Insulin gene therapy to treat diabetes
AssigneeWisconsin Alumni Research Foundation
InventorsTausif ALAM, Hans SOLLINGER
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Abstract
The present invention provides recombinant adeno-associated virus serotype 8 (AAV8) virus particles that are designed to deliver insulin-encoding polynucleotides to the liver. Also provided are the nucleic acid constructs carried by the virus particles, methods for producing the AAV8 virus particles, and methods of using the AAV8 virus particles to control blood glucose levels in a mammal.
Claims33
A nucleic acid construct for producing a recombinant AAV8 vector encoding insulin comprising:
- a) a 5′ inverted terminal repeat (ITR);
- b) a promoter enhancer;
- c) a glucose inducible regulatory element (GIRE);
- d) a liver-specific promoter;
- e) a translational enhancer;
- f) a polynucleotide encoding insulin with a modified peptidase site;
- g) an albumin 3′ untranslated region (UTR);
- and
- h) a 3′ ITR;
- wherein the vector contains only one GIRE.
The construct of any one of the preceding claims, wherein the GIRE is SEQ ID NO:4.
The construct of any one of the preceding claims, wherein the liver-specific promoter is an albumin promoter.
The construct of any one of the preceding claims, wherein the translational enhancer is a vascular endothelial growth factor (VEGF) translational enhancer.
The construct of any one of the preceding claims, wherein the polynucleotide encoding insulin comprises a furin cleavage site and can be processed by furin.
The construct of any one of the preceding claims, wherein the albumin 3′ UTR is SEQ ID NO:8.
The construct of any one of the preceding claims, wherein the 3′ ITR is SEQ ID NO:9.
The construct of any one of the preceding claims, wherein components (a)-(h) are present in the vector in the following 5′ to 3′ order:
- (a), (b), (c), (d), (e), (f), (g), and (h).
The construct of any one of the preceding claims, wherein the construct is SEQ ID NO:1.
A method of producing an AAV8 virus particle, the method comprising:
- a) transducing a host cell with:
- i. a plasmid comprising SEQ ID NO:1,
- ii. a packaging plasmid, and
- iii. a helper plasmid;
- b) collecting the supernatant and the cells from culture after a suitable time;
- and
- c) isolating virus particles.
Dependencies are inferred from the claim text.