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US-12616662-B2 - Lipid particles for nucleic acid delivery and clinical applications of same

US12616662B2US 12616662 B2US12616662 B2US 12616662B2US-12616662-B2

Abstract

Lipid particles for nucleic acid delivery and clinical applications of same are provided. Accordingly there is provided a lipid particle comprising a cationic lipid encapsulating a nucleic acid sequence, wherein said nucleic acid sequence encodes a protein having a length of at least 500 amino acids, the cationic lipid being represented by Formula I, as defined in the specification.

Inventors

  • Dan Peer
  • Daniel ROSENBLUM
  • Anna GUTKIN

Assignees

  • RAMOT AT TEL-AVIV UNIVERSITY LTD.

Dates

Publication Date
20260505
Application Date
20221117

Claims (12)

  1. 1 . A lipid particle comprising a cationic lipid encapsulating a nucleic acid sequence, wherein said nucleic acid sequence encodes a protein having a length of at least 500 amino acids, the cationic lipid being represented by Formula I: wherein: m is 0 or 1 A1 and A2 are each independently a saturated or unsaturated linear, non-branched, alkylene chain, of at least 8 carbon atoms in length; L1 is a first linking group which an alkylene of 1 to 4 carbon atoms in length; X is —O—C(═O)—or —NH—C(═O); L2 is a second linking group which is an alkylene of 1 to 4 carbon atoms in length; and R1 and R2 are each independently hydrogen, alkyl or cycloalkyl, or, alternatively, R1 and R2 form together with the nitrogen to which they are attached a heteroalicyclic ring, provided that when X is —O—C(═O)—, m is 1.
  2. 2 . A method of preparing a lipid particle for delivery of a nucleic acid sequence, the method comprising encapsulating a nucleic acid sequence in a lipid particle comprising a cationic lipid represented by Formula I: wherein: m is 0 or 1 A1 and A2 are each independently a saturated or unsaturated linear, non-branched, alkylene chain, of at least 8 carbon atoms in length; L1 is a first linking group which an alkylene of 1 to 4 carbon atoms in length; X is —O—C(—O)—or —NH—C(═O); L2 is a second linking group which is an alkylene of 1 to 4 carbon atoms in length; and R1 and R2 are each independently hydrogen, alkyl or cycloalkyl, or, alternatively, R1 and R2 form together with the nitrogen to which they are attached a heteroalicyclic ring, provided that when X is —O—C(═O)—, m is 1, wherein said nucleic acid sequence encodes a protein having a length of at least 500 amino acids.
  3. 3 . The lipid particle of claim 1 , wherein said lipid particle comprises a targeting moiety.
  4. 4 . The method of claim 2 , comprising attaching to said lipid particle a targeting moiety.
  5. 5 . The lipid particle of claim 1 , wherein said nucleic acid sequence is an mRNA.
  6. 6 . The lipid particle of claim 1 , wherein said protein is an enzyme.
  7. 7 . The lipid particle of claim 6 , wherein said enzyme is a genome editing endonuclease.
  8. 8 . The lipid particle of claim 7 , wherein said genome editing endonuclease is CRISPR-associated endonuclease.
  9. 9 . The lipid particle of claim 7 , wherein said lipid particle further encapsulates a nucleic acid sequence guiding said genome editing endonuclease to a gene of interest.
  10. 10 . The lipid particle of claim 9 , wherein said gene of interest is PLK1.
  11. 11 . The lipid particle of claim 9 , wherein said nucleic acid sequence guiding said genome editing endonuclease to said gene of interest is a CRISPR system gRNA.
  12. 12 . The lipid particle of claim 1 , wherein said particle is a nanoparticle having a size of 30-150 nm.

