TRANSGENIC PIG ISLETS AND USES THEREOF FOR TREATING DIABETES

Abstract

The present invention relates to an isolated transgenic pig beta cell wherein the PKC and the PKA pathway are constitutively activated; to a transgenic pig islet comprising said transgenic pig beta cell; and to a transgenic pig comprising said transgenic pig beta cell or said transgenic pig islet. Another object of the invention is a device comprising a transgenic pig beta cell or a transgenic pig islet of the invention. The present invention also relates to the use of said transgenic pig beta cell, said transgenic pig islet, or said device for treating a disease, disorder or condition related to the impaired function of endocrine pancreas or of beta cell.

Claims

1. An isolated transgenic pig beta cell wherein the PKC and the PKA pathway are constitutively activated.

2. The isolated transgenic pig beta cell according to claim 1, wherein the cell comprises a constitutively active acetylcholine receptor.

3. The isolated transgenic pig beta cell according to claim 1, wherein the cell comprises a constitutively active muscarinic receptor.

4. The isolated transgenic pig beta cell according to claim 1, wherein the cell comprises a constitutively active type III muscarinic receptor.

5. The isolated transgenic pig beta cell according to claim 1, wherein the cell comprises a constitutively active type III muscarinic receptor having an amino acid sequence SEQ ID NO: 4, or a sequence having at least 70% sequence identity with SEQ ID NO: 4.

6. The isolated transgenic pig beta cell according to claim 1, wherein the cell expresses GLP-1.

7. The isolated transgenic pig beta cell according to claim 1, wherein the cell expresses GLP1 having the amino acid sequence SEQ ID NO: 6, or a sequence having at least 70% sequence identity with SEQ ID NO: 6.

8. An isolated transgenic pig islet comprising a pig beta cell according to claim 1.

9. An ex vivo method for obtaining an isolated transgenic pig beta cell according to claim 1 or an isolated transgenic pig islet comprising a said isolated transgenic pig beta cell, wherein said method uses an expression vector comprising a nucleic acid sequence encoding a constitutively active type III muscarinic receptor, and an expression vector comprising a nucleic acid sequence encoding GLP-1.

10. An isolated transgenic pig beta cell or an isolated transgenic pig islet obtained by the method according to claim 9.

11. A transgenic pig comprising an isolated transgenic pig beta cell according to claim 1, or an isolated transgenic pig islet comprising a said isolated transgenic pig beta cell.

12. A device comprising an isolated transgenic pig beta cell according to claim 1 or an isolated transgenic pig islet comprising an said isolated transgenic pig beta cell.

13. The device according to claim 12, wherein said isolated transgenic pig beta cell or said isolated transgenic pig islet are encapsulated in an alginate composition.

14. A method for treating a disease, disorder or condition related to the impaired function of endocrine pancreas or beta cell in a subject in need thereof, comprising administering an isolated transgenic pig beta cell according to claim 1 or an isolated transgenic pig islet comprising a said isolated transgenic pig beta cell, or a device comprising a said isolated transgenic pig beta cell or isolated transgenic pig islet.

15. The method according to claim 14, wherein said disease, disorder or condition related to the impaired function of endocrine pancreas or beta cell is selected from the group comprising type I diabetes, type II diabetes, gestational diabetes, latent autoimmune diabetes, type 1.5 diabetes, lipoatrophic diabetes, maturity onset diabetes of the young, neonatal diabetes, prediabetes, steroid-induced diabetes, and pancreatic cancer.

16. The method according to claim 14, wherein said disease, disorder or condition related to the impaired function of endocrine pancreas or beta cell is permanent neonatal diabetes or transient neonatal diabetes.

17. The method according to claim 14, wherein said disease, disorder or condition related to the impaired function of endocrine pancreas or beta cell is an endocrine pancreas cancer.

18. The method according to claim 14, wherein said disease, disorder or condition related to the impaired function of endocrine pancreas or beta cell is an endocrine pancreatic tumor or a pancreatic neuroendocrine carcinoma.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0151] FIG. 1 is a histogram showing the insulin secretion from adult and neonate isolated pig islets. Batches of 200 islets were incubated in 1 ml krebs medium containing 15 mM glucose (G15) alone or in combination with forskolin (Fsk; 1 μM), phorbol myristate acetate (PMA; 20 nM) or both Fsk and PMA. Insulin secretion was measured in the incubation media and expressed as a percentage of total insulin content of each batch of islets. The numbers above the columns represent the fold increase of insulin secretion in test groups compared to 15 mM glucose alone. * p<0.05. Values are means±SEM for n=5-21 from 8 different adult preparations and n=11-43 from 11 different neonate preparations.

