Polynucleotide nanoparticles for the modulation of gene expression and uses thereof

• US 10,731,157 B2
• Filed: 02/23/2018
• Issued: 08/04/2020
• Est. Priority Date: 08/24/2015
• Status: Active Grant

• Current Assignee: Halo-Bio Rnai Therapeutics, Inc.
• Original Assignee: Halo-Bio Rnai Therapeutics, Inc.
• Inventors: Hauser, Todd M.
• Primary Examiner(s): Long, Scott
• Application Number:
  US15/904,224
• Publication Number:
  US 20190055555A1
• Time in Patent Office: 893 Days
• CPC Class Codes: A61K 9/14 Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles microspheres A61K9/16; microcapsules A61K9/50; nanocapsules, nanoparticles of the matrix type A61K9/51
• CPC Class Codes: A61P 35/00 Antineoplastic agents
• 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 15/82 for plant cells , e.g. plant artificial chromosomes (PACs)
• CPC Class Codes: C12N 2310/14 interfering N.A.
• CPC Class Codes: C12N 2310/16 Aptamers
• CPC Class Codes: C12N 2310/351 Conjugate
• CPC Class Codes: C12N 2310/51 in polymeric form, e.g. multimers, concatemers
• CPC Class Codes: C12N 2310/52 branched

Abstract

The present invention is directed to novel self-forming polynucleotide nanoparticles, and the use of such nanoparticles and compositions comprising the same for gene modulation in a variety of organisms.

Claims

1. An isolated polynucleotide nanoparticle comprising a contiguous polynucleotide comprising two or more MV-RNA sequences, each MV-RNA sequence joined by at least one linkage nucleotide,

wherein the contiguous polynucleotide is self-forming into the nanoparticle having a diameter of approximately 40-100 nm with approximately twice the ratio of stems near the surface of the nanoparticle than at the core of the nanoparticle.

3. The isolated polynucleotide nanoparticle of claim 1, wherein the polynucleotide nanoparticle comprises aptamer or cellular uptake sequences on one or more surface loops.

4. The isolated polynucleotide nanoparticle of claim 1, wherein the polynucleotide nanoparticle has a general structure set forth in any one of FIG. 1 (2^nd to 5^th graphics counting from left to right), FIG. 2 (2^nd to 6^th graphics counting from left to right), and FIG. 3.

5. The isolated polynucleotide nanoparticle of claim 1, wherein (i) the 5′

end of the contiguous polynucleotide hybridizes to a reverse complement sequence of the 3′

end of the contiguous polynucleotide to form a stem or (ii) the first MV-RNA in the nanoparticle comprises both the 5′

end and the 3′

end of the contiguous polynucleotide to close the nanoparticle.

6. The isolated polynucleotide nanoparticle of claim 1, wherein the at least one linkage nucleotide comprises:

(i) a stem-loop structure or (ii) dinucleotide or (iii) mononucleotide.

7. The isolated polynucleotide nanoparticle of claim 1, wherein the polynucleotide nanoparticle comprises natural or synthetic RNA or DNA.

8. The isolated polynucleotide nanoparticle of claim 1, wherein the at least one linkage nucleotide is 1-12 nucleotides.

9. The isolated polynucleotide nanoparticle of claim 1, wherein the polynucleotide nanoparticle is without a 5′

phosphate end or 3′

hydroxyl terminus.

10. The isolated polynucleotide nanoparticle of claim 1, wherein the polynucleotide nanoparticle is expressed within a host cell selected from a plant cell or human cell or yeast cell or bacterial cell, or in vitro transcription.

11. The isolated polynucleotide nanoparticle of claim 1, wherein the polynucleotide nanoparticle is expressed within a host cell and targets genes other than the host.

12. A composition comprising the isolated polynucleotide nanoparticle of claim 1, in combination with a physiologically acceptable excipient.

2. An isolated polynucleotide nanoparticle approximately 100 nm diameter with at least four times the ratio of stems on the outer surface than at the center, comprising stacked sets of three MV-RNA, wherein each stacked MV-RNA set is separated by a linking element.

13. The isolated polynucleotide nanoparticle of claim 2, wherein the polynucleotide nanoparticle contains aptamer or cellular uptake sequences on one or more surface loops.

14. The isolated polynucleotide nanoparticle of claim 2, wherein the linkage element comprises:

(i) a stem-loop structure or (ii) dinucleotide or (iii) mononucleotide cleavable by an endonuclease.

15. The isolated polynucleotide nanoparticle of claim 2, wherein the polynucleotide nanoparticle comprises natural or synthetic RNA or DNA.

16. The isolated polynucleotide nanoparticle of claim 2, wherein the linkage element sequence is 3-12 nucleotides.

17. The isolated polynucleotide nanoparticle of claim 2, wherein the polynucleotide nanoparticle is a single polynucleotide nanoparticle circularized with ribozyme cleavage.

18. The isolated polynucleotide nanoparticle of claim 2, wherein the polynucleotide nanoparticle is expressed within a host cell selected from a plant cell or human cell or yeast cell or bacterial cell, or in vitro transcription.

19. The isolated polynucleotide nanoparticle of claim 2, wherein the polynucleotide nanoparticle is expressed within a host cell and targets genes other than the host.

20. A composition comprising the isolated polynucleotide nanoparticle of claim 2, in combination with a physiologically acceptable excipient.

21. The isolated polynucleotide nanoparticle of claim 2, wherein the polynucleotide is single-stranded and the nanoparticle is self-forming.