WO2024257088A1 - Lipides de carbamate biodégradables présentant une stabilité accrue - Google Patents

Lipides de carbamate biodégradables présentant une stabilité accrue Download PDF

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WO2024257088A1
WO2024257088A1 PCT/IL2024/050567 IL2024050567W WO2024257088A1 WO 2024257088 A1 WO2024257088 A1 WO 2024257088A1 IL 2024050567 W IL2024050567 W IL 2024050567W WO 2024257088 A1 WO2024257088 A1 WO 2024257088A1
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lipid
alkyl
alkylene
alkenyl
group
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Roi MASHIACH
Anjaiah AITHA
Dan Peer
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Ramot at Tel Aviv University Ltd
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Ramot at Tel Aviv University Ltd
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Priority to IL325271A priority Critical patent/IL325271A/en
Priority to AU2024302998A priority patent/AU2024302998A1/en
Priority to CN202480051966.XA priority patent/CN121729406A/zh
Priority to KR1020267000768A priority patent/KR20260048536A/ko
Priority to EP24822968.4A priority patent/EP4727917A1/fr
Publication of WO2024257088A1 publication Critical patent/WO2024257088A1/fr
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/0008—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition
    • A61K48/0025—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition wherein the non-active part clearly interacts with the delivered nucleic acid
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C271/00—Derivatives of carbamic acids, i.e. compounds containing any of the groups, the nitrogen atom not being part of nitro or nitroso groups
    • C07C271/06—Esters of carbamic acids
    • C07C271/08—Esters of carbamic acids having oxygen atoms of carbamate groups bound to acyclic carbon atoms
    • C07C271/10—Esters of carbamic acids having oxygen atoms of carbamate groups bound to acyclic carbon atoms with the nitrogen atoms of the carbamate groups bound to hydrogen atoms or to acyclic carbon atoms
    • C07C271/20—Esters of carbamic acids having oxygen atoms of carbamate groups bound to acyclic carbon atoms with the nitrogen atoms of the carbamate groups bound to hydrogen atoms or to acyclic carbon atoms to carbon atoms of hydrocarbon radicals substituted by nitrogen atoms not being part of nitro or nitroso groups
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C12N15/88—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation using microencapsulation, e.g. using amphiphile liposome vesicle
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C12N15/90—Stable introduction of foreign DNA into chromosome
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/0008—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition
    • A61K48/0025—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition wherein the non-active part clearly interacts with the delivered nucleic acid
    • A61K48/0041—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition wherein the non-active part clearly interacts with the delivered nucleic acid the non-active part being polymeric
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/111—General methods applicable to biologically active non-coding nucleic acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/20—Type of nucleic acid involving clustered regularly interspaced short palindromic repeats [CRISPR]

Definitions

  • the present invention provides lipids and lipid nanoparticle formulations comprising these lipids, alone or in combination with other lipids. These lipid nanoparticles may be formulated with nucleic acids to facilitate their intracellular delivery both in vitro and for in vivo therapeutic applications.
  • the lipids of the present invention are characterized as particularly hydrolytically and alcoholytically stable, while maintaining their biological activity.
  • Therapeutic nucleic acids including small interfering RNA (siRNA), micro RNA (miRNA), antisense oligonucleotides, messenger RNA (mRNA), ribozymes, pDNA and immune stimulating nucleic acids act via a variety of mechanisms. Specific proteins can be downregulated by siRNA or miRNA through RNA interference (RNAi). Hematopoietic cells, such as leukocytes in general, and primary T lymphocytes and B-cells in particular, are notoriously hard to transfect with small interfering RNAs (siRNAs).
  • RNA interference RNA interference
  • siRNA and miRNA constructs can be synthesized with any nucleotide sequence directed against a target protein.
  • siRNA constructs have shown the ability to specially silence target proteins in both in vitro and in vivo models. These are currently being evaluated in clinical studies.
  • RNA messenger RNA
  • mRNA is the family of large RNA molecules which transport the genetic information from DNA to ribosome.
  • Some nucleic acids such as mRNA or plasmids, can be used to effect expression of specific cellular products. Such nucleic acids would be useful in the treatment to the of diseases related deficiency of a protein or enzyme.
  • problems associated with nucleic acids are the stability of the phosphodiester inter nucleotide link and its susceptibility to nucleases. Apart from that these nucleic acids have limited ability to cross the cell membrane.
