Codon-optimized nucleic acid encoding SMN1 protein
The present application relates to the fields of genetics, gene therapy, and molecular biology. More specifically, the present invention relates to an isolated codon-optimized nucleic acid that encodes the SMN1 protein (survival motor neuron protein), an expression cassette and a vector based thereon, as well as an AAV9 (adeno-associated virus serotype 9)-based recombinant virus for increasing the expression of the SMN1 gene in target cells, and use thereof.
1 . A codon-optimized nucleic acid that encodes a SMN1 protein (survival motor neuron protein), the SMN1 protein comprising the amino acid sequence of SEQ ID NO: 1, the nucleic acid including the nucleic acid sequence of SEQ ID NO: 2.
2 . An expression cassette that includes the codon-optimized nucleic acid of claim 1 , including the following elements in the 5′-end to 3′-end direction:
a left (first) ITR (inverted terminal repeats);
a CMV (cytomeqalovirus) enhancer;
a CMV (cytomeqalovirus) promoter;
an intron of the hBG1 gene (hemoglobin subunit gamma 1 gene);
the codon-optimized nucleic acid including the nucleic acid sequence of SEQ ID NO: 2;
an hGH1 polyadenylation signal (human growth hormone gene polyadenylation signal); and
a right (second) ITR.
3 . The expression cassette of claim 2 that includes a nucleic acid comprising the nucleic acid sequence of SEQ ID NO: 4.
4 . An expression vector that includes the codon-optimized nucleic acid of claim 1 .
5 . An AAV9 (adeno-associated virus serotype 9)-based recombinant virus for increasing the expression of the SMN1 gene in target cells, the AAV9-based recombinant virus including a capsid and the expression cassette of claim 2 .
6 . The AAV9-based recombinant virus of claim 5 , wherein the capsid comprises the AAV9 protein VP1.
7 . The AAV9-based recombinant virus of claim 6 , wherein the capsid comprises the AAV9 protein VP1 having the amino acid sequence of SEQ ID NO: 5.
8 . The AAV9-based recombinant virus of claim 6 , wherein the capsid comprises the AAV9 protein VP1 having the amino acid sequence of SEQ ID NO: 5 with one or more point mutations.
9 . The AAV9-based recombinant virus of claim 5 , wherein the capsid comprises the AAV9 protein VP1 having the amino acid sequence of SEQ ID NO: 5 or the amino acid sequence of SEQ ID NO: 5 with one or more point mutations, and wherein the expression cassette includes the following elements in the 5′-end to 3′-end direction:
a CMV enhancer;
a CMV promoter;
an intron of the hBG1 gene;
the codon-optimized nucleic acid including the nucleic acid sequence of SEQ ID NO: 2;
an hGH1 polyadenylation signal; and
a right ITR.
10 . The AAV9-based recombinant virus of claim 5 , wherein the capsid includes the AAV9 protein VP1 having the amino acid sequence of SEQ ID NO: 5 or the amino acid sequence of SEQ ID NO: 5 with one or more point mutations, and wherein the expression cassette includes a nucleic acid with SEQ ID NO: 4.
11 . A pharmaceutical composition for delivering the SMN1 gene to target cells, including the AAV9-based recombinant virus of claim 5 , in combination with one or more pharmaceutically acceptable excipients.
12 . A method for delivering the SMN1 gene to one or more target cells comprising introducing the AAV9-based recombinant virus of claim 5 into the one or more target cells.
13 . A method for delivering the SMN1 gene to one or more target cells comprising introducing the composition of claim 11 into the one or more target cells.