[0152] FIG. 2 is a histogram showing glucose-induced insulin secretion from transfected Min6 cells incubated with 1 (G1) or 15 mM (G15) glucose. The numbers above pairs of columns represent the stimulation ratio (G15/G1) in each group. * p<0.05 indicates significant difference between test groups and controls. Values are means±SEM for n=15-21 from 7 different experiments.

[0153] FIG. 3 is a combination of two histograms showing the insulin secretion from neonatal (A) and adult (B) isolated pig islets exposed to 200 MOI viral expression vectors carrying sequences coding for GLP-1 (GLP-1 Ser8), activated muscarinic receptor (M3R) or both (GLP-1+M3R) during 48 hours. Batches of 200 islets were incubated in 1 ml krebs medium containing 1 mM glucose (G1) or 15 mM glucose (G15). Insulin secretion was measured in the incubation media and expressed as a percentage of total insulin content of each batch of islets. * p<0.05. Values are means±SEM for n=38-46 from 10 different preparations.

[0154] FIG. 4 is a graph showing the insulin secretion of isolated adult pig islets exposed to 200 MOI viral expression vectors carrying sequences coding for GLP-1 (GLP-1 Ser8), activated muscarinic receptor (M3R) or both (GLP-1+M3R) during 48 hours. Batches of 600 islets were perifused in krebs medium containing 1 mM glucose (G1) then 15 mM glucose (G15) as indicated on top of the figure. Insulin secretion was then measured in the effluent fractions. Values are means±SEM for n=3-4 from 4 different preparations.

EXAMPLES

[0155] The present invention is further illustrated by the following examples.

Example 1

[0156] Isolated pig islets were cultured overnight at 37° C., 5% CO.sub.2/95% O.sub.2 in RMPI medium containing 10% heat-inactivated FCS, 100 IU/ml penicillin, 100 μg/ml streptomycin, 5 mmol/l glucose. Then, isolated pig islets were incubated for 2 hours 1 mL of Krebs-Ringer buffer at 1 mmol/L or 15 mmol/L glucose, optionally supplemented with 20 nM phorbol myristate acetate (PMA, a direct activator of the PKC pathway) and/or 1 μM forskolin (an indirect activator of the PKA pathway). Finally, insulin was quantified in recovered media and in incubated islets by radioimmuno-assay.

[0157] As shown in FIG. 1, direct PKC activation by PMA increases glucose-induced insulin secretion by 2-fold in isolated pig islets isolated from adult pigs and up to 8-fold in pig islets isolated from neonate pigs, thus improving porcine islet responsiveness to glucose. We also observed slightly smaller increases when beta-cell cAMP was elevated by forskolin to activate PKA and Epac2. Interestingly, when we exposed the transgenic pig islets to both PMA and forskolin in the presence of 15 mM glucose, we observed an unexpected synergy in the secretory response which was then augmented by almost 6-fold in adult islets and 25-fold in neonate islets. These results thus demonstrate an unexpected synergistic effect of PKC activation and PKA activation on insulin secretion.

Example 2

[0158] In order to verify the expression of transgenic proteins, the activation of the targeted pathways and the effects of such activation on glucose-induced insulin secretion, a line of murine beta-cells (MIN6) was transfected.

[0159] The expression of transgenic proteins was verified at the mRNA level by RT-PCR. The sequence of the activated muscarinic receptor differs greatly from the wild type receptor and expression of the protein can thus be validated by western blotting of transfected MIN6 cells extracts using an antibody specific to a tag that has been added to the receptor sequence. GLP-1 production was verified by measuring the amount of intracellular GLP-1 in transgenic and control cells as well as measuring GLP-1 secretion in culture media.

