a heteroaryl group; and 
         R 2  and R 3  are independently selected from the group consisting of H, C 1-14  alkyl, and C 2-14  alkenyl. 
       
     
     
         31 . The pharmaceutical composition of  claim 29 , wherein m is 5, 7, or 9. 
     
     
         32 . The pharmaceutical composition of  claim 29 , wherein the compound is of Formula (II) 
       
         
           
           
               
               
           
         
         or a salt or stereoisomer thereof, wherein 
         l is selected from 1, 2, 3, 4, and 5; 
         M 1  is a bond or M′; 
         R 4  is unsubstituted C 1-3  alkyl, or —(CH 2 ) n Q, in which n is 2, 3, or 4, and Q is OH, —NHC(S)N(R) 2 , —NHC(O)N(R) 2 , —N(R)C(O)R, —N(R)S(O) 2 R, —N(R)R 8 , —NHC(═NR 9 )N(R) 2 , —NHC(═CHR 9 )N(R) 2 , —OC(O)N(R) 2 , —N(R)C(O)OR, heteroaryl, or heterocycloalkyl; 
         M and M′ are independently selected from —C(O)O—, —OC(O)—, —C(O)N(R′)—, —P(O)(OR′)O—, —S—S—, an aryl group, and a heteroaryl group; and 
         R 2  and R 3  are independently selected from the group consisting of H, C 1-14  alkyl, and C 2-14  alkenyl. 
       
     
     
         33 . The pharmaceutical composition of  claim 30 , wherein M 1  is M′. 
     
     
         34 . The pharmaceutical composition of  claim 33 , wherein M and M′ are independently —C(O)O— or —OC(O)—. 
     
     
         35 . The pharmaceutical composition of  claim 30 , wherein l is 1, 3, or 5. 
     
     
         36 . The pharmaceutical composition of  claim 29 , wherein the compound is selected from the group consisting of Compound 1 to Compound 232, salts and stereoisomers thereof, and any combination thereof. 
     
     
         37 . (canceled) 
     
     
         38 . The pharmaceutical composition of  claim 29 , wherein the compound is Compound 18, a salt or a stereoisomer thereof, or any combination thereof. 
     
     
         39 .- 143 . (canceled) 
     
     
         144 . A method of expressing a porphobilinogen deaminase (PBGD) polypeptide in a human subject in need thereof comprising administering to the subject an effective amount of the pharmaceutical composition of  claim 29 , wherein the pharmaceutical composition is suitable for administrating as a single dose or as a plurality of single unit doses to the subject. 
     
     
         145 . A method of treating, preventing or delaying the onset of acute intermittent porphyria (AIP) signs or symptoms in a human subject in need thereof comprising administering to the subject an effective amount of the pharmaceutical composition of  claim 29 , wherein the administration treats, prevents or delays the onset of one or more of the signs or symptoms of AIP in the subject. 
     
     
         146 . (canceled) 
     
     
         147 . A method of reducing an aminolevulinate acid (ALA), a porphobilinogen (PBG) and/or a porphyrin urinary excretion level in a human subject comprising administering to the subject an effective amount of the pharmaceutical composition of  claim 29 , wherein the administration reduces the ALA, PBG and/or porphyrin urinary excretion level in the subject. 
     
     
         148 . The method of  claim 147 , wherein
 (i) the ALA urinary excretions level is reduced by at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or at least 100% as compared to the subject's baseline ALA excretion level or a reference ALA excretion level during an acute porphyria attack, for at least 24 hours, at least 48 hours, at least 72 hours, at least 96 hours, or at least 120 hours post-administration,   (ii) the PBG urinary excretions level is reduced by at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or at least 100% as compared to the subject's PBG excretion baseline level or a reference PBG excretion level during an acute porphyria attack, for at least 24 hours, at least 48 hours, at least 72 hours, at least 96 hours, or at least 120 hours post-administration, and/or   (iii) the porphyrin urinary excretions level is reduced by at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or at least 100% as compared to the subject's baseline porphyrin excretion level or a reference porphyrin excretion level during an acute porphyria attack, for at least 24 hours, at least 48 hours, at least 72 hours, at least 96 hours, or at least 120 hours post-administration.   
     
     
         149 . A method of reducing an alanine transaminase (ALT), a aspartate transaminase (AST) and/or a bilirubin serum level in a human subject comprising administering to the subject an effective amount of the pharmaceutical composition of  claim 29 , wherein the administration reduces the ALT, AST and/or bilirubin serum level in the subject. 
     
     
         150 . The method of  claim 149 , wherein
 (i) the ALT serum level is reduced by at least 90%, at least 80%, at least 70%, at least 60%, at least 50%, at least 40%, or at least 30% as compared to the subject's baseline ALT serum level or a reference ALT serum level within at least 24 hours, at least 48 hours, at least 72 hours, at least 96 hours, or at least 120 hours post-administration,   (ii) the AST serum level is reduced by at least 90%, at least 80%, at least 70%, at least 60%, at least 50%, at least 40%, or at least 30% as compared to the subject's baseline AST serum level or a reference AST serum level, for at least 24 hours, at least 48 hours, at least 72 hours, at least 96 hours, or at least 120 hours post-administration, and/or   (iii) the bilirubin serum level is reduced by at least 90%, at least 80%, at least 70%, at least 60%, at least 50%, at least 40%, or at least 30% as compared to the subject's baseline bilirubin serum level or a reference bilirubin serum level, for at least 24 hours, at least 48 hours, at least 72 hours, at least 96 hours, or at least 120 hours post-administration.   
     
     
         151 . The method of  claim 145 , wherein 12 hours after the pharmaceutical composition or polynucleotide is administered to the subject, the PBGD activity in the subject is increased at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 150%, at least 200%, at least 300%, at least 400%, at least 500%, or at least 600% compared to the subject's baseline PBGD activity. 
     
     
         152 .- 154 . (canceled) 
     
     
         155 . The method of  claim 145 , wherein 24 hours after the pharmaceutical composition or polynucleotide is administered to the subject:
 (a) the level of ALA in the subject is reduced by at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or 100% compared to the subject's baseline ALA;   (b) the level of PBG in the subject is reduced by at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or 100% compared to the subject's baseline PBG;   (c) the level of porphyrin in the subject is reduced by at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or 100% compared to the subject's baseline porphyrin;   (d) the level of ALT in the subject is reduced by at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or 100% compared to the subject's baseline ALT;   (e) the level of AST in the subject is reduced by at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or 100% compared to the subject's baseline AST; or   (f) the level of AST in the subject is reduced by at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or 100% compared to the subject's baseline AST.   
     
     
         156 .- 178 . (canceled) 
     
     
         179 . The pharmaceutical composition of  claim 29 , wherein the lipid nanoparticle comprises from about 45 mol % to about 55 mol % of ionizable lipid. 
     
     
         180 . The pharmaceutical composition of  claim 29 , wherein the lipid nanoparticle comprises from about 35 mol % to about 40 mol % of structural lipid. 
     
     
         181 . The pharmaceutical composition of  claim 29 , wherein the lipid nanoparticle comprises from amount 2 mol % to about 5 mol % PEG lipid.