Polynucleotides encoding propionyl-coa carboxylase alpha and beta subunits for the treatment of propionic acidemia

Abstract

This disclosure relates to mRNA therapy for the treatment of propionic acidemia (PA). mRNAs for use in the invention, when administered in vivo, encode human propionyl-CoA carboxylase alpha (PCCA) and/or human propionyl-CoA carboxylase beta (PCCB), and isoforms thereof, functional fragments thereof, and fusion proteins comprising PCCA and/or PCCB. mRNAs of the invention are preferably encapsulated in lipid nanoparticles (LNPs) to effect efficient delivery to cells and/or tissues in subjects, when administered thereto. mRNA therapies of the invention increase and/or restore deficient levels of propionyl-CoA carboxylase (PCC) expression and/or activity in subjects. mRNA therapies of the invention further decrease levels of disease-associated toxic metabolites associated with deficient PCCA or PCCB activity, in subjects.

1 . A pharmaceutical composition comprising a delivery agent comprising a lipid nanoparticle comprising a compound of Formula (I): 
       
         
           
           
               
               
           
         
         or an N-oxide, salt, or isomer 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 hydrogen, 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 , —N(R)S(O) 2 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)—, —OC(O)-M″-C(O)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, in which M″ is a bond, C 1-13  alkyl or C 2-13  alkenyl; 
         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 3-15  alkyl and C 3-15  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; 
         m is selected from 5, 6, 7, 8, 9, 10, 11, 12, and 13; and
 wherein 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 pharmaceutical composition comprises an mRNA comprising an open reading frame (ORF) encoding a propionyl-CoA carboxylase alpha (PCCA) polypeptide, and wherein the pharmaceutical composition when administered as a single intravenous dose to a human subject in need thereof is sufficient to: 
 (i) increase the level of PCC activity in liver tissue to within at least 2%, at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 100% of normal PCC activity level for at least 12 hours, at least 24 hours, at least 48 hours, at least 72 hours, at least 96 hours, at least 120 hours, at least 1 week, at least 2 weeks, at least 3 weeks, or at least 4 weeks post-administration; 
 (ii) increase the level of PCC activity in liver tissue at least 1.5-fold, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 10-fold, at least 20-fold, or at least 50-fold compared to the subject's baseline PCC activity level or a reference PCC activity level in a human subject having propionic academia (PA) for at least 12 hours, at least 24 hours, at least 48 hours, at least 72 hours, at least 96 hours, at least 120 hours, at least 1 week, at least 2 weeks, at least 3 weeks, or at least 4 weeks post-administration; 
 (iii) reduce plasma, serum, whole blood, and/or liver levels of propionic acid at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 100% compared to the subject's baseline plasma, serum, whole blood and/or liver propionic acid level or a reference plasma, serum, whole blood and/or liver propionic acid level in a human subject having propionic acidemia (PA) for at least 12 hours, at least 24 hours, at least 48 hours, at least 72 hours, at least 96 hours, at least 120 hours, at least 1 week, at least 2 weeks, at least 3 weeks, or at least 4 weeks post-administration; 
 (iv) reduce plasma, serum, whole blood, and/or urine levels of propionyl-L-carnitine (C3), 2-methylcitric acid (2-MC), 3-hydroxypropionic acid, (30HPA), and/or ammonia at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 100% compared to the subject's baseline plasma, serum, or urine level or a reference plasma, serum, or urine C3, 2-MC, 3OHPA, and/or ammonia level in a human subject having PA for at least 12 hours, at least 24 hours, at least 48 hours, at least 72 hours, at least 96 hours, at least 120 hours, at least 1 week, at least 2 weeks, at least 3 weeks, or at least 4 weeks post-administration; 
 (v) reduce plasma, serum, whole blood and/or liver levels of propionic acid at least 1.5-fold, at least 2-fold, at least 5-fold, at least 10-fold, at least 20-fold, or at least 50-fold as compared to the subject's baseline plasma, serum, whole blood and/or liver propionic acid level or a reference plasma, serum, whole blood and/or liver propionic acid level in a patient with PA for at least 12 hours, at least 24 hours, at least 48 hours, at least 72 hours, at least 96 hours, at least 120 hours, at least 1 week, at least 2 weeks, at least 3 weeks, or at least 4 weeks post-administration; and/or 
 (vi) reduce plasma, serum, and/or urine level of C3, 2-MC, 30HPA, and/or ammonia at least 1.5-fold, at least 2-fold at least 5-fold, at least 10-fold, at least 20-fold or at least 50-fold as compared to the subject's baseline plasma, serum, and/or urine C3, 2-MC, 3OHPA, and/or ammonia level or a reference plasma, serum, and/or urine C3, 2-MC, 30HPA, and/or ammonia level in a patient with PA for at least 12 hours, at least 24 hours, at least 48 hours, at least 72 hours, at least 96 hours, at least 120 hours, at least 1 week, at least 2 weeks, at least 3 weeks, or at least 4 weeks post-administration. 
 
