GLP-1 gene delivery for the treatment of type 2 diabetes

• US 20030220274A1
• Filed: 05/21/2002
• Published: 11/27/2003
• Est. Priority Date: 05/21/2002
• Status: Active Grant

• Current Assignee: CLSN Laboratories, Inc. (Imunon, Inc.)
• Original Assignee: EGEN, Inc. (Imunon, Inc.)
• Inventors: Lee, Minhyung, Ko, Kyungsoo, Oh, Seungjoon
• Granted Patent: US 7,374,930 B2
• Time in Patent Office: Days
• US Class Current: 514/44
• 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: A61K 47/59 obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
• CPC Class Codes: A61K 47/593 Polyesters, e.g. PLGA or polylactide-co-glycolide
• CPC Class Codes: A61K 47/645 Polycationic or polyanionic oligopeptides, polypeptides or polyamino acids, e.g. polylysine, polyarginine, polyglutamic acid or peptide TAT
• 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: C07K 14/605 Glucagons

Abstract

This patent discloses compositions and methods of use thereof to normalize the blood glucose levels of patients with type 2 diabetes. It relates particularly to a plasmid comprising a chicken β actin promoter and enhancer; a modified GLP-1 (7-37) cDNA (pβGLP1), carrying a furin cleavage site, which is constructed and delivered into a cell for the expression of active GLP-1.

Claims

1. A plasmid consisting essentially of:

an expression facilitating sequence derived from chicken β

-actin promoter and enhancer;

an expression sequence comprising ATG(start codon) followed by a sequence(CGTCAACGTCGT) coding for a furin cleavage site(FCT) and a sequence coding for an active form of a bioactive peptide that is operably linked to said expression facilitating sequence.

2. A plasmid consisting essentially of:

an expression facilitating sequence derived from chicken β

-actin promoter and enhancer;

an expression sequence comprising ATG(start codon) followed by a sequence(CGTCAACGTCGT) coding for a furin cleavage site(FCT) and a sequence coding for GLP-1(7-37) or derivatives thereof that are operably linked to said expression facilitating sequence.

3. A plamid comprising an nucleotide sequence represented by the SEQ ID NO:

1.

7. The formulation of claim 3, wherein the plasmid comprising an nucleotide sequence represented by the SEQ ID NO:

1 or having the nucleotide sequence represented by the SEQ ID NO;

2.

4. A plamid consisting essentially an nucleotide sequence represented by the SEQ ID NO:

2.

5. A transfection formulation comprising a cationic polymeric gene carrier complexed with a selected plasmid consisting essentially of:

an expression facilitating sequence derived from chicken β

-actin promoter and enhancer;

an expression sequence comprising ATG(start codon) followed by a sequence(CGTCAACGTCGT) coding for a furin cleavage site(FCT) and a sequence coding for the active form of GLP-1(7-37) or derivatives thereof that are operably linked to said expression facilitating sequence in a proper ratio.

6. The formulation of claim 5, wherein the cationic polymeric gene carrier is PAGA.

8. The formulation of claim 5, wherein the weight ratio of DNA to the cationic polymer is within a range of 1:

0.85 to 1;

2.90.

9. The formulation of claim 5, wherein the cationic polymeric gene carrier further comprising a targeting moiety.

10. A method of transfecting a cell comprising the steps of:

(a) providing a transfection formulation comprising a cationic polymeric gene carrier complexed with a selected plasmid consisting essentially of;

an expression facilitating sequence derived from chicken β

-actin promoter and enhancer;

an expression sequence comprising ATG(start codon) followed by a sequence(CGTCAACGTCGT) coding for a furin cleavage site(FCT) and a sequence coding for the active form of GLP-1(7-37) or derivatives thereof that are operably linked to said expression facilitating sequence in a proper ratio,

(b) contacting the cell with an effective amount of the transfecting formulation such that the cell internalizes the plasmid; and

(c) culturing the cell with the internalized selected plasmid DNA under conditions favorable for the growth thereof.

11. The method of claim 10, wherein the cationic polymeric gene carrier is PAGA or PEI.

12. The method of claim 11, wherein the weight ratio of DNA to PEI is preferably within a range of 1:

0.676 to 1;

2.704.

14. The method of claim 10, wherein the plasmid comprising an nucleotide sequence represented by the SEQ ID NO:

1 or having the nucleotide sequence represented by the SEQ ID NO;

2.

13. The method of claim 111, wherein the weight ratio of DNA to PAGA is preferably within a range of 1:

0.82 to 1;

2.46.

15. A method of normalizing the blood glucose levels of a warm blooded animal with type 2 diabetes, comprising the steps of:

(a) providing a transfection formulation comprising a cationic polymeric gene carrier complexed with a selected plasmid consisting essentially of;

an expression facilitating sequence derived from chicken β

-actin promoter and enhancer;

an expression sequence comprising ATG(start codon) followed by a sequence(CGTCAACGTCGT) coding for a furin cleavage site(FCT) and a sequence coding for the active form of GLP-1(7-37) or derivatives thereof that are operably linked to said expression facilitating sequence in a proper charge ratio(positive charge of the copolymer/negative charge of the nucleic acid) that is optimally effective for both in vivo and in vitro transfection,

(b) administering to the animal to be treated an effective amount of the composition such that the cell internalizes the GLP-1 gene.

16. The method of claim 15, wherein the cationic polymeric gene carrier is PAGA.

17. The method of claim 16, wherein the weight ratio of DNA to PAGA is preferably within a range of 1:

0.82 to 1;

2.46.

18. The method of claim 16, wherein plasmid comprising an nucleotide sequence represented by the SEQ ID NO:

1 or having the nucleotide sequence represented by the SEQ ID NO;

2.

19. The method of claim 16, wherein the cationic polymeric gene carrier further comprises a targeting moiety.