Cells genetically modified to comprise pancreatic islet glucokinase and uses thereof

• US 9,365,829 B2
• Filed: 02/20/2014
• Issued: 06/14/2016
• Est. Priority Date: 08/10/2007
• Status: Active Grant

• Current Assignee: University of Technology at Sydney
• Original Assignee: University of Technology at Sydney
• Inventors: Simpson, Ann Margaret, Tao, Chang
• Primary Examiner(s): EPPS -SMITH, JANET L
• Application Number:
  US14/185,716
• Publication Number:
  US 20150030574A1
• Time in Patent Office: 845 Days
• Field of Search: 424/93.21, 435/325, 435/370
• US Class Current: 1/1
• CPC Class Codes: A61K 35/12 Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells vaccines or medicinal preparations containing antigens or antibodies A61K39/00
• CPC Class Codes: A61K 35/407 Liver; Hepatocytes
• CPC Class Codes: A61K 38/28 Insulins
• CPC Class Codes: A61K 38/45 Transferases (2)
• CPC Class Codes: A61K 48/00 Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
• 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 2501/33 Insulin together with transferrin C12N2500/25; Insulin-like growth factors C12N2501/105
• CPC Class Codes: C12N 2510/00 Genetically modified cells
• CPC Class Codes: C12N 2830/20 transcription of more than one cistron
• CPC Class Codes: C12N 5/067 Hepatocytes
• CPC Class Codes: C12N 9/1205 Phosphotransferases with an alcohol group as acceptor (2.7.1), e.g. protein kinases
• CPC Class Codes: C12Y 207/01002 Glucokinase (2.7.1.2)

Abstract

The present invention relates generally to a population of cells genetically modified to produce insulin in a glucose responsive manner and uses thereof. More particularly, the present invention relates to a population of cells genetically modified to produce insulin in response to physiologically relevant levels of glucose and uses thereof. The cells of the present invention are useful in a wide variety of applications, in particular in the context of therapeutic and prophylactic regimes directed to the treatment of diabetes and/or the amelioration of symptoms associated with diabetes, based on the transplantation of the cells of the present invention into mammals requiring treatment. Also facilitated is the design of in vitro based screening systems for testing the therapeutic effectiveness and/or toxicity of potential adjunctive treatment regimes.

Claims

1. An isolated genetically modified mammalian hepatocyte, wherein said genetically modified mammalian hepatocytes are transfected with a vector expressing a nucleic acid molecule encoding human pancreatic islet glucokinase, and produces insulin in response to extracellular glucose levels that are equivalent to physiological glucose levels.

2. The isolated genetically modified hepatocyte of claim 1, wherein said genetically modified hepatocyte is a human cell.

3. The isolated genetically modified hepatocyte of claim 1, wherein said hepatocyte further comprises a nucleic acid molecule encoding insulin.

4. The isolated genetically modified hepatocyte of claim 3 wherein said hepatocyte is an Huh7ins cell.

5. The isolated genetically modified hepatocyte of claim 1, wherein said hepatocyte expresses glucokinase in response to an extracellular glucose concentration in the range of 3-8 mM.

6. The isolated genetically modified hepatocyte of claim 5 wherein said hepatocyte expresses glucokinase in response to an extracellular glucose concentration in the range of 3.5-7 mM.

7. The isolated genetically modified hepatocyte of claim 6 wherein said hepatocyte expresses glucokinase in response to an extracellular glucose concentration in the range of 4-6 mM.

8. The isolated genetically modified hepatocyte of claim 7 wherein said hepatocyte expresses glucokinase in response to an extracellular glucose concentration in the range of 4-5 mM.

9. The isolated genetically modified hepatocyte of claim 1, wherein said human pancreatic islet glucokinase is encoded by SEQ ID NO:

2.

10. The isolated genetically modified hepatocyte of claim 9 wherein said hepatocyte comprises a vector which expresses SEQ ID NO:

2.

11. The isolated genetically modified hepatocyte of claim 10 wherein said vector is a bicistronic vector.

12. The isolated genetically modified hepatocyte of claim 11 wherein said bicistronic vector is pIRESpuro3.

13. The isolated genetically modified hepatocyte of claim 12 wherein said bicistronic vector corresponds to SEQ ID NO:

3.

14. The isolated genetically modified hepatocyte of claim 12 wherein said hepatocyte is an Huh7ins cell.

15. A method of therapeutically or prophylactically treating a condition in a mammal, which condition is characterised by the aberrant production of functional insulin, said method comprising introducing into said mammal an effective number of the isolated genetically modified cells according to claim 1.

16. The method of claim 15 wherein said mammal is a human.

17. The method of claim 15 wherein said aberrant production of functional insulin is an inadequate production of functional insulin.

18. The method of claim 17 wherein said inadequate production of functional insulin is an inadequate production of functional insulin in response to an extracellular glucose concentration of greater than 3 mM.

19. The method of claim 18 wherein said glucose concentration is 4-8 mM.

20. The method of claim 19 wherein said glucose concentration is 4-6 mM.

21. The method of claim 15 wherein said condition is diabetes.

22. The method of claim 21 wherein said diabetes is type 1 diabetes, IDDM, gestational diabetes, slowly progressive IDDM, latent autoimmune diabetes or type 2 diabetes.

23. A method of reducing glucose levels in a mammal said method comprising introducing into said subject an effective number of the isolated genetically modified cells according to claim 1.

24. The method according to claim 23 wherein said mammal is a human.