       
     
     
         2 . A pharmaceutical composition comprising a delivery agent comprising a lipid nanoparticle comprising a compound of Formula (I): 
       
         
           
           
               
               
           
         
         or an N-oxide, salt, or isomer 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 hydrogen, 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 , —N(R)S(O) 2 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)—, —OC(O)-M″-C(O)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, in which M″ is a bond, C 1-13  alkyl or C 2-13  alkenyl; 
         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 3-15  alkyl and C 3-15  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; 
         m is selected from 5, 6, 7, 8, 9, 10, 11, 12, and 13; and
 wherein 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 pharmaceutical composition comprises an mRNA comprising an open reading frame (ORF) encoding a propionyl-CoA carboxylase beta (PCCB) polypeptide, and wherein the pharmaceutical composition when administered as a single intravenous dose to a human subject in need thereof is sufficient to: 
 (i) increase the level of PCC activity in liver tissue to within at least 2%, at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 100% of normal PCC activity level for at least 12 hours, at least 24 hours, at least 48 hours, at least 72 hours, at least 96 hours, at least 120 hours, at least 1 week, at least 2 weeks, at least 3 weeks, or at least 4 weeks post-administration; 
 (ii) increase the level of PCC activity in liver tissue at least 1.5-fold, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 10-fold, at least 20-fold, or at least 50-fold compared to the subject's baseline PCC activity level or a reference PCC activity level in a human subject having propionic academia (PA) for at least 12 hours, at least 24 hours, at least 48 hours, at least 72 hours, at least 96 hours, at least 120 hours, at least 1 week, at least 2 weeks, at least 3 weeks, or at least 4 weeks post-administration; 
 (iii) reduce plasma, serum, whole blood, and/or liver levels of propionic acid at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 100% compared to the subject's baseline plasma, serum, whole blood and/or liver propionic acid level or a reference plasma, serum, whole blood and/or liver propionic acid level in a human subject having PA for at least 12 hours, at least 24 hours, at least 48 hours, at least 72 hours, at least 96 hours, at least 120 hours, at least 1 week, at least 2 weeks, at least 3 weeks, or at least 4 weeks post-administration; 
 (iv) reduce plasma, serum, whole blood, and/or urine levels of propionyl-L-carnitine (C3), 2-methylcitric acid (2-MC), 3-hydroxypropionic acid, (30HPA), and/or ammonia at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 100% compared to the subject's baseline plasma, serum, or urine level or a reference plasma, serum, or urine C3, 2-MC, 3OHPA, and/or ammonia level in a human subject having PA for at least 12 hours, at least 24 hours, at least 48 hours, at least 72 hours, at least 96 hours, at least 120 hours, at least 1 week, at least 2 weeks, at least 3 weeks, or at least 4 weeks post-administration; 
 (v) reduce plasma, serum, whole blood and/or liver levels of propionic acid at least 1.5-fold, at least 2-fold, at least 5-fold, at least 10-fold, at least 20-fold, or at least 50-fold as compared to the subject's baseline plasma, serum, whole blood and/or liver propionic acid level or a reference plasma, serum, whole blood and/or liver propionic acid level in a patient with PA for at least 12 hours, at least 24 hours, at least 48 hours, at least 72 hours, at least 96 hours, at least 120 hours, at least 1 week, at least 2 weeks, at least 3 weeks, or at least 4 weeks post-administration; and/or 
 (vi) reduce plasma, serum, and/or urine level of C3, 2-MC, 30HPA, and/or ammonia at least 1.5-fold, at least 2-fold at least 5-fold, at least 10-fold, at least 20-fold or at least 50-fold as compared to the subject's baseline plasma, serum, and/or urine C3, 2-MC, 3OHPA, and/or ammonia level or a reference plasma, serum, and/or urine C3, 2-MC, 30HPA, and/or ammonia level in a patient with PA for at least 12 hours, at least 24 hours, at least 48 hours, at least 72 hours, at least 96 hours, at least 120 hours, at least 1 week, at least 2 weeks, at least 3 weeks, or at least 4 weeks post-administration. 
 
