Liver-specific nucleic acid regulatory elements and methods and use thereof
The disclosure presents nucleic acid regulatory elements that are able to enhance liver-specific expression of genes, methods employing these regulatory elements and uses of these elements. Expression cassettes and vectors containing these nucleic acid regulatory elements are also disclosed. The nucleic acid regulatory elements, methods employing these regulatory elements and uses of these elements are particularly useful for applications using gene therapy, more particularly liver-directed gene therapy, and for vaccination purposes.
1 . A nucleic acid regulatory element comprising SEQ ID NO: 6 or a sequence having at least 95% identity to SEQ ID NO: 6, or consisting of a functional fragment of SEQ ID NO: 6 comprising at least 200 contiguous nucleotides from SEQ ID NO: 6;
wherein the nucleic acid regulatory element has a maximal length of 600 nucleotides;
wherein the nucleic acid regulatory element forms part of a nucleic acid expression cassette, wherein the nucleic acid regulatory element is operably linked to a promoter and a transgene; and
wherein the nucleic acid regulatory element allows enhancing promotor-driven transgene expression relative to transgene expression from the promoter alone.
2 . The nucleic acid regulatory element according to claim 1 , further comprising at least one liver-specific regulatory element different from the nucleic acid regulatory element comprising SEQ ID NO: 6 or the sequence having at least 95% identity to SEQ ID NO: 6.
3 . The nucleic acid regulatory element according to claim 2 , comprising a nucleic acid regulatory element comprising SEQ ID NO: 6 or a sequence having at least 95% identity to SEQ ID NO: 6, and at least one nucleic acid regulatory element comprising SEQ ID NO: 22 or a sequence having at least 95% identity to SEQ ID NO: 22.
4 . The nucleic acid regulatory element according to claim 1 , wherein the transgene encodes a therapeutic protein or an immunogenic protein.
5 . The nucleic acid regulatory element according to claim 1 , wherein the promoter is a liver-specific promoter.
6 . The nucleic acid regulatory element according to claim 5 , wherein the liver-specific promoter comprises SEQ ID NO: 27, wherein the nucleic acid regulatory element further comprises a liver-specific regulatory element comprising SEQ ID NO: 24, and wherein the nucleic acid regulatory element comprises the combination of the transthyretin enhancer (TTRe) and minimal transthyretin promoter (TTRm) nucleic acids of SEQ ID NO: 28.
7 . The nucleic acid regulatory element according to claim 1 , further comprising a minute virus of mouse (MVM) intron.
8 . The nucleic acid regulatory element according to claim 1 , further comprising a transcriptional termination signal derived from the bovine growth hormone polyadenylation signal (BGHpA).
9 . A vector comprising the nucleic acid regulatory element according to claim 1 .
10 . The vector according to claim 9 , comprising SEQ ID NO: 38.
11 . A pharmaceutical composition comprising the nucleic acid regulatory element according to claim 1 , and a pharmaceutically acceptable carrier.
12 . A method of treating a liver disease in a subject in need thereof, comprising administering a therapeutically effective amount of the nucleic acid regulatory element according to claim 1 .
13 . The method according to claim 12 , wherein the liver disease is hemophilia A or hemophilia B.
14 . An in vitro or ex vivo method for expressing a transgene product in liver cells comprising:
i) introducing the nucleic acid regulatory element according to claim 1 into the liver cells; and
ii) expressing the transgene product in the liver cells.
15 . The nucleic acid regulatory element according to claim 1 , wherein the functional fragment comprises at least 250 contiguous nucleotides from SEQ ID NO:6.
16 . The nucleic acid regulatory element according to claim 1 , wherein the promoter is a heterologous promoter.
17 . The nucleic acid regulatory element according to claim 1 , wherein the promoter is a liver-specific promoter.