Search

US-12622981-B2 - Immolative cell-penetrating complexes for nucleic acid delivery

US12622981B2US 12622981 B2US12622981 B2US 12622981B2US-12622981-B2

Abstract

There are provided herein, inter alia, complexes, compositions and methods for the delivery of therapeutic, diagnostic and imaging agents, including nucleic acid, into a cell. The complexes, compositions and methods may facilitate complexation, protection, delivery and release of oligonucleotides and polyanionic cargos into target cells, tissues, and organs both in vitro and in vivo.

Inventors

  • Robert M Waymouth
  • Paul Wender
  • Jessica R. Vargas
  • Tim R. Blake
  • Colin J. McKinlay
  • Ronald Levy
  • Ole Audun Werner Haabeth
  • Nancy Benner
  • Katherine Near

Assignees

  • THE BOARD OF TRUSTEES OF THE LELAND STANFORD JUNIOR UNIVERSITY

Dates

Publication Date
20260512
Application Date
20240312

Claims (20)

  1. 1 . A method of transfecting a nucleic acid into a cell in vivo, the method comprising contacting the cell in vivo with a cationic amphipathic polymer non-covalently bound to a nucleic acid, the cationic amphipathic polymer having the formula: wherein Ring A is a substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl; z5 is 1-10 CART has the formula: -L 1 -[(LP 1 ) z1 -(IM) z2 -(LP 2 ) z3 ] z4 -L 2 -R 2A wherein L 1 and L 2 are independently a bond, —C(O)O—, —O—, —S—, —NH—, -C(O)NH—, —NHC(O)—, —S(O) 2 —, —S(O)NH—, —NHC(O)NH—, substituted or unsubstituted C 1 -C 20 alkylene or heteroalkylene, substituted or unsubstituted C 3 -C 8 cycloalkylene or heterocycloalkylene, substituted or unsubstituted C 6 -C 10 arylene or heteroarylene; LP 1 and LP 2 are independently a bond or a lipophilic polymer domain, wherein at least one of LP 1 and LP 2 is the lipophilic polymer domain, wherein the lipophilic polymer domain has the formula: and wherein R is independently hydrogen, unbranched C 1 -C 30 alkyl, which may be fully saturated, mono- or polyunsaturated, or cholesterol; IM is a pH-sensitive immolation domain of formula (1), (2), or (3): z1 and z3 are independently 0 to 30 and at least one of z1 and z3 is not 0; z4 is 1 to 100; and z2 is 2 to 50.
  2. 2 . The method of claim 1 , wherein z1 or z3 is 0.
  3. 3 . The method of claim 2 , wherein R is independently stearyl, oleyl, linoleyl, dodecyl, or nonenyl.
  4. 4 . The method of claim 3 , wherein Ring A is a substituted or unsubstituted aryl, optionally wherein the aryl is benzyl, phenyl or naphthalenyl.
  5. 5 . The method of claim 4 , wherein L 1 is substituted or unsubstituted C 1 -C 20 alkylene, substituted or unsubstituted C 1 -C 20 heteroalkylene, substituted or unsubstituted C 3 -C 8 cycloalkylene, substituted or unsubstituted C 3 -C 8 heterocycloalkylene, substituted or unsubstituted C 6 -C 10 arylene, or substituted or unsubstituted C 6 -C 10 heteroarylene.
  6. 6 . The method of claim 5 , wherein L 1 is
  7. 7 . The method of claim 1 , wherein the nucleic acid is RNA or DNA.
  8. 8 . The method of claim 7 , wherein the nucleic acid is messenger RNA, small interference RNA, short hairpin RNA, micro RNA, guide RNA, CRISPR RNA, transactivating RNA, plasmid DNA, minicircle DNA, or genomic DNA.
  9. 9 . The method of claim 8 , wherein the cell is a mammalian cell or a human cell.