  • lipids e.g., cationic lipids
  • cationic lipids have proved to be excellent carriers of nucleic acids to treat different diseases in gene therapy applications.
  • Lipid nanoparticles formed from cationic lipids and other co-lipids such as cholesterol, DSPC and PEGylated lipids encapsulated oligonucleotides which protect them from degradation and facilitate the cellular uptake.
  • WO 2014007398 discloses a cationic lipid that facilitates introduction of a nucleic acid into a cell, wherein the nucleic acid has an activity of suppressing the expression of a target gene by utilizing RNA interference (RNAi).
  • RNAi RNA interference
  • WO 2022168085 discloses lipids for lipid nanoparticles (LNPs) preparation. These lipid nanoparticles protect nucleic acids from degradation and/or clearance from circulation and intracellular release.
  • Biodegradable lipids and nanoparticles containing the same show reverse correlation between lipid and particle stability and their activity in delivering active agents, such as nucleic acids to the target sites. Once the stability reaches a certain threshold, they become inactive as vesicles. The challenge to balance between stability and degradability is one of the key limitations of such nanocarriers.
  • the present invention relates to novel lipids which can be used in lipid nanoparticle preparation. These lipid nanoparticles protect nucleic acids from degradation, clearance from circulation and intracellular release.
  • the nucleic acid encapsulated lipid nanoparticles advantageously are well-tolerated and provide an adequate therapeutic index, such that patient treatment at an effective dose of the nucleic acid is not associated with unacceptable toxicity and/or risk to the patient.
  • the present invention also provides the methods of chemical synthesis of these lipids, lipid nanoparticle preparation and formulations with nucleic acids.
  • the present invention relates to novel lipids, and formulations of such lipids with siRNA and pDNA.
  • the lipids of the present invention include a carbamate moiety, and were found to have increased hydrolytic and alcoholytic stability from degradation compared to counterparts, which lack the carbamate moiety. Moreover, it was surprisingly found that the present carbamate lipids, despite the increase in stability do not suffer a decrease in their biological activity. Specifically, in the typical case in the art, once the stability of lipids within an LNP reaches a certain threshold, they become inactive in delivering agents, such as RNA, to various organs. It was surprisingly found that lipids according to the present invention can overcome this stability-activity barrier.
  • lipid represented by the structure of Formula (V) or a salt thereof: each one of n and m is individually selected from the group consisting of: 1, 2, 3, 4 and 5;
  • R 1 is selected from the group consisting of: C 3-9 alkylene-CO 2 -C 8-18 alkyl, C 3-9 alkylene- CO 2 -C 8-18 alkenyl, C 3-9 alkenylene-CO 2 -C 8-18 alkyl, C 3-9 alkenylene-CO 2 -C 8-18 alkenyl, C 3-9 alkylene-O 2 C-C 8-18 alkyl, C 3-9 alkylene-O 2 C-C 8-18 alkenyl, C 3-9 alkenylene-O 2 C-C 8- 18 alkyl and C 3-9 alkenylene-O 2 C-C 8-18 alkenyl;
  • R 2 is selected from the group consisting of: C 10-20 alkyl, C 10-20 alkenyl, C 10-20 alkynyl, C 3-9 alkylene-CO 2 -C 8-18 alkyl, C 3-9 alkylene-CO 2 -C 8-18 alkenyl, C 3-9 alkenylene-CO 2 -C 8-18 alkyl, C 3-9 alkenylene-CO 2 -C 8-18 alkenyl, C 3-9 alkylene-O 2 C-C 8-18 alkyl, C 3-9 alkylene- O 2 C-C 8-18 alkenyl, C 3-9 alkenylene- O 2 C-C 8-18 alkyl and C 3-9 alkenylene-O 2 C-C 8-18 alkenyl; and each one of R 3 and R 4 is individually, a C 1-4 alkyl or wherein R 3 and R 4 together with the nitrogen atom to which they are bound form a 5-6 membered ring
  • n is 2, 3 or 4. Each possibility represents a separate embodiment of the invention. According to some embodiments, n is 2.
  • n is 2, 3 or 4. Each possibility represents a separate embodiment of the invention. According to some embodiments, m is 2.
  • each one of n and m is 2, and the lipid is represented by the structure of Formula (III) or a salt thereof:
  • each one of R 3 and R 4 is individually, a Cl -4 alkyl.
