MRNA therapy for argininosuccinate synthetase deficiency
The present invention provides, among other things, methods of treating Argininosuccinate Synthetase Deficiency (ASD), including administering to a subject in need of treatment a composition comprising an mRNA encoding argininosuccinate synthetase (ASS1) at an effective dose and an administration interval such that at least one symptom or feature of ASD is reduced in intensity, severity, or frequency or has delayed in onset. In some embodiments, the mRNA is encapsulated in a liposome comprising one or more cationic lipids, one or more non-cationic lipids, one or more cholesterol-based lipids and one or more PEG-modified lipids.
1. A method of treating Argininosuccinate Synthetase Deficiency (ASD), the method comprising
administering intravenously to a subject with the ASD a composition comprising an mRNA-loaded liposome at an effective dose and administration interval to treat the ASD;
wherein the administering results in reduced ammonia levels in a blood sample from the treated subject as compared to a baseline ammonia level before treatment;
wherein the mRNA encodes an human argininosuccinate synthetase (ASS1) and comprises a nucleotide sequence at least 90% identical to
SEQ ID NO: 3
AUGAGCAGCAAGGGCAGCGUGGUGCUGGCCUACAGCGGCGGCCUGGACACC
AGCUGCAUCCUGGUGUGGCUGAAGGAGCAGGGCUACGACGUGAUCGCCUAC
CUGGCCAACAUCGGCCAGAAGGAGGACUUCGAGGAGGCCCGCAAGAAGGCC
CUGAAGCUGGGCGCCAAGAAGGUGUUCAUCGAGGACGUGAGCCGCGAGUUC
GUGGAGGAGUUCAUCUGGCCCGCCAUCCAGAGCAGCGCCCUGUACGAGGAC
CGCUACCUGCUGGGCACCAGCCUGGCCCGCCCCUGCAUCGCCCGCAAGCAG
GUGGAGAUCGCCCAGCGCGAGGGCGCCAAGUACGUGAGCCACGGCGCCACC
GGCAAGGGCAACGACCAGGUGCGCUUCGAGCUGAGCUGCUACAGCCUGGCC
CCCCAGAUCAAGGUGAUCGCCCCCUGGCGCAUGCCCGAGUUCUACAACCGC
UUCAAGGGCCGCAACGACCUGAUGGAGUACGCCAAGCAGCACGGCAUCCCC
AUCCCCGUGACCCCCAAGAACCCCUGGAGCAUGGACGAGAACCUGAUGCAC
AUCAGCUACGAGGCCGGCAUCCUGGAGAACCCCAAGAACCAGGCCCCCCCC
GGCCUGUACACCAAGACCCAGGACCCCGCCAAGGCCCCCAACACCCCCGAC
AUCCUGGAGAUCGAGUUCAAGAAGGGCGUGCCCGUGAAGGUGACCAACGUG
AAGGACGGCACCACCCACCAGACCAGCCUGGAGCUGUUCAUGUACCUGAAC
GAGGUGGCCGGCAAGCACGGCGUGGGCCGCAUCGACAUCGUGGAGAACCGC
UUCAUCGGCAUGAAGAGCCGCGGCAUCUACGAGACCCCCGCCGGCACCAUC
CUGUACCACGCCCACCUGGACAUCGAGGCCUUCACCAUGGACCGCGAGGUG
CGCAAGAUCAAGCAGGGCCUGGGCCUGAAGUUCGCCGAGCUGGUGUACACC
GGCUUCUGGCACAGCCCCGAGUGCGAGUUCGUGCGCCACUGCAUCGCCAAG
AGCCAGGAGCGCGUGGAGGGCAAGGUGCAGGUGAGCGUGCUGAAGGGCCAG
GUGUACAUCCUGGGCCGCGAGAGCCCCCUGAGCCUGUACAACGAGGAGCUG
GUGAGCAUGAACGUGCAGGGCGACUACGAGCCCACCGACGCCACCGGCUUC
AUCAACAUCAACAGCCUGCGCCUGAAGGAGUACCACCGCCUGCAGAGCAAG
GUGACCGCCAAGUGA;
wherein the liposome has a diameter less than about 100 nm; and
wherein the liposome comprises one or more cationic lipids, one or more non-cationic lipids, one or more cholesterol-based lipids and one or more PEG-modified lipids.
