Methods and compositions for treating a premature termination codon-mediated disorder
The invention relates generally to modified tRNAs and the use of modified tRNAs to express in a mammalian cell a functional gene product encoded by a gene containing a premature termination codon and/or to treat a disease mediated by a premature termination codon, e.g., Dravet syndrome.
1 . A tRNA comprising the nucleotide sequence of SEQ ID NO: 20, 19, 21, or 2, wherein each thymine in SEQ ID NO: 20, 19, 21, or 2 is replaced by uracil.
2 . An expression vector comprising a nucleotide sequence encoding the tRNA of claim 1 .
3 . The expression vector of claim 2 , wherein the expression vector comprises:
(a) 1, 2, 3, 4, or more than 4 copy numbers of the nucleotide sequence encoding the tRNA; and/or
(b) the nucleotide sequence set forth in any one of SEQ ID NOs: 869-888 that is immediately 5′ to the nucleotide sequence encoding the tRNA.
4 . The expression vector of claim 2 , wherein the expression vector is a viral vector.
5 . The expression vector of claim 4 , wherein the viral vector:
(a) is a DNA virus vector; and/or
(b) is an adeno-associated virus (AAV) vector.
6 . A pharmaceutical composition comprising a nucleic acid encoding the tRNA of claim 1 and a pharmaceutically acceptable excipient.
7 . The pharmaceutical composition of claim 6 , wherein the composition does not comprise an aminolipid particle, an aminolipid delivery compound, or a nanoparticle.
8 . A method of expressing in a mammalian cell a functional gene product encoded by a gene containing a premature termination codon, the method comprising introducing into the cell an effective amount of a nucleic acid encoding the tRNA of claim 1 , wherein expression of the tRNA permits an arginine amino acid to be incorporated into the gene product at a position that would otherwise result in a truncated gene product caused by the premature termination codon, and wherein the premature termination codon is TGA.
9 . The method of claim 8 , wherein the cell contains:
(a) less truncated gene product than a cell without the tRNA; and/or
(b) a greater amount of functional gene product than a cell without the tRNA.
10 . The method of claim 8 , wherein the gene is a dystrophin gene or an SCN1A gene.
11 . A method of increasing in a cell voltage-gated sodium channel activity encoded by a SCN1A gene containing a premature termination codon, the method comprising introducing into the cell an effective amount of a nucleic acid encoding the tRNA of claim 1 , wherein expression of the tRNA permits an arginine amino acid to be incorporated into the SCN1A gene product at a position that would otherwise result in a truncated SCN1A gene product caused by the premature termination codon, and wherein the premature termination codon is TGA.
12 . The method of claim 8 , wherein:
(a) the cell is a human cell; and/or
(b) the tRNA becomes aminoacylated in the cell.
13 . A method of treating a premature termination codon-mediated disorder in a subject in need thereof, wherein the subject has a gene with a premature termination codon and the premature termination codon is TGA, the method comprising administering to the subject an effective amount of a nucleic acid encoding the tRNA of claim 1 , thereby to treat the disorder in the subject.
14 . The method of claim 13 , wherein the disorder is Duchenne Muscular Dystrophy or Dravet syndrome.
15 . The method of claim 13 , wherein the subject is a human.
16 . The tRNA of claim 1 , comprising the nucleotide sequence of SEQ ID NO: 20.
17 . The tRNA of claim 1 , comprising the nucleotide sequence of SEQ ID NO: 19.
18 . The tRNA of claim 1 , comprising the nucleotide sequence of SEQ ID NO: 21.
19 . The tRNA of claim 1 , comprising the nucleotide sequence of SEQ ID NO: 2.