Nanolipoprotein particles and related compositions methods and systems for loading RNA

Patent metadata

Field	Value
Publication number	US-12083223-B2
Application number	US-201815969311-A
Country	US
Kind code	B2
Filing date	May 2, 2018
Priority date	May 2, 2017
Publication date	Sep 10, 2024
Grant date	Sep 10, 2024

Abstract

Nanolipoprotein particles comprising at least a scaffold protein component and a membrane lipid component and related complexes, compositions, methods and systems are described, in which the membrane lipid component comprises at least one or more membrane forming lipids and one or more cationic lipids.

First claim

What is claimed is:

1. A method of producing a nanolipoprotein (NLPs)-polynucleotide complex, the method comprising:
contacting a polynucleotide having at least 200 bases with a plurality of cationic nanolipoprotein particles, each cationic nanolipoprotein particle comprising one or more cationic lipids and a membrane forming lipid arranged in a discoidal membrane lipid bilayer stabilized by a scaffold protein, the discoidal membrane lipid bilayer having a diameter from 5 to 25 nm,

the contacting performed to allow binding bewteen the polynucleotide and the nanolipoprotein particles thus providing nanolipoprotein particles loaded with the polynucleotide,
wherein the polynucleotide having at least 200 bases is, an RNA, or an analog or a fragment thereof.

2. The method of claim 1 , further comprising before contacting the polynucleotide with the plurality of nanoparticles,
contacting one or more membrane forming lipids and one or more cationic lipids with one or more scaffold proteins to provide the plurality of nanolipoprotein particles in which the one or more cationic lipids are comprised within a membrane forming lipid bilayer stabilized by the one or more scaffold proteins.

3. The method of claim 2 , wherein the one or more cationic lipids are in a molar concentration of about 1 to about 60 mol %.

4. The method of claim 2 , wherein the one or more cationic lipids are in molar concentration of about 5 to about 40 mol %.

5. The method of claim 2 , wherein a molar percent ratio between a total lipid comprising the one or more membrane forming lipids and the one or more cationic lipid and the scaffold protein ranges from 20:1 to 240:1.

6. The method of claim 2 , wherein the membrane forming lipid is in amount from 99 to 40% and the cationic lipid is in an amount from 1 to 60% with respect to a total lipid concentration.

7. The method of claim 1 , wherein the polynucleotide has a number of bases of at least 5,000 bases.

8. The method of claim 1 , wherein the polynucleotide has a number of bases from about 10,000 bases to about 15,000 bases.

9. The method of claim 1 , wherein the polynucleotide has a number of bases larger than 15,000.

10. The method of claim 1 , wherein the polynucleotide is a RNA replicon.

11. A cationic-nanolipoprotein (NLPs)-polynucleotide complex, comprising
a polynucleotide molecule having at least 200 bases attached to one or more cationic NLPs,
wherein each cationic NLP comprises one or more cationic lipids and a membrane forming lipid arranged in a discoidal membrane lipid bilayer stabilized by a scaffold protein, the discoidal membrane lipid bilayer having a diameter from 5 to 25 nm, and
wherein the polynucleotide is, an RNA, or an analog or a fragment thereof.

12. The cationic NLPs polynucleotide complex of claim 11 , wherein the cationic NLP is a plurality of cationic NLPs.

13. The cationic NLPs polynucleotide complex of claim 11 , wherein the polynucleotide has a number of bases of at least 5,000 bases.

14. The cationic-NLPspolynucleotide-complex of claim 11 wherein the polynucleotide has a number of bases from about 10,000 bases to about 15,000 bases.

15. The cationic-NLPs-polynucleotide-complex of claim 11 , wherein the polynucleotide has a number of bases larger than 15,000.

16. The cationic-NLPs-polynucleotide-complex of claim 11 , wherein the polynucleotide is an RNA replicon.

17. The cationic-NLPs-polynucleotide-complex of claim 11 , further comprising an adjuvant, a functional polymer, a membrane-associated protein for targeted delivery, one or more additive, one or more telodendrimers, or a combination thereof.

18. A method of delivering a polynucleotide to a target environment, the comprising
contacting the target environment with the cationic-nanolipoprotein (NLPs)-polynucleotide complex of claim 11 .