2. The method of claim 1 , wherein the one or more cationic lipids comprise a cationic lipid selected from the group consisting of C12-200, MC3, DLinDMA, DLinkC2DMA, cKK-E12, ICE (Imidazol-based), HGT5000, HGT5001, DODAC, DDAB, DMRIE, DOSPA, DOGS, DODAP, DODMA and DMDMA, DODAC, DLenDMA, DMRIE, CLinDMA, CpLinDMA, DMOBA, DOcarbDAP, DLinDAP, DLincarbDAP, DLinCDAP, KLin-K-DMA, DLin-K-XTC2-DMA, HGT4003, and combination thereof.
3. The method of claim 1 , wherein the one or more cholesterol-based lipids are at least one of cholesterol and PEGylated cholesterol.
4. The method of claim 1 , wherein the one or more cationic lipids constitutes about 30-50% of the liposome by weight.
5. The method of claim 1 , wherein the ratio of the one or more cationic lipids: the one or more non-cationic lipids: the one or more cholesterol-based lipids: the one or more PEG-modified lipids is approximately 40:30:25:5 by molar ratio.
6. The method of claim 1 , wherein the liposome comprises a combination selected from:
cKK-E12, DOPE, cholesterol and DMG-PEG2K;
C 12 -200, DOPE, cholesterol and DMG-PEG2K;
HGT4003, DOPE, cholesterol and DMG-PEG2K; or ICE, DOPE, cholesterol and DMG-PEG2K.
7. The method of claim 1 , wherein the effective dose ranges from about 0.01 to 5.0 mg/kg body weight.
8. The method of claim 1 , wherein the composition is administered once a week.
9. The method of claim 1 , wherein the administering of the composition results in a reduced citrulline level in the subject as compared to a baseline citrulline level before the treatment.
10. The method of claim 1 , wherein the mRNA is codon optimized.
11. The method of claim 1 , wherein the mRNA comprises one or more modified nucleotides.
12. The method of claim 1 , wherein the mRNA is unmodified.
13. The method of claim 1 , wherein the mRNA comprises a nucleotide sequence identical to
SEQ ID NO: 3
AUGAGCAGCAAGGGCAGCGUGGUGCUGGCCUACAGCGGCGGCCUGGACACC
AGCUGCAUCCUGGUGUGGCUGAAGGAGCAGGGCUACGACGUGAUCGCCUAC
CUGGCCAACAUCGGCCAGAAGGAGGACUUCGAGGAGGCCCGCAAGAAGGCC
CUGAAGCUGGGCGCCAAGAAGGUGUUCAUCGAGGACGUGAGCCGCGAGUUC
GUGGAGGAGUUCAUCUGGCCCGCCAUCCAGAGCAGCGCCCUGUACGAGGAC
CGCUACCUGCUGGGCACCAGCCUGGCCCGCCCCUGCAUCGCCCGCAAGCAG
GUGGAGAUCGCCCAGCGCGAGGGCGCCAAGUACGUGAGCCACGGCGCCACC
GGCAAGGGCAACGACCAGGUGCGCUUCGAGCUGAGCUGCUACAGCCUGGCC
CCCCAGAUCAAGGUGAUCGCCCCCUGGCGCAUGCCCGAGUUCUACAACCGC
UUCAAGGGCCGCAACGACCUGAUGGAGUACGCCAAGCAGCACGGCAUCCCC
AUCCCCGUGACCCCCAAGAACCCCUGGAGCAUGGACGAGAACCUGAUGCAC
AUCAGCUACGAGGCCGGCAUCCUGGAGAACCCCAAGAACCAGGCCCCCCCC
GGCCUGUACACCAAGACCCAGGACCCCGCCAAGGCCCCCAACACCCCCGAC