       
     
     
         3 . A pharmaceutical composition comprising a delivery agent comprising a lipid nanoparticle comprising a compound of Formula (I): 
       
         
           
           
               
               
           
         
         or an N-oxide, salt, or isomer 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 hydrogen, 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 , —N(R)S(O) 2 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)—, —OC(O)-M″-C(O)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, in which M″ is a bond, C 1-13  alkyl or C 2-13  alkenyl; 
         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 3-15  alkyl and C 3-15  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; 
         m is selected from 5, 6, 7, 8, 9, 10, 11, 12, and 13; and
 wherein 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 pharmaceutical composition comprises an mRNA comprising an open reading frame (ORF) encoding a propionyl-CoA carboxylase alpha (PCCA) polypeptide and an mRNA comprising an open reading frame (ORF) encoding a propionyl-CoA carboxylase beta (PCCB) polypeptide, and wherein the pharmaceutical composition when administered as a single intravenous dose to a human subject in need thereof is sufficient to: 
 (i) increase the level of PCC activity in liver tissue to within at least 2%, at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 100% of normal PCC activity level for at least 12 hours, at least 24 hours, at least 48 hours, at least 72 hours, at least 96 hours, at least 120 hours, at least 1 week, at least 2 weeks, at least 3 weeks, or at least 4 weeks post-administration; 
 (ii) increase the level of PCC activity in liver tissue at least 1.5-fold, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 10-fold, at least 20-fold, or at least 50-fold compared to the subject's baseline PCC activity level or a reference PCC activity level in a human subject having propionic academia (PA) for at least 12 hours, at least 24 hours, at least 48 hours, at least 72 hours, at least 96 hours, at least 120 hours, at least 1 week, at least 2 weeks, at least 3 weeks, or at least 4 weeks post-administration; 
 (iii) reduce plasma, serum, whole blood, and/or liver levels of propionic acid at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 100% compared to the subject's baseline plasma, serum, whole blood and/or liver propionic acid level or a reference plasma, serum, whole blood and/or liver propionic acid level in a human subject having PA for at least 12 hours, at least 24 hours, at least 48 hours, at least 72 hours, at least 96 hours, at least 120 hours, at least 1 week, at least 2 weeks, at least 3 weeks, or at least 4 weeks post-administration; 
 (iv) reduce plasma, serum, whole blood, and/or urine levels of propionyl-L-carnitine (C3), 2-methylcitric acid (2-MC), 3-hydroxypropionic acid, (30HPA), and/or ammonia at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 100% compared to the subject's baseline plasma, serum, or urine level or a reference plasma, serum, or urine C3, 2-MC, 3OHPA, and/or ammonia level in a human subject having PA for at least 12 hours, at least 24 hours, at least 48 hours, at least 72 hours, at least 96 hours, at least 120 hours, at least 1 week, at least 2 weeks, at least 3 weeks, or at least 4 weeks post-administration; 
 (v) reduce plasma, serum, whole blood and/or liver levels of propionic acid at least 1.5-fold, at least 2-fold, at least 5-fold, at least 10-fold, at least 20-fold, or at least 50-fold as compared to the subject's baseline plasma, serum, whole blood and/or liver propionic acid level or a reference plasma, serum, whole blood and/or liver propionic acid level in a patient with PA for at least 12 hours, at least 24 hours, at least 48 hours, at least 72 hours, at least 96 hours, at least 120 hours, at least 1 week, at least 2 weeks, at least 3 weeks, or at least 4 weeks post-administration; and/or 
 (vi) reduce plasma, serum, and/or urine level of C3, 2-MC, 30HPA, and/or ammonia at least 1.5-fold, at least 2-fold at least 5-fold, at least 10-fold, at least 20-fold or at least 50-fold as compared to the subject's baseline plasma, serum, and/or urine C3, 2-MC, 3OHPA, and/or ammonia level or a reference plasma, serum, and/or urine C3, 2-MC, 30HPA, and/or ammonia level in a patient with PA for at least 12 hours, at least 24 hours, at least 48 hours, at least 72 hours, at least 96 hours, at least 120 hours, at least 1 week, at least 2 weeks, at least 3 weeks, or at least 4 weeks post-administration. 
 