  • R 3 is a C 1-2 alkyl. According to some embodiments, R 3 is According to some embodiments, R 4 is a C 1-2 alkyl. According to some embodiments, R 4 is CH 3 .
  • each one of n and m is 2, each one of R 3 and R 4 is individually CH 3 , and the lipid is represented by the structure of Formula (IV) or a salt thereof:
  • R 1 is selected from the group consisting of: C 3-9 alkylene- CO 2 -C 8-18 alkyl, C 3-9 alkylene-CO 2 -C 8-18 alkenyl, C 3-9 alkenylene-CO 2 -C 8-18 alkyl, C 3-9 alkylene-O 2 C-C 8-18 alkyl, C 3-9 alkylene-O 2 C-C 8-18 alkenyl, C 3-9 alkenylene-O 2 C-C 8-18 alkyl. According to some embodiments, R 1 is selected from the group consisting of: C 3-9 alkylene-CO 2 -C 8-18 alkyl and C 3-9 alkylene-O 2 C-C 8-18 alkyl.
  • R 1 is selected from the group consisting of: C 6-9 alkylene-CO 2 -C 14-18 alkyl, C 6-9 alkylene-O 2 C-C 14-18 alkyl. According to some embodiments, wherein R 1 is C 6-9 alkylene-CO 2 -C 14-18 alkyl.
  • R 2 is selected from the group consisting of: C 10-20 alkenyl, C 3-9 alkylene-CO 2 -C 8-18 alkyl and C 3-9 alkylene-O 2 C-C 8-18 alkyl. According to some embodiments, wherein R 2 is selected from the group consisting of: C 16-20 alkenyl, C 6-9 alkylene-CO 2 -C 14-18 alkyl and C 6-9 alkylene-O 2 C-C 14-18 alkyl.
  • each one of R 1 and R 2 is individually selected from the group consisting of: C 3-9 alkylene-CO 2 -C 8-18 alkyl, C 3-9 alkylene-CO 2 -C 8-18 alkenyl, C 3-9 alkenylene-CO 2 -C 8-18 alkyl, C 3-9 alkylene-O 2 C-C 8-18 alkyl, C 3-9 alkylene-O 2 C-C 8-18 alkenyl, C 3-9 alkenylene-O 2 C-C 8-18 alkyl.
  • each one of R 1 and R 2 is individually selected from the group consisting of: C 3-9 alkylene-CO 2 -C 8-18 alkyl and C 3-9 alkylene-O 2 C-C 8-18 alkyl. According to some embodiments, each one of R 1 and R 2 is C 6-9 alkylene-CO 2 -C 14-18 alkyl. According to some embodiments, each one of the alkenyl groups is unsubstituted.
  • each one of the alkenylene groups is unsubstituted.
  • each one of the alkyl and alkylene groups is unsubstituted.
  • the lipid is selected from the group consisting of:
  • the lipid is selected from the group consisting of: EA- 405C, EA-506C, EA-513C, EA-524C and salts thereof. According to some embodiments, the lipid is EA-524C or a salt thereof.
  • the lipid is hydrolytically stable, alcoholytically stable or both, for at least one week.
  • particle comprising the lipid disclosed herein and a membrane stabilizing lipid.
  • the membrane stabilizing lipid is selected from the group consisting of cholesterol, phospholipids, cephalins, sphingolipids and glycoglycerolipids. Each possibility represents a separate embodiment of the invention. According to some embodiments, the membrane stabilizing lipid comprises cholesterol.
  • the particle further comprises one or more additional components selected from the group consisting of a PEG-lipid conjugate, a neutral lipid and a charged lipid.
  • the additional component comprises 1,2-Distearoyl-sn- glycero-3-phosphocholine (DSPC).
  • the additional component comprises 1 ,2-Dimyristoyl-sn- glyceryl -methoxy polyethylene glycol (DMG-PEG).
  • the particle comprises the lipid, cholesterol, 1,2- Distearoyl-sn-glycero-3-phosphocholine (DSPC) and 1,2-Dimyristoyl-sn-gly cerylmethoxy polyethylene glycol (DMG-PEG).
  • DSPC 1,2- Distearoyl-sn-glycero-3-phosphocholine
  • DMG-PEG 1,2-Dimyristoyl-sn-gly cerylmethoxy polyethylene glycol
  • the particle further comprises a nucleic acid encapsulated within a particle comprising the lipid.
