US-20260124319-A1 - NANOPARTICLES COMPRISING P52K PROTEIN AND ONE OR MORE NUCLEIC ACIDS AND, OR PROTEINS OF INTEREST AND METHODS OF USE THEREOF FOR DELIVERY TO CELLS

Abstract

Nanoparticles comprising p52K and nucleic acids and/or proteins of interest which self-assemble to form a p52K nucleic acid and/or p52K protein complexes are disclosed. The nanoparticles find use, for example, in the delivery of the aforementioned nucleic acids and or proteins into cells using a pharmaceutically acceptable carrier.

Inventors

• Matthew J. CHARMAN
• Matthew D. Weitzman

Assignees

• THE CHILDREN'S HOSPITAL OF PHILADELPHIA

Dates

Publication Date

20260507

Application Date

20230724

Claims (20)

1. 1 . A nanoparticle formulation for delivery of one or more heterologous molecules of interest to a cell, comprising a self-assembling p52K protein which forms a heterologous molecule-p52K complex, in a pharmaceutically acceptable carrier.
2. 2 . The nanoparticle formulation of claim 1 , wherein said p52K protein is encoded by a nucleic acid and comprises at least one ITR sequence at the 5′end, the 3′ end or both.
3. 3 . The nanoparticle formulation of claim 1 , wherein said ITR region is present at a) both the 5′ and 3′ ends of the p52K the nucleic acid of interest, or b) the 5′end of the nucleic acid of interest.
4. 4 . (canceled)
5. 5 . The nanoparticle formulation of claim 1 , wherein said 52K protein comprises an IDR region at the N terminus, the C terminus or both, said IDR facilitating self-assembly of said nucleic acid-p52 complex.
6. 6 . The nanoparticle formulation of claim 1 , further comprising protein VII or one or more polypeptides.
7. 7 . (canceled)
8. 8 . The nanoparticle formulation of claim 1 , which is encapsulated in a liposome or micelle.
9. 9 . The nanoparticle formulation of claim 8 , wherein said liposome or micelle further comprises targeting ligands on the surface thereof.
10. 10 . The nanoparticle formulation of claim 8 wherein the liposome or micelle comprises a hydrophobic group selected from the group consisting of lipophilic alkyl groups, cholesterol, and combinations thereof.
11. 11 . The nanoparticle formulation of claim 1 , wherein said nucleic acid of interest encodes a virus genome or a metabolic gene.
12. 12 . (canceled)
13. 13 . The nanoparticle formulation of claim 1 , wherein said nucleic acid of interest encodes an inhibitory RNA molecule selected from an siRNA, an shRNA or a ribozyme.
14. 14 . The nanoparticle formulation of claim 1 , wherein said at least one heterologous molecule includes a nucleic acid of interest encoding a guide RNA suitable for use with CRISPR Cas and a Cas9 enzyme.
15. 15 . The nanoparticle formulation of claim 1 , wherein said nucleic acid of interest is modified to limit recognition by an innate immune response.
16. 16 . The nanoparticle formulation of claim 1 , wherein said nucleic acid of interest expresses a chimeric antigen receptor.
17. 17 . The nanoparticle formulation of claim 1 , wherein said at least one heterologous molecule includes a nucleic acid of interest said guide RNA targets a site for single base editing and an enzyme suitable for use in base editing.
18. 18 . (canceled)
19. 19 . The nanoparticle formulation of claim 1 , wherein said at least one heterologous molecules is a nucleic acid encoding a protein which upon administration to a subject in need thereof, ameliorates symptoms for an indicated disorder listed in Table 2.
20. 20 . A kit comprising a nanoparticle formulation of claim 1 and instructions for use thereof.

