Methods of inducing insulin production
The present invention provides a method of inducing insulin production in a cell by up-regulating a target gene involved in insulin production in said cell using an saRNA (short activating ribonucleic acid) which specifically down-regulates a target antisense RNA transcript present in said cell, wherein (i) said target RNA transcript is complementary to a sequence located on the coding strand of the target gene between 1000 nucleotides upstream and 1000 nucleotides downstream of the transcription start site of the target gene; and (ii) said sRNA is a single or double stranded RNA molecule up to 30 nucleotides in length comprising a sequence of at least 13 nucleotides which has at least 95% complementarity to a region of the target transcript. Also provided are certain saRNA molecules and uses thereof, particular medical uses, and induced cells and uses thereof.
1. A method of inducing insulin production in a cell by up-regulating a target gene involved in insulin production in said cell, wherein the target gene is selected from the group consisting of insulin, PDX1, Neurogenin 3 (Ngn3), Rfx6, MafA, GLP1 receptor, FOXA2, HNF1A, Hlxb9, Hnf6, Ptf1a, Neuro D, betacellulin, ABCC8, glucokinase (GCK), GLUT2, and Nkx6-1, comprising
(i) designing an saRNA (short activating ribonucleic acid) which specifically targets a target antisense RNA transcript present in said cell, wherein said target RNA transcript is complementary to a sequence located on the coding strand of the target gene between 500 nucleotides upstream and 500 nucleotides downstream of the transcription start site of the target gene; and
said saRNA is a single or double stranded RNA molecule up to 30 nucleotides in length comprising a sequence of at least 13 nucleotides which has at least 95% complementarity to a region of the target transcript;
(ii) synthesizing said saRNA;
(iii) administering said saRNA to said cell; and
(iv) performing analysis to confirm said saRNA up-regulates said target gene.
2. A method according to claim 1 , wherein the cell is induced to produce insulin in a glucose-responsive manner.
3. A method according to claim 1 , wherein the cell is induced to secrete insulin.
4. A method according to claim 1 , wherein said target RNA transcript overlaps the transcription start site and/or a promoter region of the target gene.
5. A method according to claim 1 , wherein said saRNA is a double-stranded RNA of 19 to 25 base pairs in length with 1-5 unpaired bases on each 3′ end forming a 3′ overhang.
6. A method according to claim 1 , wherein said saRNA can down-regulate said target RNA transcript by RNA-induced silencing complex (RISC)-associated cleavage.
7. A method according to claim 1 , wherein said saRNA possesses none of the motifs aaaa, cccc, gggg, and uuuu and/or has a GC-percentage of at least 20% and no more than 75%.
8. A method according to claim 1 , wherein the cell which is induced to produce insulin is an adult or embryonic stem cell, a pluripotent cell, a multipotent cell or a somatic cell.
9. A method according to claim 1 , wherein the cell which is induced to produce insulin is a liver cell or a pancreatic cell.
10. A method of treating a condition associated with aberrant insulin production, uptake or utilisation, said method comprising administering to a subject in need thereof a pharmaceutically effective amount of an saRNA, wherein said saRNA is capable of inducing insulin production in a cell by up-regulating a target gene involved in insulin production in said cell by specifically targeting a target RNA transcript present in said cell, wherein the target gene is selected from the group consisting of insulin, PDX1, Neurogenin 3 (Ngn3), Rfx6, MafA, GLP1 receptor, FOXA2, HNF1A, Hlxb9, Hnf6, Ptf1a, Neuro D, betacellulin, ABCC8, glucokinase (GCK), GLUT2, and Nkx6-1, and wherein
(i) said target RNA transcript is complementary to a sequence located on the coding strand of the target gene between 500 nucleotides upstream and 500 nucleotides downstream of the transcription start site of the target gene;
(ii) said sRNA is a single or double stranded RNA molecule up to 30 nucleotides in length comprising a sequence of at least 13 nucleotides which has at least 95% complementarity to a region of the target transcript; and
(iii) an analysis has been performed to confirm that said saRNA up-regulates said target gene.
11. The method of claim 7 , wherein the GC-percentage of said saRNA is between 20% and 55%.
12. The method of claim 1 , wherein the analysis is mRNA analysis.