ORNITHINE TRANSCARBAMYLASE (OTC) CONSTRUCTS AND METHODS OF USING THE SAME
The present disclosure provides, among other things, polynucleotide constructs, compositions, and methods of treating ornithine transcarbamylase deficiency, including administering to a subject in need thereof a composition comprising a polynucleotide construct comprising a 5′ UTR, a codon optimized mRNA encoding an ornithine transcarbamylase, and a 3′ UTR.
1 . A polynucleotide construct comprising, from 5′ to 3′:
(a) a 5′ UTR comprising the sequence of SEQ ID NO: 2;
(b) an mRNA sequence comprising an open reading frame (ORF) encoding a functional human ornithine transcarbamylase (OTC), wherein ORF comprises a codon optimized sequence at least about 95% identical to SEQ ID NO: 1; and
(c) a 3′ UTR comprising the sequence of SEQ ID NO: 3.
2 . A polynucleotide construct comprising an mRNA sequence comprising an open reading frame (ORF) encoding a functional human ornithine transcarbamylase (OTC), wherein the mRNA sequence comprises a sequence having no more than five nucleic acids different from SEQ ID NO: 4.
3 . The polynucleotide construct of claim 2 comprising, from 5′ to 3′:
(a) a 5′ UTR;
(b) the mRNA sequence comprising the ORF encoding the OTC; and
(c) a 3′ UTR.
4 . The polynucleotide construct of claim 3 , wherein the 5′ UTR comprises the sequence of SEQ ID NO: 2.
5 . The polynucleotide construct of claim 3 or 4 , wherein the 3′ UTR comprises the sequence of SEQ ID NO: 3.
6 . The polynucleotide construct of any one of claims 1 - 5 , wherein the functional OTC comprises the amino acid sequence of SEQ ID NO:7.
7 . The polynucleotide construct of any one of claims 1 - 6 , wherein the OFR sequence comprises SEQ ID NO: 1.
8 . The polynucleotide construct of any one of claims 1 - 7 , wherein the mRNA sequence has no more than four, three, two, or one nucleic acids different from SEQ ID NO: 4.
9 . The polynucleotide construct of any one of claims 1 - 8 which comprises the sequence of SEQ ID NO: 4.
10 . The polynucleotide construct of any one of claims 1 - 9 which further comprises a 5′ terminal cap.
11 . The polynucleotide construct of claim 10 , wherein the 5′ terminal cap is a Cap1.
12 . The polynucleotide construct of any one of claims 1 - 11 which further comprises a polyA tail.
13 . The polynucleotide construct of claim 12 , wherein the polyA tail is between 80 and 1000 nucleic acids long.
14 . The polynucleotide construct of claim 12 , wherein polyA tail is between 100 and 500 nucleic acids long.
15 . The polynucleotide construct of any one of claims 1 - 14 , wherein the mRNA comprises at least one chemically modified uridine.
16 . The polynucleotide construct of claim 15 , wherein at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or about 100% of the uridines are chemically modified.
17 . The polynucleotide construct of claim 15 or 16 , wherein the chemically modified uridine is selected from the group consisting of pseudouridine (ψ), N1-methyl pseudouridine (N1-me-ψ), and a combination thereof.
18 . A composition comprising:
(a) a polynucleotide construct of any one of claims 1 - 17 ; and
(b) a delivery agent.
19 . The composition of claim 18 , wherein the delivery agent comprises a lipid nanoparticle (LNP), a liposome, a polymer, a micelle, a plasmid, a virus, or any combination thereof.
20 . The composition of claim 19 , wherein the LNP is selected from the group consisting of PEG2000-C-DMA:13-B43:Cholesterol:DSPC, PEG2000-S:13-B43:Cholesterol:DSPC, PEG2000-S:18-B6:Cholesterol:DSPC, and PEG750-C-DLA:18-B6:Cholesterol:DSPC.
21 . The composition of claim 19 or 20 , wherein the polynucleotide construct is encapsulated in the LNP.
22 . The composition of claim 21 , wherein the polynucleotide construct is fully encapsulated in the LNP.
23 . The composition of claim 22 , wherein at least 95% of the polynucleotide construct is encapsulated in the LNP.
24 . The composition of any one of claims 18 - 23 which further comprises a pharmaceutically acceptable carrier.
25 . A method for increasing the amount of OTC expression in a cell comprising administering to the cell a composition comprising the polynucleotide construct of any one of claims 1 - 17 or the composition of any one of claims 18 - 24 .
26 . The method of claim 25 , wherein the cell is a liver cell.
27 . A method for treating or reducing the symptoms associated with ornithine transcarbamylase deficiency (OTCD) comprising administering to a subject in need thereof a therapeutically effective amount of a composition comprising the polynucleotide construct of any one of claims 1 - 17 or the composition of any one of claims 18 - 24 .
28 . A method for treating or reducing the risk of hyperammonemia in a subject with OTCD comprising administering to a subject in need thereof a therapeutically effective amount of a composition comprising the polynucleotide construct of any one of claims 1 - 17 or the composition of any one of claims 18 - 24 .
29 . An expression cassette comprising a sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 8.
30 . The expression cassette of claim 29 which further comprises a promoter.
31 . The expression cassette of claim 30 , wherein the promoter is a T7 promoter.
32 . A plasmid comprising the expression cassette of any one of claims 29 - 31 .
33 . A host cell comprising the expression cassette of any one of claims 29 - 31 , or the plasmid of claim 32 .
34 . Use of the polynucleotide construct of any one of claims 1 - 17 , or the composition of any one of claims 18 - 24 , or the expression cassette of claim any one of claims 29 - 31 , or the plasmid of claim 32 , or the host cell of claim 33 , for the manufacture of a medicament for the treatment of OTCD in a subject in need thereof.
35 . Use of the polynucleotide construct of any one of claims 1 - 17 , or the composition of any one of claims 18 - 24 , or the expression cassette of claim any one of claims 29 - 31 , or the plasmid of claim 32 , or the host cell of claim 33 , for the manufacture of a medicament for the treatment of or for reducing the risk of hyperammonemia in a subject with OTCD.
36 . A method for the in vivo delivery of a nucleic acid, the method comprising:
administering to a mammalian subject the polynucleotide construct of any one of claims 1 - 17 , or the composition of any one of claims 18 - 24 , or the expression cassette of any one of claims 29 - 31 , or the plasmid of claim 32 , or the host cell of claim 33 .
37 . A method for treating a disease or disorder in a mammalian subject in need thereof, the method comprising: administering to the mammalian subject a therapeutically effective amount of the polynucleotide construct of any one of claims 1 - 17 , or the composition of any one of claims 18 - 24 , or the expression cassette of any one of claims 29 - 31 , or the plasmid of claim 32 , or the host cell of claim 33 .
38 . The method of claim 37 , wherein the disease or disorder is a urea cycle disorder.