AUCCUGGAGAUCGAGUUCAAGAAGGGCGUGCCCGUGAAGGUGACCAACGUG
AAGGACGGCACCACCCACCAGACCAGCCUGGAGCUGUUCAUGUACCUGAAC
GAGGUGGCCGGCAAGCACGGCGUGGGCCGCAUCGACAUCGUGGAGAACCGC
UUCAUCGCCAUGAAGAGCCGCGGCAUCUACGAGACCCCCGCCGGCACCAUC
CUGUACCACGCCCACCUGGACAUCGAGGCCUUCACCAUGGACCGCGAGGUG
CGCAAGAUCAAGCAGCGCCUGGGCCUGAAGUUCGCCGAGCUGGUGUACACC
GGCUUCUGGCACAGCCCCGAGUGCGAGUUCGUGCGCCACUGCAUCGCCAAG
AGCCAGGAGCGCGUGGAGGGCAACGUGCAGGUGAGCGUGCUGAAGGGCCAG
GUGUACAUCCUGGGCCGCGAGAGCCCCCUGAGCCUGUACAACGAGGAGCUG
GUGAGCAUGAACGUGCAGGGCGACUACGAGCCCACCGACGCCACCGGCUUC
AUCAACAUCAACAGCCUGCGCCUGAAGGAGUACCACCGCCUGCAGAGCAAG
GUGACCGCCAAGUGA.
14. A method of treating Argininosuccinate Synthetase Deficiency (ASD), the method comprising
administering intravenously to a subject with the ASD a composition comprising an mRNA molecule encoding an human argininosuccinate synthetase (ASS1) encapsulated in a liposome at an effective dose and an administration interval such that the administering of the composition results in reduced ammonia levels in a blood sample from the treated subject as compared to a baseline ammonia level before treatment,
wherein the liposome has a diameter of less than about 100 nm, and
wherein the liposome comprises cKK-E12:
15. The method of claim 14 , wherein the mRNA comprises a nucleotide sequence at least 90% identical to
SEQ ID NO: 3
AUGAGCAGCAAGGGCAGCGUGGUGCUGGCCUACAGCGGCGGCCUGGACACC
AGCUGCAUCCUGGUGUGGCUGAAGGAGCAGGGCUACGACGUGAUCGCCUAC
CUGGCCAACAUCGGCCAGAAGGAGGACUUCGAGGAGGCCCGCAAGAAGGCC
CUGAAGCUGGGCGCCAAGAAGGUGUUCAUCGAGGACGUGAGCCGCGAGUUC
GUGGAGGAGUUCAUCUGGCCCGCCAUCCAGAGCAGCGCCCUGUACGAGGAC
CGCUACCUGCUGGGCACCAGCCUGGCCCGCCCCUGCAUCGCCCGCAAGCAG
GUGGAGAUCGCCCAGCGCGAGGGCGCCAAGUACGUGAGCCACGGCGCCACC
GGCAAGGGCAACGACCAGGUGCGCUUCGAGCUGAGCUGCUACAGCCUGGCC
CCCCAGAUCAAGGUGAUCGCCCCCUGGCGCAUGCCCGAGUUCUACAACCGC
UUCAAGGGCCGCAACGACCUGAUGGAGUACGCCAAGCAGCACGGCAUCCCC
AUCCCCGUGACCCCCAAGAACCCCUGGAGCAUGGACGAGAACCUGAUGCAC
AUCAGCUACGAGGCCGGCAUCCUGGAGAACCCCAAGAACCAGGCCCCCCCC
GGCCUGUACACCAAGACCCAGGACCCCGCCAAGGCCCCCAACACCCCCGAC
AUCCUGGAGAUCGAGUUCAAGAAGGGCGUGCCCGUGAAGGUGACCAACGUG
AAGGACGGCACCACCCACCAGACCAGCCUGGAGCUGUUCAUGUACCUGAAC
GAGGUGGCCGGCAAGCACGGCGUGGGCCGCAUCGACAUCGUGGAGAACCGC
UUCAUCGCCAUGAAGAGCCGCGGCAUCUACGAGACCCCCGCCGGCACCAUC
CUGUACCACGCCCACCUGGACAUCGAGGCCUUCACCAUGGACCGCGAGGUG
CGCAAGAUCAAGCAGCGCCUGGGCCUGAAGUUCGCCGAGCUGGUGUACACC
GGCUUCUGGCACAGCCCCGAGUGCGAGUUCGUGCGCCACUGCAUCGCCAAG
AGCCAGGAGCGCGUGGAGGGCAACGUGCAGGUGAGCGUGCUGAAGGGCCAG
GUGUACAUCCUGGGCCGCGAGAGCCCCCUGAGCCUGUACAACGAGGAGCUG
GUGAGCAUGAACGUGCAGGGCGACUACGAGCCCACCGACGCCACCGGCUUC
AUCAACAUCAACAGCCUGCGCCUGAAGGAGUACCACCGCCUGCAGAGCAAG
GUGACCGCCAAGUGA.