       
     
     
         4 .- 26 . (canceled) 
     
     
         27 . The pharmaceutical composition of  claim 3 , wherein the human subject has propionic acidemia (PA). 
     
     
         28 . The pharmaceutical composition of  claim 3 , wherein the human subject is on a protein restricted diet. 
     
     
         29 . The pharmaceutical composition of  claim 3 , wherein the human subject is not on a protein restricted diet. 
     
     
         30 .- 67 . (canceled) 
     
     
         68 . A method of expressing a propionyl-CoA carboxylase alpha (PCCA) polypeptide in a human subject in need thereof, comprising administering to the subject an effective amount of the pharmaceutical composition of  claim 1 . 
     
     
         69 . A method of expressing a propionyl-CoA carboxylase beta (PCCB) polypeptide in a human subject in need thereof, comprising administering to the subject an effective amount of the pharmaceutical composition of  claim 2 . 
     
     
         70 . A method of expressing a propionyl-CoA carboxylase alpha (PCCA) polypeptide and a propionyl-CoA carboxylase beta (PCCB) polypeptide in a human subject in need thereof, comprising administering to the subject an effective amount of the pharmaceutical compositions of  claim 3 . 
     
     
         71 .- 72 . (canceled) 
     
     
         73 . A method of treating, preventing, or delaying the onset and/or progression of propionic academia (PA) in a human subject in need thereof, comprising administering to the subject an effective amount of the pharmaceutical composition of  claim 3 . 
     
     
         74 .- 77 . (canceled) 
     
     
         78 . A method of reducing propionic acid blood level in a human subject in need thereof, comprising administering to the subject an effective amount of the pharmaceutical composition of  claim 3 . 
     
     
         79 .- 82 . (canceled) 
     
     
         83 . A method of reducing C3, 2-MC, 30HPA, and/or ammonia plasma, serum, whole blood, and/or liver level in a human subject in need thereof, comprising administering to the subject an effective amount of the pharmaceutical composition of  claim 3 . 
     