19. The method of claim 18 , wherein the target environment in a cell or a tissue.

20. A method of delivering a polynucleotide to a subject, the method comprising
administering to the subject the cationic nanolipoprotein (NLPs)-polynucleotide complex of claim 11 , to deliver the polynucleotide to the subject, wherein the polynucleotide is a RNA replicon.

21. A method of inducing expression of a protein in a subject, the method comprising
administering to the subject the cationic nanolipoprotein (NLPs)-polynucleotide complex of claim 11 comprising a polynucleotide encoding for the protein to induce expression of the protein in the subject, wherein the polynucleotide is a RNA replicon.

22. A method of stimulating a humoral and a cellular immune response in a subject, the method comprising
administering to the subject the cationic nanolipoprotein (NLPs)-polynucleotide complex of claim 11 comprising a polynucleotide encoding a recombinant antigen stimulating both the humoral and the cellular branch of the immune system of the subject,
wherein the polynucleotide is a RNA replicon.

23. A pharmaceutical composition comprising the cationic-nanolipoprotein-polynucleotide complex of claim 11 and a pharmaceutically acceptable vehicle.

24. A composition comprising the cationic-nanolipoprotein-polynucleotide complex of claim 11 and an acceptable vehicle.

25. The cationic-NLPspolynucleotide-complex of claim 11 , wherein the polynucleotide has a number of bases from 200 to 5,000 bases.

26. The cationic-NLPspolynucleotide-complex of claim 11 , wherein the polynucleotide has a number of bases from 200 to 10,000 bases.

27. The cationic-NLPspolynucleotide-complex of claim 11 , wherein the polynucleotide has a number of bases from 200 to 15,000 bases.

28. The cationic-NLPspolynucleotide-complex of claim 11 , wherein the polynucleotide has a number of bases from 5,000 to 10,000 bases.

29. The cationic-NLPspolynucleotide-complex of claim 11 , wherein the NLPs are at molar ratios ranging from 10 to 500 NLPs per polynucleotide molecule.

30. The cationoc-NLPspolynucleotide-complex of claim 11 , wherein the polynucleotide has a number of bases from 10,000 to 15,000 bases and wherein the polynucleotide and the NLP are at molar ratios ranging from 50 500 NLPs per polynucleotide molecules.

31. The cationic-NLPspolynucleotide-complex of claim 11 , wherein the polynucleotide has a number of bases from 10,000 to 15,000 bases and wherein the polynucleotide and the NLP are molar ratios ranging from 50 to 150 NLPs per RNA molecules.

32. The cationic-NLPspolynucleotide-complex of claim 11 , wherein the polynucleotide has a number of bases from 10,000 to 15,000 bases and wherein the polynucleotide and the NLPs are molar ratios ranging from 10 to 100 NLPs per RNA molecules.

33. The cationic-NLPspolynucleotide-complex of claim 11 , wherein the polynucleotide has a number of bases form 10,000 to 15,000 bases and wherein the polynucleotide and the NLPs are molar ratios ranging from 10 to 25 NLPs per RNA molecules.

34. The cationic-NLPspolynucleotide-complex of claim 11 , wherein the polynucleotide is an RNA encoding a recombinant antigen stimulating both the humoral and the cellular branch of the immune system of the subject.

35. The cationic-NLPspolynucleotide-complex of claim 11 , wherein the cationic lipid is in molar concentration of about 5 to about 40 mol %.

36. The cat

Assignees

• L Livermore Nat Security Llc

Inventors

• Kamrud Kurt I
• Wang Nathaniel S
• Felderman Martina
• Hubby Bolyn
• Gouvis Heather D
• Fischer Nicholas O
• Coleman Matthew A
• Evans Angela Clare
• He Wei
• Rasley Amy
• Blanchette Craig D

Classifications

• A61K47/6917

  the form being a lipoprotein vesicle, e.g. HDL or LDL proteins · CPC title

• C12N15/88

  using microencapsulation, e.g. using {amphiphile} liposome vesicle · CPC title

• A61K9/1274 Primary

  Non-vesicle bilayer structures, e.g. liquid crystals, tubules, cubic phases or cochleates; Sponge phases · CPC title

• A61K48/0008

  characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition · CPC title

• A61K9/1272 Primary

  comprising non-phosphatidyl surfactants as bilayer-forming substances, e.g. cationic lipids or non-phosphatidyl liposomes coated or grafted with polymers (lipids as modifying agents {A61K47/543}) · CPC title