  10. 10 . A cationic amphipathic polymer of the formula: wherein Ring A is a substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl; z5 is 1-10 CART has the formula: -L 1 -[(LP 1 ) z1 -(LP 3 ) z1a -(IM) z2 -(LP 2 ) z3 -(LP 4 ) z3b ] z4 -L 2 -H wherein, L 1 and L 2 are independently a bond, —C(O)O—, —O—, —S—, —NH—, —C(O) NH—, —NHC(O)—, —S(O) 2 —, —S(O)NH—, —NHC(O)NH—, substituted or unsubstituted C 1 -C 20 alkylene or heteroalkylene, substituted or unsubstituted C 3 -C 8 cycloalkylene or heterocycloalkylene, substituted or unsubstituted C 6 -C 10 arylene or heteroarylene; LP 1 , LP 2 , LP 3 and LP 4 are independently a bond or a lipophilic polymer domain, wherein at least two of LP 1 , LP 2 , LP 3 and LP 4 are a lipophilic polymer domain; z1, z1a, z3, and z3b are independently 0 to 100, wherein at least two of z1, z1a, z3, and z3b are not 0; z4 is 2 to 100; and z2 is 2 to 100; wherein the lipophilic polymer domain has the formula: wherein R is unbranched C 1 -C 30 alkyl, which may be fully saturated, monounsaturated, polyunsaturated, or cholesterol; and wherein IM is a pH-sensitive immolation domain of formula (1), (2), or (3):
  11. 11 . The cationic amphipathic polymer of claim 10 , wherein z3 and z3b are each 0.
  12. 12 . The cationic amphipathic polymer of claim 11 , wherein LP 1 and LP 3 are each the lipophilic polymer domain.
  13. 13 . The cationic amphipathic polymer of claim 12 , wherein each R is independently stearyl, oleyl, linoleyl, dodecyl, or nonenyl.
  14. 14 . The cationic amphipathic polymer of claim 13 , wherein Ring A is a substituted or unsubstituted aryl, optionally wherein the aryl is benzyl, phenyl or naphthalenyl.
  15. 15 . The cationic amphipathic polymer of claim 14 , wherein L 1 is substituted or unsubstituted C 1 -C 20 alkylene, substituted or unsubstituted C 1 -C 20 heteroalkylene, substituted or unsubstituted C 3 -C 8 cycloalkylene, substituted or unsubstituted C 3 -C 8 heterocycloalkylene, substituted or unsubstituted C 6 -C 10 arylene, or substituted or unsubstituted C 6 -C 10 heteroarylene.
  16. 16 . The cationic amphipathic polymer of claim 15 , wherein L 1 is
  17. 17 . A complex comprising the cationic amphipathic polymer of claim 10 noncovalently bound to a nucleic acid.
  18. 18 . The complex of claim 17 , wherein the nucleic acid is RNA or DNA.
  19. 19 . The complex of claim 17 , wherein the nucleic acid is messenger RNA, small interference RNA, short hairpin RNA, micro RNA, guide RNA, CRISPR RNA, transactivating RNA, plasmid DNA, minicircle DNA, or genomic DNA.
  20. 20 . A pharmaceutical composition comprising the complex of claim 17 , and a pharmaceutically acceptable excipient.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS This application is a continuation of U.S. patent application Ser. No. 15/662,190, filed on Jul. 27, 2017, which claims the benefit of priority under 35 U.S.C. § 119 to U.S. Provisional Application No. 62/367,555, filed Jul. 27, 2016, the disclosures of which are hereby incorporated by reference herein in their entireties and for all purposes. STATEMENT AS TO RIGHTS TO DISCLOSURES MADE UNDER FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT This invention was made with Government support under contract DE-SC0005430 awarded by the Department of Energy, under contract 1306730 awarded by the National Science Foundation and under contracts CA031841 and CA031845 awarded by the National Institutes of Health. The Government has certain rights in the invention. REFERENCE TO A “SEQUENCE LISTING,” A TABLE, OR A COMPUTER PROGRAM LISTING APPENDIX SUBMITTED AS AN ASCII FILE The present application contains a sequence listing entitled 6045-0521C_Sequence Listing.xml, created on Mar. 12, 2024, and is 2.67 kilobytes in size. The sequence listing is submitted electronically along with the filing of the present application and is hereby incorporated by reference in its