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 the steps of:
a) treating a mammal with a vector, comprising:
i) at least one glucose inducible regulatory element (GIRE);
ii) at least one liver-specific promoter;
iii) a gene encoding insulin with a modified peptidase site;
iv) at least one albumin 3′untranslated region (UTR); and
v) at least one human growth hormone (HGH) intron having the sequence of residues 475-754 of SEQ ID NO:2; and
b) measuring the mammal's insulin levels, 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 further comprises at least one translational enhancer.
4. The method of claim 1 , wherein the vector comprises 2-4 GIREs.
5. The method of claim 1 , wherein the at least one liver specific promoter is an albumin promoter.
6. The method of claim 5 , wherein the albumin promoter is residues 147-474 of SEQ ID NO:2.
7. The method of claim 1 , wherein the gene encoding insulin with a modified peptidase site is residues 919-1251 of SEQ ID NO:2.
8. The method of claim 1 , wherein the gene encoding insulin with a modified peptidase site is modified human insulin cDNA.
9. The method of claim 8 , wherein the modified human insulin cDNA is modified at the B and C peptide of human insulin from KRR to RTKR and at the C and A peptide from LQKR to RQKR.
10. The method of claim 1 , wherein the at least one albumin 3′ UTR is residues 1252-2125 of SEQ ID NO:2.
11. The method of claim 2 , wherein the at least one promoter enhancer is the alpha-fetoprotein enhancer.
12. The method of claim 2 , wherein the at least one promoter enhancer is residues 15-265 of SEQ ID NO:3.
13. The method of claim 3 , wherein the at least one translational enhancer is vascular endothelial growth factor (VEGF) translational enhancer.
14. The method of claim 13 , wherein the vascular endothelial growth factor (VEGF) translational enhancer is residues 755-918 of SEQ ID NO:2.
15. The method of claim 1 , wherein the treating step comprises exposing the mammal to a virus infective for the mammal, wherein the virus comprises the vector and whereby the mammal is infected by the virus.
16. The method of claim 15 , wherein the virus is adenovirus.
17. The method of claim 15 , wherein the exposing comprising exposing at least one hepatocyte of the mammal to the virus.
18. The method of claim 15 , wherein the exposing comprises direct injection.
19. The method of claim 15 , wherein the exposing comprises vascular injection.
20. A method of controlling blood glucose levels in a mammal, comprising the steps of:
a) treating a mammal with an adenoviral 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) at least one translational enhancer; and
b) observing the mammal's insulin levels, wherein the mammal's insulin levels are controlled by the glucose-regulated synthesis of insulin by said vector.