[0160] To verify the effect of the transgenic proteins on insulin secretion, transfected and control MIN6 cells were challenged by an increase of glucose concentration during static incubation experiments. Insulin secretion was measured and the stimulation index i.e., the ratio between insulin secretion at high glucose and insulin secretion at low glucose, of transgenic and control cells was calculated. In brief, 2.Math.10.sup.5 cells were seeded in 12-well plates and cultivated for 48h before they were transfected with one the plasmids carrying the GLP-1 (7-37) gene (GLP-1), the mutated GLP-1 (7-A8S-37) gene (GLP-1 Ser8) or the constitutively activated muscarinic receptor gene (M3). Control cells were simply exposed to Lipofectamine without any plasmidic DNA. 48h after transfection, cells were starved then incubated for 2h in a 1 mL Krebs-Ringer buffer containing either 1 or 15 mM glucose. Insulin secretion was measured in the incubation media and expressed as a percentage of total insulin content of the cells.

[0161] FIG. 2 shows that transgenic expression of GLP-1 in Min6 cells causes a slight non-significant increase of glucose-induced insulin secretion (stimulation index 2.6 vs 2.2 in control cells). Interestingly, the transgene encoding a modified GLP-1 (GLP-1 Ser8) with a larger half-life induced a greater significant increase of the secretory response to 15 mM glucose (stimulation index 3.2 vs 2.2 in control cells). Expression of a constitutively activated type-3 muscarinic receptor (M3) in Min6 cells significantly increased insulin secretion induced by 15 mM glucose (stimulation index 3.9 vs 2.2 in controls).

[0162] These results thus show that the used plasmids successfully induce expression of the molecules of interest and that this expression increases the secretory response to glucose.

Example 3

[0163] The effect of transgenic activation of PKA and PKC pathways was evaluated in isolated pig islets. For this purpose, the GLP-1 Ser8 and M3R sequences were inserted in a pENTCMV adenoviral vector to permit expression of transgenic GLP-1 (GLP-1 Ser8) and activated muscarinic receptor (M3R) in primary islet cells. For co-expression of GLP-1 and M3R, the two sequences were inserted in the same bicistronic vector to study the effect of concomitant activation of PKA and PKC on pig islet insulin secretion. Neonatal pig islets cultivated in HAM F10 (panel A) and adult pig islets cultivated in RPMI (panel B) were exposed to GLP-1, M3R or GLP-1+M3R viral expression vectors at a multiplicity of infection of 200 (MOI=200) during 48 hours before glucose challenge. Islets were then incubated for 2 hours in 1 mL of Krebs-Ringer buffer containing 1 mM or 15 mM glucose. Insulin was then quantified in recovered media and in incubated islets by radioimmunoassay.

[0164] As shown in FIG. 3, stimulation index in control islets was at 3.3 for neonatal pig islets and 3 for adults. Islets expressing GLP-1 Serb showed improved, however non-significantly different from controls insulin secretion with a stimulation index of 4.4 in neonates but there was no effect on secretion in adults (stimulation index: 2.7). Insulin secretion was further increased and the difference compared to controls was significant when islets were infected with M3R expression vector resulting in a stimulation index of 5.4 and 4.1 in neonates and adults respectively. Finally, and as expected from the results shown in example 1, neonatal islets co-expressing GLP-1 and M3R showed a synergetic response in terms of insulin secretion since their stimulation index was 7.3. This effect was also observed although to a less degree in adult islets (stimulation index: 5.5).

[0165] The effect of transgenic PKA and PKC activation on glucose-induced insulin secretion was also tested during dynamic islet perifusion experiments. Control and virus-treated adult islets were placed in perifusion chambers sealed with 0.2 um filters. Islets were first perifused with 1 mM glucose (G1) krebs medium during 30 minutes for equilibrium then during 10 minutes in G1 with media collection every 2 minutes followed by 30 minutes stimulation with G15. As shown in FIG. 4 and in agreement with what we observed in static incubations, GLP-1 expression had virtually no effect on acute insulin secretion. M3R expression increased both phases of glucose-induced insulin secretion but this increase was greater when adult pig islets co-expressed GLP-1 and M3R.

[0166] These results thus confirm data obtained using pharmacological activation of PKA and PKC pathways in pig islet cells. Our conclusion is that concomitant activation of both pathways in pig beta-cells would be the best strategy to obtain functionally-enhanced pig islets.