Description

CROSS-REFERENCE TO RELATED APPLICATION This application is a § 371 of International Application No. PCT/US2023/028473, filed Jul. 24, 2023, which claims priority to U.S. Provisional Patent Application No. 63/391,374 filed Jul. 22, 2022, the entire contents of each being incorporated herein by reference. GRANT STATEMENT This invention was made with government support under grant numbers R01-AI145266, R01-AI121321 and R01-AI118891 awarded by the National Institute of Allergy and Infectious Diseases. The government has certain rights in the invention. INCORPORATION BY REFERENCE OF MATERIAL SUBMITTED IN ELECTRONIC FORM The Contents of the electronic sequence listing (CHOP-139-US01.xml; Size: 33,040 bytes; and Date of Creation: Jan. 5, 2026) is herein incorporated by reference in its entirety. FIELD OF THE INVENTION This invention relates to the fields of nanoparticle production and delivery of nucleic acids encoding proteins of interest. Specifically, the invention provides nanoparticles comprising p52K, and optionally, other accessory or viral proteins, which self-assemble upon binding to nucleic acids, forming a p52K nucleic acid complex. The nanoparticles find use, for example in the delivery of the nucleic acids and optionally other proteins into cells. BACKGROUND OF THE INVENTION Several publications and patent documents are cited throughout the specification in order to describe the state of the art to which this invention pertains. Each of these citations is incorporated herein by reference as though set forth in full. Biomolecular condensates (BMCs) formed by liquid-liquid phase-separation (LLPS) play fundamental roles in compartmentalizing and regulating cellular processes1,2. During infection, viruses hijack host cells as they replicate and ultimately package genomes into progeny particles for viral spread and transmission. The emerging importance of BMCs as a way of organizing and regulating cellular processes has led to suggestions that viral membrane-less sub-cellular compartments are BMCs, with evidence presented for and against LLPS as the driver of viral BMC formation3-5,7,10. Particular attention has been focused on RNA viruses which replicate in the cytoplasm, such as SARS-CoV-2 and respiratory syncytial virus (RSV)3,4,6,8,11. The importance of BMCs is supported by evidence that condensate hardening drugs can attenuate the spread of RSV infection in vivo6. The ability of specific purified viral proteins to undergo phase-separation in vitro further implicates BMCs in multiple different viral processes, including the compaction of viral RNA genomes that may facilitate packaging3,5,8,9,11. However, progress towards demonstrating definitive mechanistic roles for phase-separation in virus infected cells has ultimately been hampered by the lack of tractable model systems in which phase-separation can be abolished and restored so that its contribution to productive infection can be determined. SUMMARY OF THE INVENTION In accordance with the present invention, a nanoparticle formulation for delivery of at least one heterologous molecule of interest to a cell for expression and or production, therein is provided. An exemplary particle comprises self-assembling p52K protein or functional fragment thereof which forms a complex with said heterologous molecule of interest, forming a molecule-p52K complex, in a pharmaceutically acceptable carrier. In certain aspects, the nanoparticle formulation comprises a p52K protein encoded by a nucleic acid and comprises at least one ITR sequence at the 5′end, the 3′ end or both. In other aspects, the ITR region is present at both the 5′ and 3′ ends of the p52K. The heterologous molecule can be a nucleic acid of interest which can also comprise an ITR region present at the 5′end, the 3′ end or both ends. In certain aspects, the p52K protein comprises an IDR region and the N terminus, the C terminus or both, said IDR facilitating self-assembly of said nucleic acid-p52 complex. The nanoparticle formulations described above can also comprise viral proteins, such as protein VII. The nanoparticle can also contain one or more polypeptides having enzymatic, or metabolic functions. In certain aspects, the nanoparticle formulation is encapsulated in a liposome or micelle. The liposome or micelle can further comprise targeting ligands on the surface thereof. The liposome or micelle can further comprise a hydrophobic group selected from the group consisting of lipophilic alkyl groups, cholesterol, and combinations thereof. The at least one heterologous molecule of interest can include, without limitation, nucleic acid encoding a virus genome, a nucleic acid of interest encodes a metabolic gene, a nucleic acid encoding an inhibitory RNA molecule selected from an siRNA, an shRNA or a ribozyme, a nucleic acid encoding a guide RNA suitable for use with CRISPR Cas and a Cas9 enzyme, a nucleic acid modified to limit recognition by an innate immune response, a nucleic acid which express