Hepatocyte based insulin gene therapy for diabetes
A method and vectors for controlling blood glucose levels in a mammal are disclosed. In one embodiment, the method comprises the steps of: treating the hepatocyte cells of a patient with a first, second or third vector, wherein the first vector comprises a promoter enhancer, glucose inducible regulatory elements, a liver-specific promoter, a gene encoding human insulin with modified peptidase and an albumin 3′UTR and lacks an HGH intron, wherein the second vector comprises an HGH intron, glucose inducible regulatory elements, a liver-specific promoter, a gene encoding human insulin with modified peptidase site and an albumin 3′UTR and lacks a promoter enhancer, wherein the third vector comprises an HGH intron, glucose inducible regulatory elements, a liver-specific promoter, a gene encoding human insulin with modified peptidase site, an albumin 3′UTR and a promoter enhancer and observing the patient's insulin levels, wherein the patient's insulin levels are controlled.
1. A method of controlling blood glucose levels in a mammal, comprising
treating a mammal with a vector comprising:
i) at least one glucose inducible regulatory element (GIRE);
ii) an albumin promoter having the sequence of residues 380-706 of SEQ ID NO: 1;
iii) a gene encoding insulin with a modified peptidase site;
iv) at least one albumin 3′ untranslated region (UTR); and
v) a vascular endothelial growth factor (VEGF) translational enhancer;
wherein the mammal's insulin levels are controlled by the glucose-regulated synthesis of insulin by said vector.
2. The method of claim 1 , wherein the vector comprises at least one promoter enhancer.
3. The method of claim 1 , wherein the vector comprises 2-4 GIREs.
4. The method of claim 1 , wherein the gene encoding insulin with a modified peptidase site is residues 871-1203 of SEQ ID NO:1.
5. The method of claim 1 , wherein the gene encoding insulin with a modified peptidase site is modified human insulin cDNA.
6. The method of claim 5 , wherein the modified human insulin cDNA is modified at the B and C peptide of human insulin from KTRR (SEQ ID NO: 4) to RTKR (SEQ ID NO: 5) and at the C and A peptide from LQKR (SEQ ID NO: 6) to RQKR (SEQ ID NO: 7).
7. The method of claim 1 , wherein the at least one albumin 3′ UTR comprises a 3′ UTR of human albumin.
8. The method of claim 1 , wherein the at least one albumin 3′ UTR is residues 1204-2077 of SEQ ID NO:1.
9. The method of claim 2 , wherein the at least one promoter enhancer is an alpha-fetoprotein enhancer.
10. The method of claim 2 , wherein the at least one promoter enhancer is residues 15-265 of SEQ ID NO:1.
11. The method of claim 1 , wherein the VEGF translational enhancer is residues 707-870 of SEQ ID NO:1.
12. The method of claim 1 , wherein the vector is a viral vector.
13. The method of claim 1 , wherein treating comprises exposing at least one hepatocyte of the mammal to the vector.
14. The method of claim 13 , wherein the exposing comprises direct injection.
15. The method of claim 13 , wherein the exposing comprises vascular injection.
16. The method of claim 1 , wherein the mammal is human.
17. The method of claim 1 , wherein the mammal is a cat or a dog.