Sugar alcohol-based compositions for delivering nucleic acid-based drugs in vivo and in vitro

• US 9,387,251 B2
• Filed: 08/12/2014
• Issued: 07/12/2016
• Est. Priority Date: 08/10/2012
• Status: Active Grant

• Current Assignee: David Ts Wu, Shi-Lung Lin
• Original Assignee: David Ts Wu, Shi-Lung Lin
• Inventors: Lin, Yi-Wen, Lin, Shi-Lung
• Primary Examiner(s): Schultz, Doug
• Application Number:
  US14/457,829
• Publication Number:
  US 20140350085A1
• Time in Patent Office: 700 Days
• Field of Search: None
• US Class Current: 1/1
• CPC Class Codes: A61K 31/7105 Natural ribonucleic acids, i.e. containing only riboses attached to adenine, guanine, cytosine or uracil and having 3'-5' phosphodiester links
• CPC Class Codes: A61K 31/713 Double-stranded nucleic acids or oligonucleotides
• CPC Class Codes: A61K 47/26 Carbohydrates, e.g. sugar alcohols, amino sugars, nucleic acids, mono-, di- or oligo-saccharides; Derivatives thereof, e.g. polysorbates, sorbitan fatty acid esters or glycyrrhizin
• CPC Class Codes: C12N 15/111 General methods applicable to biologically active non-coding nucleic acids
• CPC Class Codes: C12N 15/113 Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing when used in plants C12N15/8218
• CPC Class Codes: C12N 2310/141 MicroRNAs, miRNAs
• CPC Class Codes: C12N 2320/32 Special delivery means, e.g. tissue-specific
• CPC Class Codes: C12N 2330/50 Biochemical production, i.e. in a transformed host cell
• CPC Class Codes: C12N 2330/51 Specially adapted vectors
• CPC Class Codes: C12N 2740/15041 Use of virus, viral particle or viral elements as a vector

Abstract

This invention relates to a composition and its use for formulating nucleic acid-based drugs/vaccines with sugar alcohol compositions into complexes for both in-vitro and in-vivo delivery. Particularly, the present invention includes the ingredients and processes necessary for formulating therapeutic and pharmaceutical nucleic acid compositions, such as miRNA, microRNA precursors, shRNAs, siRNAs, ribozymes, antisense RNAs/DNAs, RNA-DNA hybrids and DNA vectors/vaccines, with glycylated sugar alcohols/sugars into delivery complexes, which can then be absorbed by cells in vivo and in vitro via active endocytosis. Also, the present invention discloses that chemical compounds containing sugar alcohol- and/or sugar-like structures can protect nucleic acids, in particular miRNAs, shRNAs, siRNAs and ribozymes, from degradation in vivo as well as in vitro. Therefore, the present invention is also a formula and method for preserving the structural integrity and functional efficacy of these nucleic acid-based drugs and/or vaccines in vivo and in vitro.

Claims

1. A method for formulating nucleic acid compositions with sugars and sugar alcohols into stable complexes for both in-vitro and in-vivo delivery into mammalian cells, comprising:

(a) providing at least a nucleic acid composition with at least a negative charge;

(b) preparing at least one sugar or sugar alcohol composition modified by glycylation to form a glycylated sugar or sugar alcohol composition;

wherein said glycylation is a chemical reaction that replaces the hydroxyl (HO—

) groups of a sugar alcohol or sugar with glycyl (NH₂CH₂COO—

) groups and thus results in the formation of an ether (R—

O—

R) linkage between each OH-removed carbon of the sugar alcohol or sugar and the glycyl group and wherein said glycylation reaction occurs under a pH value ranged from 2.5 to 8.0 at a temperature over 45°

C. for at least 30 minutes;

wherein the sugar alcohol has a formula of H(HCHO)_n+1H, and the sugar has a formula of H(HCHO)_nHCO, and wherein the optimal concentration of said glycyl group is ranged from 0.05 to 3.0 M for mixing with per 1.0 M of sugar alcohol in the glycylation reaction; and

(c) mixing the nucleic acid composition from (a) and the glycylated sugar or sugar alcohol composition from (b) together under a condition below pH 8.0 to form at least a delivery complex.

2. The method as defined in claim 1, wherein said nucleic acid composition is RNA.

3. The method as defined in claim 2, wherein said nucleic acid composition is small hairpin RNAs (shRNA).

4. The method as defined in claim 1, wherein said sugar or sugar alcohol composition is glycerol (glycerin).

5. The method as defined in claim 1, wherein said at least one modified sugar or sugar alcohol composition by glycylation contains a mixture of diglycylglycerol and triglycylglyceride.

