Nanoparticle-mediated gene delivery, genomic editing and ligand-targeted modification in various cell populations

• US 10,526,616 B2
• Filed: 09/23/2014
• Issued: 01/07/2020
• Est. Priority Date: 09/23/2013
• Status: Active Grant

• Current Assignee: Rensselaer Polytechnic Institute
• Original Assignee: Rensselaer Polytechnic Institute
• Inventors: Kotha, Shiva Prasad, Watson, Andre Ronald, Pandit, Vaibhav A.
• Primary Examiner(s): Schultz, James D
• Application Number:
  US15/024,264
• Publication Number:
  US 20160230189A1
• Time in Patent Office: 1,932 Days
• Field of Search: None
• CPC Class Codes: A61K 47/6455 Polycationic oligopeptides, polypeptides or polyamino acids, e.g. for complexing nucleic acids
• CPC Class Codes: A61K 47/6923 the form being an inorganic particle, e.g. ceramic particles, silica particles, ferrite or synsorb
• CPC Class Codes: A61K 47/6929 the form being a nanoparticle, e.g. an immuno-nanoparticle
• CPC Class Codes: A61K 48/0041 the non-active part being polymeric
• CPC Class Codes: A61P 19/10 for osteoporosis
• CPC Class Codes: C12N 15/85 for animal cells
• CPC Class Codes: C12N 15/88 using microencapsulation, e.g. using amphiphile liposome vesicle
• CPC Class Codes: C12N 2800/95 Protection of vectors from inactivation by agents such as antibodies or enzymes, e.g. using polymers
• CPC Class Codes: C12N 9/22 Ribonucleases RNAses, DNAses catalytic nucleic acids C12N15/113

Abstract

An improved nanoparticle for transfecting cells is provided. The nanoparticle includes a core polyplex and a silica coating on the core polyplex and, optionally, a polymer attached to an outer surface of the silica coating, where the polyplex includes an anionic polymer, a cationic polymer, a cationic polypeptide, and a polynucleotide. Also provided is an improved method of modifying intracellular polynucleotides. The method includes contacting a cell with a nanoparticle that includes a core polyplex and a silica coating on the core polyplex and, optionally, a polymer attached to an outer surface of the silica coating, where the polyplex includes an anionic polymer, a cationic polymer, a cationic polypeptide, and a polynucleotide.

Claims

1. A nanoparticle comprising:

a core polyplex comprising a cationic polymer, a cationic polypeptide, a polynucleotide, and one or more anionic polymers, wherein the one or more anionic polymers is selected from the group consisting of poly(D-glutamic acid), a glycosaminoglycan, a glycoprotein, a polysaccharide, poly(mannuronic acid), poly(guluronic acid), heparin, heparin sulfate, chondroitin, chondroitin sulfate, keratan, keratan sulfate, aggrecan, poly(glucosamine), and any combination of two or more of the foregoing;

a silica coating on the core polyplex; and

poly(L-arginine), a vasoactive endothelial growth factor peptide, or both attached to an outer surface of said silica coating;

wherein

the cationic polymer and the one or more anionic polymers are not covalently bound to each other.

2. The nanoparticle of claim 1 wherein the anionic polymer is poly(D-glutamic acid).

3. The nanoparticle of claim 1 wherein the cationic polymer is selected from the group consisting of poly(ethylenimine) and poly(L-arginine).

4. The nanoparticle of claim 1 wherein the cationic polypeptide is a histone tail peptide.

5. The nanoparticle of claim 4 wherein the histone tail peptide is human H3 histone tail peptide.

6. The nanoparticle of claim 1 wherein the anionic polymer is poly(D-glutamic acid), the cationic polymer is selected from the group consisting of poly(ethylenimine) and poly(L-arginine), and the cationic polypeptide is a histone tail peptide.

7. The nanoparticle of claim 6 wherein the polynucleotide comprises a nucleotide sequence that encodes a nuclease.

8. The nanoparticle of claim 7 wherein the nuclease is a TALEN.

9. The nanoparticle of claim 8 wherein the TALEN is capable of inducing a break at a site-specific locus of DNA, wherein the break results in a change of expression of a protein encoded by a gene.

10. The nanoparticle of claim 9 wherein the change is a decrease and the gene encodes a sclerostin protein.

11. A nanoparticle comprising:

a core polyplex comprising a cationic polymer, a cationic polypeptide, a polynucleotide, and one or more anionic polymers, wherein the one or more anionic polymers is selected from the group consisting of poly(D-glutamic acid), a glycosaminoglycan, a glycoprotein, a polysaccharide, poly(mannuronic acid), poly(guluronic acid), heparin, heparin sulfate, chondroitin, chondroitin sulfate, keratan, keratan sulfate, aggrecan, poly(glucosamine), and any combination of two or more of the foregoing; and

a silica coating on the core polyplex;

wherein

the cationic polymer and the one or more anionic polymers are not covalently bound to each other.

12. The nanoparticle of claim 11 wherein the anionic polymer is poly(D-glutamic acid).

13. The nanoparticle of claim 11 wherein the cationic polymer is selected from the group consisting of poly(ethylenimine) and poly(L-arginine).

14. The nanoparticle of claim 11 wherein the cationic polypeptide is a histone tail peptide.

15. The nanoparticle of claim 14 wherein the histone tail peptide is human H3 histone tail peptide.

16. The nanoparticle of claim 11 wherein the anionic polymer is poly(D-glutamic acid), the cationic polymer is selected from the group consisting of poly(ethylenimine) and poly(L-arginine), and the cationic polypeptide is a histone tail peptide.

17. The nanoparticle of claim 16 wherein the polynucleotide comprises a nucleotide sequence that encodes a nuclease.

18. The nanoparticle of claim 17 wherein the nuclease is a TALEN.

19. The nanoparticle of claim 18 wherein the TALEN is capable of inducing a break at a site-specific locus of DNA, wherein the break results in a change of expression of a protein encoded by a gene.

20. The nanoparticle of claim 19 wherein the change is a decrease and the gene encodes a sclerostin protein.

21. A nanoparticle of claim 16, further comprising a polymer attached to an outer surface of said silica coating.

22. A nanoparticle of claim 21, wherein said polymer attached to an outer surface of said silica coating comprises one or more of poly(L-arginine) or a vasoactive endothelial growth factor peptide.

23. A nanoparticle comprising:

a core polyplex comprising poly(D-glutamic acid), a cationic polymer comprising poly(ethylenimine) or poly(L-arginine), or both, a polynucleotide, and a histone tail peptide,

a silica coating on the core polyplex, and

poly(L-arginine), a vasoactive endothelial growth factor peptide, or both attached to an outer surface of said silica coating;

wherein

the poly(D-glutamic acid) and the cationic polymer are not covalently bound to each other.