METHOD OF IN VIVO ADMINISTRATION OF THE CODING SEQUENCE OF THE SIRT6 GENE VIA ADENO-ASSOCIATED VIRUS

Abstract

Methods for in vivo administration of the coding sequence of the sirt6 gene. In particular, methods that include the administration of adeno-associated virus vectors or recombinant adeno-associated virus vectors including the coding sequence of the sirt6 gene.

Claims

1. A method for extending the lifespan of a subject comprising the administration of an effective number of non-integrating extra copies of the coding sequence of the sirt6 gene or a variant thereof to said subject.

2. The method according to claim 1, wherein the extra copies of the coding sequence are comprised in adeno-associated virus vectors (AAV) or recombinant adeno-associated virus vectors (rAAV).

3. The method according to claim 2, wherein the adeno-associated virus vectors or recombinant adeno-associated virus vectors are selected from the group consisting of AAV serotype 1, serotype 2, serotype 3, serotype 4, and serotype 5.

4. The method according to claim 2, wherein the adeno-associated virus vectors or recombinant adeno-associated virus vectors are administered by systemic intravenous infusion.

5. The method according to claim 2, wherein the adeno-associated virus vectors or recombinant adeno-associated virus vectors are administered by intra muscular injection.

6. The method according to claim 2, wherein the adeno-associated virus vectors or recombinant adeno-associated virus vectors are administered by intramuscular injection coupled with electroporation.

7. The method according to claim 2, wherein the adeno-associated virus vectors or recombinant adeno-associated virus vectors are administered by inhalation.

8. The method according to claim 2, wherein the adeno-associated virus vectors or recombinant adeno-associated virus vectors are administered by topical administration.

9. The method according to claim 2, wherein the adeno-associated virus vectors or recombinant adeno-associated virus vectors are administered by local administration to an organ.

10. The method according to claim 2, wherein the non-integrating extra copies of the coding sequence of sirt6 are operably linked to at least one promotor.

11. The method according to claim 2, wherein the non-integrating extra copies of the coding sequence of sirt6 are operably linked to at least one enhancer.

12. The method according to claim 1, wherein said sirt6 gene or variant thereof is from human origin.

13. The method according to claim 1, wherein said sirt6 gene or variant thereof is from a mammal different than human.

14. The method according to claim 1, wherein said sirt6 gene or variant thereof is from Strongylocentrotus franciscans, Arctica islandica, Balaena mysticetus, Cypinus carpio or Turritopsis dohrnii Jellyfish.

15. The method according to claim 1, wherein said sirt6 gene or variant thereof is synthetic.

16. The method according to claim 1, wherein the subject is a human.

17. The method according to claim 1, wherein the subject is an animal.

18. A method for increasing health span of a subject comprising the administration of non-integrating extra copies of the coding sequence of the sirt6 gene or a variant thereof to said subject.

19. The method according to claim 18, wherein increasing health span means preventing aged-associated diseases such as neurodegenerative diseases, type 2 diabetes or cardio-vascular diseases.

20. A method for delaying the onset of age-related diseases of a subject comprising the administration of non-integrating extra copies of the coding sequence of the sirt6 gene or a variant thereof to said subject.

Description

DETAILED DESCRIPTION

[0010] In one embodiment, the present invention is directed to methods for extending the lifespan by providing non-integrating extra copies of the coding sequence of a regulator human gene SIRT6 (wild type or variants of the coding sequence of the gene).

[0011] The present disclosure relates to methods and compositions for extending lifespan. In one aspect of the invention, a method for extending the life of an organism is provided. The method includes administering an effective number of extra copies of regulator gene SIRT6 through adeno-associated virus vectors. The SIRT6 gene limited size (8,941 base pairs with a coding DNA sequence of thousand base pairs) allows a reliable significant episomal expression of the coding sequence of the SIRT6 by adeno-associated virus vectors (including self-complementary adeno associated virus and CpG-depleted Adeno-Associated Virus). The expression of the coding sequence of the SIRT6 gene was assessed both in vitro and in vivo. The in vitro assessment of the coding sequence of the SIRT6 gene expression was performed using deacetylation assays with SIRT6 proteins. The acetylation status of SIRT6 targets was assayed with acetyl-specific antibodies. In vitro deacetylation reactions were performed as described in (Gil et al., 2013; Michishita et al., 2008). Histone acetylation levels after reactions were detected using western blot analysis with acetylation specific antibodies. The in vitro expression of the coding sequence of the SIRT6 gene was performed using UV induced damage to human cell lines and SIRT6 knockout Human cell lines. In addition, an in vivo assessment of the coding sequence of the SIRT6 gene expression was performed in mice (CD-1 mice from Charles River Laboratories) and SIRT6 knockout mice.