entirety. BACKGROUND There is a need for new materials and strategies that enable or enhance the delivery of therapeutic agents, diagnostic probes and/or research tools across the plasma membrane of cells and other biological barriers, as required for a wide range of clinical, diagnostic and/or research applications. The delivery of such cargo, e.g., nuclei acids, has considerable clinical potential in connection with vaccination strategies for infectious diseases, cancer immunotherapy, protein therapy and gene editing. Provided herein are solutions to these and other problems in the art. BRIEF SUMMARY In a first aspect, there is provided a cell-penetrating complex including a nucleic acid non-covalently bound to a cationic amphipathic polymer, the cationic amphipathic polymer including a pH-sensitive immolation domain. In another aspect, there is provided a nanoparticle composition including a plurality of cell-penetrating complexes as disclosed herein. In another aspect, there is provided a cationic amphipathic polymer of the formula: H-L1-[(LP1)z1-(IM)z2-(LP2)z3]z4-L2-H (I), wherein: L1 and L2 are independently a bond, —C(O)O—, —O—, —S—, —NH—, —C(O)NH—, —NHC(O)—, —S(O)2—, —S(O)NH—, substituted or unsubstituted alkylene, substituted or unsubstituted heteroalkylene, substituted or unsubstituted cycloalkylene, substituted or unsubstituted heterocycloalkylene, substituted or unsubstituted arylene, or substituted or unsubstituted heteroarylene; LP1 and LP2 are independently a bond or a lipophilic polymer domain, wherein at least one of LP1 or LP2 is a lipophilic polymer domain; IM is the pH-sensitive immolation domain; z1, 23 and z4 are independently integers from 0 to 100, wherein at least one of z1 or z3 is not 0; and z2 is an integer from 2 to 100. In embodiments, L1 is substituted or unsubstituted alkylene. In embodiments, L1 is peptide (e.g., an amino acid sequence). In another aspect, there is provided a cationic amphipathic polymer of the formula: H-L1-[(LP1)z1-(LP3)z1a-(IM)z2-(LP2)z3-(LP4)z3b]z4-L2-H (I), wherein: L1 and L2 are independently a bond, —C(O)O—, —O—, —S—, —NH—, —C(O)NH—, —NHC(O)—, —S(O)2—, —S(O)NH—, substituted or unsubstituted alkylene, substituted or unsubstituted heteroalkylene, substituted or unsubstituted cycloalkylene, substituted or unsubstituted heterocycloalkylene, substituted or unsubstituted arylene, or substituted or unsubstituted heteroarylene; LP1, LP2, LP3 and LP4 are independently a bond or a lipophilic polymer domain, wherein at least one of LP1, LP2, LP3 and LP4 is a lipophilic polymer domain; IM is the pH-sensitive immolation domain; z1, z1a, z3, z3b and z4 are independently integers from 0 to 100, wherein at least one of z1 or z3 is not 0; and z2 is an integer from 2 to 100. In embodiments, L1 is substituted or unsubstituted alkylene. In embodiments, L1 is peptide (e.g., an amino acid sequence). In another aspect, there is provided a method of transfecting a nucleic acid into a cell, the method including contacting a cell with the complex as disclosed herein. In another aspect, there is provided a cationic amphipathic polymer of the formula: R1A-[L1-[(LP1)z1-(IM)z2-(LP2)z3]z4-L2-R2A]z5, wherein R1A is hydrogen, halogen, —CCl3, —CBr3, —CF3, —CI3, CHCl2, —CHBr2, —CHF2, —CHI2, —CH2Cl, —CH2Br, —CH2F, —CH2I, —CN, —OH, —NH2, —COOH, —CONH2, —NO2, —SH, —SO3H, —SO4H, —SO2NH2, —NHNH2, —ONH2, —NHC(O)NHNH2, —NHC(O)NH2, —NHSO2H, —NHC(O)H, —NHC(O)OH, —NHOH, —OCCl3, —OCF3, —OCBr3, —OCI3, —OCHCl2, —OCHBr2, —OCHI2, —OCHF2, —OCH2Cl, —OCH2Br, —OCH2I, —OCH2F, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl, R2A is