Hepatocyte Based Insulin Gene Therapy For Diabetes

• US 20140170123A1
• Filed: 12/18/2013
• Published: 06/19/2014
• Est. Priority Date: 06/07/2011
• Status: Active Grant

• Current Assignee: Wisconsin Alumni Research Foundation (University of Wisconsin System)
• Original Assignee: Wisconsin Alumni Research Foundation (University of Wisconsin System)
• Inventors: Alam, Tausif, Sollinger, Hans
• Granted Patent: US 9,732,356 B2
• Time in Patent Office: Days
• US Class Current: 424/93.21
• CPC Class Codes: A61K 35/407 Liver; Hepatocytes
• CPC Class Codes: A61K 38/00 Medicinal preparations containing peptides peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00
• CPC Class Codes: A61P 3/10 for hyperglycaemia, e.g. antidiabetics
• CPC Class Codes: C07K 14/62 Insulins
• CPC Class Codes: C12N 15/85 for animal cells
• CPC Class Codes: C12N 15/86 Viral vectors
• CPC Class Codes: C12N 2710/10042 virus or viral particle as vehicle, e.g. encapsulating small organic molecule
• CPC Class Codes: C12N 2830/002 inducible enhancer/promoter combination, e.g. hypoxia, iron, transcription factor
• CPC Class Codes: C12N 2830/15 chimeric enhancer/promoter combination
• CPC Class Codes: C12N 2830/42 being an intron or intervening sequence for splicing and/or stability of RNA
• CPC Class Codes: C12N 2840/105 enhancing translation

Abstract

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.

Claims

1. A method for obtaining glucose-regulated expression of insulin ex vivo in mammalian hepatocytes, wherein the method comprises delivering a first, second or third genetic vector for glucose-regulated synthesis of insulin into an isolated mammalian hepatocyte and wherein the insulin levels in the blood of the mammal stays within 0.5 μ

U-100 μ

U/ml and the blood glucose concentration of the mammal stays within 80-150 mg/dl for at least 10 days after transplant of the mammalian hepatocytes into a mammal.

wherein the first vector comprises a promoter enhancer, one to five glucose inducible regulatory elements, a liver-specific promoter, a gene encoding insulin with modified peptidase sites and an albumin 3′

UTR and lacks a human growth hormone (HGH) intron,

wherein the second vector comprises a HGH intron, glucose inducible regulatory elements, a liver-specific promoter, a gene encoding insulin with modified peptidase sites and an albumin 3′

UTR and lacks a promoter enhancer,

wherein the third vector comprises a HGH intron, glucose inducible regulatory elements, a liver-specific promoter, a gene encoding insulin with modified peptidase sites, an albumin 3′

UTR and a promoter enhancer, and

wherein glucose-regulated expression of insulin occurs.

2. The method of claim 1 additionally comprising the step of transplanting the hepatocytes back into a mammal.

3. The method of claim 1, wherein the genetic vector is delivered by exposing the cells to a virus infective for the cells, wherein the virus comprises the genetic construct, and whereby at least a portion of the cells are infected by the virus under suitable conditions and at a sufficient multiplicity.

4. The method of claim 1 wherein the mammal is human.

5. The method of claim 1 wherein the insulin is human insulin.

11. The method of claim 1 wherein the vector is in a minicircle format.

6. A vector suitable for controlling blood glucose levels in a mammal,

wherein the vector comprises a promoter enhancer, glucose inducible regulatory elements, a liver-specific promoter, a gene encoding insulin with modified peptidase sites and an albumin 3′

UTR and lacks a HGH intron or

wherein the vector comprises an HGH intron, glucose inducible regulatory elements, a liver-specific promoter, a gene encoding insulin with modified peptidase sites and an albumin 3′

UTR and lacks a promoter enhancer or

wherein the vector comprises an HGH intron, glucose inducible regulatory elements, a liver-specific promoter, a gene encoding human insulin with modified peptidase sites, an albumin 3′

UTR and a promoter enhancer.

7. The vector of claim 6 wherein additionally comprising a VEGF-Enhancer.

8. The vector of claim 6 wherein the promoter is an albumin promoter.

9. The vector of claim 6 wherein the insulin is human insulin.

10. A method of controlling blood glucose levels in a mammal, comprising the steps of:

treating a mammal with a first, second or third vector,

wherein the first vector comprises a promoter enhancer, 1-5 glucose inducible regulatory elements, a liver-specific promoter, a gene encoding insulin with modified peptidase sites and an albumin 3′

UTR and lacks a HGH intron and

wherein the second vector comprises an HGH intron, glucose inducible regulatory elements, a liver-specific promoter, a gene encoding insulin with modified peptidase sites 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 insulin with modified peptidase sites and an albumin 3′

UTR and a promoter enhancer, and

observing the mammal'"'"'s insulin levels, wherein the insulin levels in the blood of the mammal stays within 0.5 μ

U-100 μ

U/ml and the blood glucose concentration of the mammal stays within 80-150 mg/dl for at least 10 days after transplant of the mammalian hepatocytes into a mammal.

12. The method of claim 10 wherein the vector is in a minicircle format.

13. The method of claim 10 wherein the mammal is human.

14. The method of claim 13 wherein the insulin is human insulin.

15. The method of claim 10 wherein the mammal'"'"'s cholesterol level decreases after treatment.

16. The method of claim 10 wherein the mammal'"'"'s triglyceride level decreases after treatment.

17. The method of claim 10 wherein the mammal is a cat.

18. The method of claim 10 wherein the mammal is a dog.

19. The method of claim 10 wherein the mammal is selected for the group consisting of hamsters, gerbils, rats, mice, rabbits, guinea pigs, chinchillas and ferrets.

20. The method of claim 10 wherein the mammal is a non-human mammal.

21. The method of claim 10 wherein the patient has a decrease in the plasma level of a compound selected from the group of AST, ALT, and alkaline phosphatase after treatment.