     
         84 .- 87 . (canceled) 
     
     
         88 . The method of  claim 68 , wherein:
 (i) the propionic acid blood and/or liver level is reduced at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 100% as compared to the subject's baseline propionic acid blood and/or liver level or a reference propionic acid blood and/or liver level in a patient with PA, for at least 24 hours, at least 48 hours, at least 72 hours, at least 96 hours, at least 120 hours, 6 days, 1 week, 8 days, 9 days, 10 days, 11 days, 12 days, 14 days, 18 days, or 21 days after a single administration;   (ii) the propionic acid plasma, serum, and/or urine level is reduced at least 20%, at least 30%, at least 40%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 100% as compared to the subject's baseline propionic acid plasma, serum, and/or urine level or a reference propionic acid plasma, serum, and/or urine level in a patient with PA, for at least 24 hours, at least 48 hours, at least 72 hours, at least 96 hours, at least 120 hours, 6 days, 1 week, 8 days, 9 days, 10 days, 11 days, 12 days, 14 days, 18 days, or 21 days after a single administration;   (iii) the propionic acid blood and/or liver level is reduced to at least within 10-fold, at least within 5-fold, at least within 2-fold, or at least within 1.5-fold as compared to a normal propionic acid blood and/or liver level within at least 24 hours, at least 48 hours, at least 72 hours, at least 96 hours, at least 120 hours, 6 days, 1 week, 8 days, 9 days, 10 days, 11 days, 12 days, 14 days, 18 days, or 21 days after a single administration;   (iv) the propionic acid plasma, serum, and/or urine level is reduced to at least within 10-fold, at least within 5-fold, at least within 2-fold, or at least within 1.5-fold, as compared to a normal propionic acid plasma, serum, and/or urine level, for at least 24 hours, at least 48 hours, at least 72 hours, at least 96 hours, at least 120 hours, 6 days, 1 week, 8 days, 9 days, 10 days, 11 days, 12 days, 14 days, 18 days, or 21 days after a single administration;   (v) the C3, 2-MC, 30HPA, and/or ammonia plasma, serum, whole blood, and/or liver level is reduced at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 100% as compared to the subject's baseline plasma, serum, whole blood and/or liver C3, 2-MC, 30HPA, and/or ammonia or a reference C3, 2-MC, 30HPA, and/or ammonia plasma, serum, whole blood, and/or liver level in a patient with PA, for at least 24 hours, at least 48 hours, at least 72 hours, at least 96 hours, at least 120 hours, 6 days, 1 week, 8 days, 9 days, 10 days, 11 days, 12 days, 14 days, 18 days, or 21 days after a single administration; and/or   (vi) the C3, 2-MC, 30HPA, and/or ammonia plasma, serum, whole blood, and/or liver level is reduced to at least within 10-fold, at least within 5-fold, at least within 2-fold, or at least within 1.5-fold as compared to a normal C3, 2-MC, 30HPA, and/or ammonia plasma, serum, whole blood, and/or liver level within at least 24 hours, at least 48 hours, at least 72 hours, at least 96 hours, at least 120 hours, 6 days, 1 week, 8 days, 9 days, 10 days, 11 days, 12 days, 14 days, 18 days, or 21 days after a single administration.   
     
     
         89 . A method of increasing PCC activity in a human subject in need thereof, comprising administering to the subject an effective amount of the pharmaceutical composition of  claim 3 . 
     
     
         90 .- 93 . (canceled) 
     
     
         94 . The method of  claim 68 , wherein:
 (i) the level of PCC activity in the subject is increased at least 1.5-fold, at least 2-fold, at least 5-fold, at least 10-fold, at least 20-fold, or at least 50-fold as compared to a reference PCC activity level in a subject having PA for at least 24 hours, at least 48 hours, at least 72 hours, at least 96 hours, or at least 120 hours after a single administration; and/or   (ii) 12 hours after a single administration of the pharmaceutical composition or polynucleotide is administered to the subject, the PCC 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 PCC activity.   
     
     
         95 . The method of  claim 89 , wherein the PCC activity is increased in the liver or blood of the subject. 
     
     
         96 . The method of  claim 68 , wherein the administration to the subject is about once a week, about once every two weeks, or about once a month. 
     
     
         97 . The method of  claim 68 , wherein the pharmaceutical composition or polynucleotide is administered intravenously.