[0012] In one embodiment, the present invention is directed to methods for increasing health span by providing non-integrating extra copies of the coding sequence of a regulator gene SIRT6.

[0013] The present disclosure relates to methods and compositions for delaying the onset of age-related diseases in individuals in need thereof. In one embodiment, the method applies to Human. In another embodiment, the method applies to animals (veterinary use). In one aspect of the invention, a method for extending the life of an organism is provided. The method includes administering an effective number of extra copies of the coding sequence of the regulator gene SIRT6 through adeno-associated virus vectors. The SIRT6 gene limited size SIRT6 (8,941 base pairs with a coding DNA sequence of thousand base pairs) allows a reliable significant episomal expression of SIRT6 by adeno-associated virus vectors.

[0014] In one embodiment of the present invention, the adeno-associated virus can be administered by a systemic intravenous infusion.

[0015] In a further aspect, the invention relates to the use of a intra muscular injection of the invention for extending lifespan and for delaying the onset of age-related diseases in individuals in need thereof.

[0016] In a further aspect, the invention relates to the use of intramuscular injections coupled with electroporation. In a further aspect, the invention relates to the use of an inhaled formulation for delivery to the lung via nebulization. In a further aspect, the invention relates to the use of a topical formulation for delivery to the skin fibroblasts (and other cells from the skin).

[0017] In a further aspect, the present invention relates to a composition comprising of the coding sequence of the SIRT6 gene and adeno-associated virus administered locally to an organ for delaying the onset of age-related damage to the organ.

[0018] In one embodiment of the present invention, the adeno associated virus use a CMV promotor gene. In a further aspect, the invention relates to the use of different promotor genes in order to direct expression of the SIRT6 protein to different tissues.

[0019] In one embodiment of the present invention, the coding sequence of the sirtuin 6 gene is from Human origin (wild type and several variants of coding sequence of the human sirtuin 6 gene).

[0020] In a further aspect, the invention relates to the use of the coding sequence of the sirtuin 6 gene from Strongylocentrotus franciscans. In a further aspect, the invention relates to the use of the coding sequence of the sirtuin 6 gene from Strongylocentrotus franciscans. In a further aspect, the invention relates to the use of the coding sequence of the sirtuin 6 gene from Arctica islandica. In a further aspect, the invention relates to the use of the coding sequence of the sirtuin 6 gene from Balaena mysticetus. In a further aspect, the invention relates to the use of the coding sequence of the sirtuin 6 gene from Cypinus carpio. In a further aspect, the invention relates to the use of the coding sequence of the sirtuin 6 gene from Turritopsis dohrnii Jellyfish. In a further aspect, the invention relates to the use of the coding sequence of the sirtuin 6 gene from different mammal with longer lifespan than human. In a further aspect, the invention relates to the use of synthetic sirtuin 6 gene derived from different mammal with longer lifespan than human.

[0021] In one embodiment, the present invention is directed to methods for extending the lifespan by providing non-integrating extra copies of the coding sequence of a regulator gene SIRT6.

[0022] In one embodiment, the present invention is directed to methods for extending the Health span (preventing aged-associated diseases such as neurodegenerative diseases, type 2 diabetes or cardio-vascular diseases) by providing non-integrating extra copies of the coding sequence of a regulator gene SIRT6.

[0023] The present disclosure relates to methods and compositions for extending lifespan. In one aspect of the invention, a method for extending the life of an organism is provided. The method includes administering an effective number of extra copies of regulator gene SIRT6 through adeno-associated virus vectors. The coding sequence of the SIRT6 gene limited size (thousand base pairs) allows a reliable significant episomal expression of SIRT6 by adeno-associated virus vectors.

[0024] The present disclosure also relates to a method for extending the lifespan of a subject comprising the administration of a composition comprising adeno-associated virus vectors or recombinant adeno-associated virus vectors comprising an effective number of non-integrating extra copies of the coding sequence of the sirt6 gene or a variant thereof to said subject operably linked to at least one enhancer and at least one promoter.