Liver-specific gene expression cassettes, and methods of use
View Patent ↗In one aspect, the present invention provides nucleic acid expression cassettes that are predominantly expressed in the mammalian liver. The present invention also provides vectors comprising a nucleic acid expression cassette that is predominantly expressed in the mammalian liver. The present invention also provides methods of ameliorating the symptoms of a disease, the methods including the steps of introducing into the liver of a mammalian subject a vector comprising a nucleic acid expression cassette that encodes a polypeptide, and expressing a therapeutic amount of the polypeptide in the liver.
1. A nucleic acid expression cassette capable of expressing human Factor IX, wherein the cassette is predominantly expressed in the mammalian liver, said cassette comprising:
(a) a hepatic locus control element consisting of SEQ ID NO:4 or SEQ ID NO:9;
(b) a heterologous hepatic promoter located 3′ to the hepatic locus control element, said promoter consisting of SEQ ID NO: 5;
(c) a Factor IX coding sequence located 3′ to the hepatic promoter, said coding sequence comprising SEQ ID NO:2;
(d) a polyadenylation signal located 3′ to the intron sequence, said polyadenylation signal consisting of SEQ ID NO:6; and
(e) an intron located 3′ to the hepatic promoter and 5′ to the polyadenylation signal, wherein said intron consists of SEQ ID NO:1,
wherein elements (a), (b), (c), (d) and (e) are operably linked to express the polypeptide encoded by the coding sequence.
2. The expression cassette of claim 1 , wherein said cassette directs expression of a therapeutic amount of Factor IX in liver cells for a period of at least 100 days, and further wherein said cassette is expressed in an animal or as a component of a recombinant adeno-associated virus (AAV) vector.
3. The expression cassette of claim 1 , wherein said cassette directs expression of a therapeutic amount of the polypeptide in liver cells for a period of at least 300 days, and further wherein said cassette is expressed in an animal or as a component of a recombinant adeno-associated virus (AAV) vector.
4. The expression cassette of claim 1 , wherein said cassette directs expression of a therapeutic amount of the polypeptide in liver cells for a period of at least 500 days, and further wherein said cassette is expressed in an animal or as a component of a recombinant adeno-associated virus (AAV) vector.
5. A nucleic acid expression cassette capable of expressing human Factor IX, wherein the cassette is predominantly expressed in the mammalian liver, said cassette comprising:
(a) a hepatic locus control element consisting of SEQ ID NO:4 or SEQ ID NO:9;
(b) a hepatic promoter located 3′ to the hepatic locus control element, said promoter consisting of SEQ ID NO:5;
(c) a Factor IX coding sequence located 3′ to the hepatic promoter, said coding sequence comprising SEQ ID NO:2;
(d) a polyadenylation signal located 3′ to the intron sequence, said polyadenylation signal consisting of SEQ ID NO:6; and
(e) an intron located 3′ to the hepatic promoter and 5′ to the polyadenylation signal, wherein said intron consists of SEQ ID NO:1, and
(f) an untranslated region located 3′ to the coding region and the intron, said untranslated region consisting of SEQ ID NO:7;
wherein elements (a), (b), (c), (d) and (e) are operably linked to express the polypeptide encoded by the coding sequence.
6. The expression cassette of claim 5 , wherein said cassette directs expression of a therapeutic amount of Factor IX in liver cells for a period of at least 100 days, and further wherein said cassette is expressed in an animal or as a component of a recombinant adeno-associated virus (AAV) vector.
7. The expression cassette of claim 5 , wherein said cassette directs expression of a therapeutic amount of the polypeptide in liver cells for a period of at least 300 days, and further wherein said cassette is expressed in an animal or as a component of a recombinant adeno-associated virus (AAV) vector.
8. The expression cassette of claim 5 , wherein said cassette directs expression of a therapeutic amount of the polypeptide in liver cells for a period of at least 500 days, and further wherein said cassette is expressed in an animal or as a component of a recombinant adeno-associated virus (AAV) vector.