6. The method as defined in claim 1, wherein said modified sugar or sugar alcohol composition by glycylation is positively charged.

7. The method as defined in claim 1, wherein said modified sugar or sugar alcohol composition is in solution and has a concentration ranged from about 0.1 μ

M to about 10 M.

8. The method as defined in claim 1, wherein a maximal solubility of said nucleic acid composition in the modified sugar or sugar alcohol composition in a solution is up to 15 mg/mL.

9. The method as defined in claim 1, wherein said modified sugar or sugar alcohol composition protects said nucleic acid compositions from degradation.

10. The method as defined in claim 1, wherein said modified sugar or sugar alcohol composition enhances the delivery efficiency of said nucleic acid compositions into mammalian cells in vitro as well as in vivo.

11. The method as defined in claim 1, wherein said condition is a pH value below 8.0.

12. The method as defined in claim 11, wherein said condition is a pH value between about pH 2.5 and about pH 7.0.

13. The method as defined in claim 1, wherein said deliver complex is formed by the ionic or electrostatic affinity occurring between the modified sugar or sugar alcohol and the nucleic acid composition.

14. The method as defined in claim 1, wherein said nucleic acid composition is cosmetic, pharmaceutical or therapeutic DNA or RNA.

15. The method as defined in claim 1, wherein said glycylation is a chemical reaction in that at least one hydroxyl group of the sugar or the sugar alcohol is replaced by at least one glycyl group from a glycine or a glycylated amino acid.

16. A method for delivering nucleic acids into cells in vitro as well as in vivo, comprising:

mixing at least a glycerol (glycerin) composition with hydroxyl group, at least one amino acid with a structure of glycyl group, and at least a nucleic acid composition, wherein said mixing forms at least one glycylated glycerol (glycerin) composition which interacts with said nucleic acid composition via ionic or electrostatic affinity and forms a delivery complex at a pH value below 8.0,

wherein said glycylated glycerol (glycerin) composition is formed by a glycylation reaction that replaces the hydroxyl (HO—

) groups of said glycerol (glycerin) composition with glycyl (NH₂CH₂COO—

) groups and thus results in the formation of an ether (R—

O—

R) linkage between each OH-removed carbon of the glycerol (glycerin) composition and the glycyl group and wherein said glycylation reaction occurs under a pH value ranged from 2.5 to 8.0 at a temperature over 45°

C. for at least 30 minutes; and

wherein the optimal concentration of said glycyl group in the glycylation reaction is ranged from 0.05 to 3.0 M for mixing with per 1.0 M of sugar alcohol in the glycylation reaction.

17. The method of claim 16, wherein said amino acid with said structure of glycyl group is glycine or a glycylated amino acid.

18. The method of claim 16, wherein said nucleic acid composition is RNA.

19. The method of claim 18, wherein said nucleic acid composition is small hairpin RNAs (shRNA).

20. The method of claim 16, further comprising mixing 0.3 M fructose and 0.9% (w/v) sodium chloride (NaCl) in a solution form with said glycylated glycerol (glycerin) composition.

21. The method of claim 16, wherein said mixture is further in a solution having a pH value below pH 8.0.

22. The method of claim 21, wherein said pH value is pH 2.5 to pH 7.0.

23. The method of claim 16, wherein said at least one glycylated glycerol (glycerin) composition is selected from the group consisting of monoglycylglycerol, diglycylglycerol and triglycylglyceride.

24. The method of claim 16, wherein a concentration of said glycylated glycerol (glycerin) composition is ranged from about 0.1 μ

M to about 10 M.

25. The method of claim 16, wherein said glycylated glycerol (glycerin) composition is positively charged.

26. The method of claim 16, wherein said nucleic acid composition is negatively charged.

27. The method of claim 16, wherein a maximal solubility of said nucleic acid composition in a solution containing the mixture is up to 15 mg/mL.

28. The method of claim 16, wherein said glycylated glycerol (glycerin) composition protects said nucleic acid compositions from degradation.

29. The method of claim 16, wherein said glycylated glycerol (glycerin) composition enhances the delivery efficiency of said nucleic acid compositions into cells in vitro as well as in vivo.

30. The method of claim 16, wherein said nucleic acid composition in said delivery complex is cosmetic, pharmaceutical and therapeutic DNA or RNA.