Nucleic acid particles, methods and use thereof

• US 9,737,557 B2
• Filed: 07/28/2015
• Issued: 08/22/2017
• Est. Priority Date: 02/26/2013
• Status: Active Grant

• Current Assignee: Massachusetts Institute of Technology
• Original Assignee: Massachusetts Institute of Technology
• Inventors: Hammond, Paula T., Lee, Jong Bum, Roh, Young Hoon
• Primary Examiner(s): EPPS -SMITH, JANET L
• Application Number:
  US14/811,263
• Publication Number:
  US 20160151404A1
• Time in Patent Office: 756 Days
• Field of Search: 424490, 435 912, 514 44 A
• CPC Class Codes: A61K 31/713 Double-stranded nucleic acids or oligonucleotides
• CPC Class Codes: A61K 45/06 Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
• CPC Class Codes: A61K 9/167 with an outer layer or coating comprising drug; with chemically bound drugs or non-active substances on their surface with further drug-free outer coating A61K9/5073
• CPC Class Codes: A61K 9/50 Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals A61K9/2081 takes precedence; particles with a single coating comprising drug A61K9/167
• CPC Class Codes: A61K 9/5089 Processes
• 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 15/88 using microencapsulation, e.g. using amphiphile liposome vesicle
• CPC Class Codes: C12N 2310/14 interfering N.A.
• CPC Class Codes: C12N 2320/32 Special delivery means, e.g. tissue-specific
• CPC Class Codes: C12P 19/34 Polynucleotides, e.g. nucleic acids, oligoribonucleotides
• CPC Class Codes: C12Q 1/6844 Nucleic acid amplification reactions

Abstract

The present invention provides, among other things, a particle which includes a core comprised of self-assembled one or more nucleic acid molecules, the core being characterized by an ability to adopt at least two configurations: a first configuration having a first greatest dimension greater than 2 μm and; a second configuration having a second greatest dimension less than 500 nm, wherein addition of a film coating converts the core from its first configuration to its second configuration. Methods of making and using of provided particles are also disclosed.

Claims

1. A particle, comprising:

a core, comprising one or more self-assembled nucleic acid molecules in a crystalline structure comprising a lamellar sheet, wherein addition of a film coating to the particle converts the core from a first configuration to a second configuration, wherein the first configuration has a first greatest dimension that is greater than 2 μ

m and;

the second configuration has a second greatest dimension that is less than 500 nm.

2. The particle of claim 1, wherein the core contains a single nucleic acid molecule.

3. The particle of claim 1, wherein the core comprises a plurality of nucleic acid molecules.

4. The particle of claim 3, wherein the nucleic acid molecules have different nucleic acid sequences.

5. The particle of claim 3, wherein all nucleic acid molecules within the core have substantially the same nucleic acid sequence.

6. The particle of claim 3, wherein the nucleic acid molecules within the core have sequences that share at least one common sequence element.

7. The particle of claim 1, wherein at least one nucleic acid molecule within the core has a nucleotide sequence that comprises multiple copies of at least a first sequence element.

8. The particle of claim 1, wherein at least one nucleic acid molecule within the core has a nucleotide sequence that comprises multiple copies of each of at least a first and a second sequence element.

9. The particle of claim 8, wherein the at least one nucleic acid molecule has a nucleotide sequence that comprises alternating copies of the first and second sequence elements.

10. The particle of claim 8, wherein the at least one nucleic acid molecule has a nucleotide sequence that comprises multiple copies of each of three or more sequence elements.

11. The particle of claim 1, wherein at least one nucleic acid molecule has a nucleotide sequence that includes one or more sequence elements found in a natural source.

12. The particle of claim 11, wherein the at least one nucleic acid molecule has a nucleotide sequence that includes a first sequence element that is found in a first natural source and a second sequence element that is found in a second natural source.

13. The particle of claim 12, wherein the first and second natural sources are the same.

14. The particle of claim 12, wherein the first and second natural sources are different.

15. The particle of claim 1, wherein at least one nucleic acid molecule in the core has a nucleotide sequence that represents an assemblage of sequence elements found in one or more source nucleic acid molecules.

16. The particle of claim 15, wherein the at least one nucleic acid molecule has a nucleotide sequence that represents an assemblage of at least two different sequence elements found in two different source nucleic acid molecules.