16. The method of claim 14 , wherein the mRNA comprises a nucleotide sequence identical to
SEQ ID NO: 3
AUGAGCAGCAAGGGCAGCGUGGUGCUGGCCUACAGCGGCGGCCUGGACACC
AGCUGCAUCCUGGUGUGGCUGAAGGAGCAGGGCUACGACGUGAUCGCCUAC
CUGGCCAACAUCGGCCAGAAGGAGGACUUCGAGGAGGCCCGCAAGAAGGCC
CUGAAGCUGGGCGCCAAGAAGGUGUUCAUCGAGGACGUGAGCCGCGAGUUC
GUGGAGGAGUUCAUCUGGCCCGCCAUCCAGAGCAGCGCCCUGUACGAGGAC
CGCUACCUGCUGGGCACCAGCCUGGCCCGCCCCUGCAUCGCCCGCAAGCAG
GUGGAGAUCGCCCAGCGCGAGGGCGCCAAGUACGUGAGCCACGGCGCCACC
GGCAAGGGCAACGACCAGGUGCGCUUCGAGCUGAGCUGCUACAGCCUGGCC
CCCCAGAUCAAGGUGAUCGCCCCCUGGCGCAUGCCCGAGUUCUACAACCGC
UUCAAGGGCCGCAACGACCUGAUGGAGUACGCCAAGCAGCACGGCAUCCCC
AUCCCCGUGACCCCCAAGAACCCCUGGAGCAUGGACGAGAACCUGAUGCAC
AUCAGCUACGAGGCCGGCAUCCUGGAGAACCCCAAGAACCAGGCCCCCCCC
GGCCUGUACACCAAGACCCAGGACCCCGCCAAGGCCCCCAACACCCCCGAC
AUCCUGGAGAUCGAGUUCAAGAAGGGCGUGCCCGUGAAGGUGACCAACGUG
AAGGACGGCACCACCCACCAGACCAGCCUGGAGCUGUUCAUGUACCUGAAC
GAGGUGGCCGGCAAGCACGGCGUGGGCCGCAUCGACAUCGUGGAGAACCGC
UUCAUCGCCAUGAAGAGCCGCGGCAUCUACGAGACCCCCGCCGGCACCAUC
CUGUACCACGCCCACCUGGACAUCGAGGCCUUCACCAUGGACCGCGAGGUG
CGCAAGAUCAAGCAGCGCCUGGGCCUGAAGUUCGCCGAGCUGGUGUACACC
GGCUUCUGGCACAGCCCCGAGUGCGAGUUCGUGCGCCACUGCAUCGCCAAG
AGCCAGGAGCGCGUGGAGGGCAACGUGCAGGUGAGCGUGCUGAAGGGCCAG
GUGUACAUCCUGGGCCGCGAGAGCCCCCUGAGCCUGUACAACGAGGAGCUG
GUGAGCAUGAACGUGCAGGGCGACUACGAGCCCACCGACGCCACCGGCUUC
AUCAACAUCAACAGCCUGCGCCUGAAGGAGUACCACCGCCUGCAGAGCAAG
GUGACCGCCAAGUGA.