Description

RELATED APPLICATIONS This application is a Continuation (CON) of PCT Patent Application No. PCT/IL2021/050571 having International filing date of May 19, 2021, which claims the benefit of priority under 35 USC § 119(e) of U.S. Provisional Patent Application No. 63/026,785 filed on May 19, 2020. The contents of the above applications are all incorporated by reference as if fully set forth herein in their entirety. SEQUENCE LISTING STATEMENT The XML file, entitled 94609SequenceListing.xml, created on Nov. 17, 2022, comprising 11,460 bytes, submitted concurrently with the filing of this application is incorporated herein by reference. FIELD AND BACKGROUND OF THE INVENTION The present invention, in some embodiments thereof, relates to lipid particles for nucleic acid delivery and clinical applications of same. Polynucleotide- or protein-based therapies are limited by the difficult cellular internalization of these macromolecules. Several delivery systems have been developed and proposed to improve cell permeability of these molecular therapies. Examples of these systems range from cell electroporation to viral delivery, the use of lipid particles (e.g. liposomes, lipid nanoparticles), inorganic compounds (cationic polymers, nanotubes, nanoparticles) and cell-penetrating peptides (CPPs). Genome editing is a powerful tool that can be used to induce targeted mutagenesis, deletions or insertions of cellular DNA sequences, and facilitate recombination at a predetermined genetic sequences within a genome; and has numerous therapeutic and biotechnological applications. Current genome editing agents include, for example, meganucleases, engineered zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and CRISPR/Cas system. However, delivering functional genome editing agents to a cell can be problematic. For example, due to the large size of the CRISPR/Cas system Cas9 nuclease (160 kDa, 4300b), its encapsulation, cell delivery and translation using both viral and non-viral delivery systems remains a challenge. Most in-vivo studies of gene editing have relied on adeno-associated virus (AAV) to deliver CRISPR/Cas components locally or to the liver. Nevertheless, AAV applications are limited by the virus small carrying capacity, immune responses and current lack of targeting [e.g. Senís, E. et al. (2014). Biotechnology Journal 9, 1402-1412]. Lipid nanoparticles (LNPs) are the only clinically approved non-viral delivery system for nucleic acids. These LNPs, based on ionizable lipids, are gaining much attention in the field of RNA therapeutics. However, LNPs formulations that were optimized for siRNA do not efficiently deliver large nucleic acids sequences that should be translated into functional proteins (e.g., mRNAs, plasmids) [see e.g. Tam, Y. K., et al. (2016) Journal of Drug Targeting 24, 774-779, Oberli, M. A. et al. (2017) Nano Letters 17, 1326-1335]. Additional Background art includes: Ramishetti S. et al. (2020) Adv Mater. January 30:e1906128; andInternational Patent Application Publication Nos. WO2016/189532, WO2018/015881 and WO2018/087753. SUMMARY OF THE INVENTION According to an aspect of some embodiments of the present invention there is provided a lipid particle comprising a cationic lipid encapsulating a nucleic acid sequence, wherein the nucleic acid sequence encodes a protein having a length of at least 500 amino acids, the cationic lipid being represented by Formula I: wherein: m is 0 or 1 A1 and A2 are each independently a saturated or unsaturated linear, non-branched, alkylene chain, of at least 8 carbon atoms in length; L1 is a first linking group which an alkylene of 1 to 4 carbon atoms in length; X is —O—C(═O)— or —NH—C(═O); L2 is a second linking group which is an alkylene of 1 to 4 carbon atoms in length; and R1 and R2 are each independently hydrogen, alkyl or cycloalkyl, or, alternatively, R1 and R2 form together with the nitrogen to which they are attached a heteroalicyclic ring, provided that when X is —O—C(═O)—, m is 1. According to an aspect of some embodiments of the present invention there is provided a method of preparing a lipid particle for delivery of a nucleic acid sequence, the method comprising encapsulating a nucleic acid sequence in a lipid particle comprising a cationic lipid represented by Formula I: wherein: m is 0 or 1 A1 and A2 are each independently a saturated or unsaturated linear, non-branched, alkylene chain, of at least 8 carbon atoms in length; L1 is a first linking group which an alkylene of 1 to 4 carbon atoms in length; X is —O—C(═O)— or —NH—C(═O); L2 is a second linking group which is an alkylene of 1 to 4 carbon atoms in length; and R1 and R2 are each independently hydrogen, alkyl or cycloalkyl, or, alternatively, R1 and R2 form together with the nitrogen to which they are attached a heteroalicyclic ring, provided that when X is —O—C(═O)—, m is 1, wherein the nucleic acid sequence encodes a protein having a length of at least 500 ami