17. The particle of claim 1, wherein at least a portion of the nucleic acid molecules within a core is cleavable.

18. The particle of claim 1, wherein the nucleic acid molecules within a core comprise single-stranded, double-stranded, triple-stranded nucleic acids or combination thereof.

19. The particle of claim 1, wherein the nucleic acid molecules within a core are arranged in a crystalline structure comprising lamellar sheets.

20. The particle of claim 1, wherein the nucleic acid molecules within a core comprise a stem-loop or linear structure.

21. The particle of claim 1, wherein the core comprises about 1×

10³ to 1×

10⁸ copies of a sequence element.

22. The particle of claim 1, wherein the core comprises at least 1×

10⁶ copies of a sequence element.

23. The particle of claim 1, wherein the nucleic acid molecules have a molecular weight of at least about 1×

10¹⁰ g/mol, about 1×

10⁹ g/mol, about 1×

10⁸ g/mol, about 1×

10⁷ g/mol, about 1×

10⁶ g/mol, or about 1×

10⁵ g/mol.

24. The particle of claim 1, wherein the core has a negative or positive surface charge.

25. The particle of claim 1, further comprising one or more agents for delivery within the core.

26. The particle of claim 25, wherein the agent is a chemotherapeutic agent selected from the group consisting of doxorubicin, carboplatin, cisplatin, cyclophosphamide, docetaxel, erlotinib, etoposide, fluorouracil, gemcitabine, imatinib mesylate, irinotecan, methotrexate, paclitaxel, sorafinib, sunitinib, topotecan, vincristine, and vinblastine.

27. The particle of claim 1, wherein the second greatest dimension of the core is less than 500 nm, less than 200 nm, less than 100 nm, less than 50 nm, less than 20 nm or less than 10 nm.

28. The particle of claim 1, further comprising a film coated on the core, and wherein the core is in the second configuration.

29. The particle of claim 28, wherein the film comprises at least one material selected from the group consisting of an organic material and an inorganic material.

30. The particle of claim 28, wherein the film comprises a polymer.

31. The particle of claim 30, wherein the film comprises a lipid.

32. The particle of claim 28, wherein the film comprises at least one polyelectrolyte layer.

33. The particle of claim 32, wherein the polyelectrolyte layer is degradable or non-degradable.

34. The particle of claim 32, wherein the polyelectrolyte layer is or comprises a polycation or polyanion.

35. The particle of claim 34, wherein the polycation is one or more member of the group consisting of polyethylenimine, poly(L-lysine) (PLL), and poly(lactic acid) (PLA).

36. The particle of claim 28, wherein the film comprises a layer-by-layer (LBL) film.

37. The particle of claim 36, wherein the LBL film comprises multiple polyelectrolyte layers.

38. The particle of claim 37, wherein the LBL film comprises multiple polyelectrolyte layers of alternating charges.

39. The particle of claim 28, wherein the film further comprises one or more agents.

40. The particle of claim 28, wherein the particle has a surface charge.

41. A method for forming the particle of claim 1 comprising:

assembling one or more nucleic acid molecules into a core with a crystalline structure comprising lamellar sheets.

42. A method for forming the particle of claim 1 comprising:

assembling one or more nucleic acid molecules into a core, wherein the core has a first greatest dimension greater than 2 pm, and coating the core with a film, wherein the coated core has a second greatest dimension less than 500 nm.

43. The method of claim 42, further comprising forming the nucleic acid molecules via rolling circle amplification (RCA), rolling circle transcription (RCT) or both.

44. The method of claim 43, wherein the step of forming comprises using a circular nucleic acid template.

45. The method of claim 44, wherein the step of forming comprises hybridizing the circular nucleic acid template with a primer.

46. The method of claim 45, wherein the primer is complementary to a portion of the circular nucleic acid template.

47. The method of claim 44, wherein the step of forming further comprises amplifying the circular nucleic acid template using an enzyme.

48. The method of claim 47, wherein the enzyme is Φ

29 DNA polymerase, T7 polymerase or both.

49. The method of claim 42, wherein the step of coating comprises mixing the core in a coating solution.

50. The method of claim 49, wherein the coating solution comprises polyethylenimine.

51. The method of claim 42, wherein the step of coating further comprises sequentially assembling additional layers.