  • the nucleic acid is selected from the group consisting of small interfering RNA (siRNA), micro RNA (miRNA), antisense oligo nucleotides, messenger RNA (mRNA), ribozymes, pDNA, CRISPR mRNA, gRNA, circular RNA and immune stimulating nucleic acids.
  • siRNA small interfering RNA
  • miRNA micro RNA
  • mRNA messenger RNA
  • ribozymes pDNA
  • CRISPR mRNA CRISPR mRNA
  • gRNA circular RNA
  • immune stimulating nucleic acids RNA comprising an open reading frame encoding a polypeptide that comprises a SARS-CoV-2 spike protein or an immunogenic fragment or variant thereof.
  • composition comprising a plurality of particles as disclosed herein and a pharmaceutically acceptable carrier, diluent or excipient.
  • the composition is a liposomal composition.
  • a method of gene silencing comprising the step of contacting a cell with a composition comprising a plurality of particles according to the present invention, and a pharmaceutically acceptable carrier, diluent or excipient.
  • the cell is a cancer cell.
  • a method for administering a therapeutic agent comprising the step of preparing a composition comprising a lipid according to the present invention, and a therapeutic agent, and administering the composition to a subject in need thereof.
  • the method further comprises encapsulating the therapeutic agent within a particle comprising the lipid.
  • the therapeutic agent is RNA comprising an open reading frame encoding a polypeptide that comprises a SARS-CoV-2 spike protein or an immunogenic fragment or variant thereof.
  • a method of treating a leukocyte associated condition comprising the step of administering to a subject in need thereof a composition comprising a plurality of particles according to the present invention and a pharmaceutically acceptable carrier, diluent or excipient.
  • the leukocyte associated condition is selected from the group consisting of cancer, infection, autoimmune diseases, neurodegenerative diseases and inflammation.
  • Figure 1A-B depict the expression level of GFP by transfecting Z138 cells and primary T Cells with mRNA-NLPs.
  • Figure 1A is a bar chart showing quantitative analysis of the GFP fluorescence intensity following transfection of mRNA-LNPs, wherein the LNPs are EA- 519, EA-402, EA-506C, EA-405 and EA-506CN.
  • Figure IB is a line chart showing quantitative analysis of the percentage of Z 138 expressing GFP following the transfection of mRNA-LNP, EA-519 (broken line + squares), EA-402 (solid line + empty diamonds), EA-506C (broken line + triangles), EA-405 (broken line + circles) and EA-506CN (solid line + full diamonds).
  • Figure 2A is a bar chart showing quantitative analysis of the GFP fluorescence intensity following Z138cells transfection of mRNA-LNPS wherein the lipids are EA-2C, EA-502C, EA-506C, and EA-405C. From left to right the different groups of lipids are at a concentration of 0.25 pg/ml, 0.5 pg/ml, 1 pg/ml, and 2 pg/ml.
  • Figure 2B is a line chart showing quantitative analysis of the percentage of Z138 cells expressing GFP following the transfection of mRNA-LNP; EA-2C (solid line + triangle facing down), EA-502C (solid line + triangle facing up), EA-506C (solid line + circles), and EA-405C (solid line + triangle facing up).
  • Figure 3 is a bar graph showing quantitative analysis of LUC expression in HeLa human cells after being transfected with the lipids EA-506 (clear frame), EA-506C (horizontal lines), EA-2 (vertical lines), and EA2-C (solid fill), as described in Example 2.
  • Left group of bars are lipids at a concentration of 0.25 pg/ml
  • middle group of bars are lipids at a concentration of 0.125 pg/ml
  • right group of bars are lipids at a concentration of 0.065 pg/ml.
  • Figure 4A is a bar chart showing quantitative analysis of the GFP fluorescence intensity following primary T-cells transfection of mRNA-LNPs comprising EA2C, EA-502C, EA- 506C, and EA-405C lipids. From left to right the different groups of lipids are at a concentration of 0.25 pg/ml, 0.5 pg/ml, 1 pg/ml, 2 pg/ml, and 4 pg/ml.
  • Figure 4B is a line chart showing quantitative analysis of the percentage of primary T-cells expressing GFP following the transfection of mRNA-LNP comprising EA-2C (broken line - triangles facing down), EA-502C (broken line - circles), EA-506C (solid line - circles), and EA- 405C (solid line - squares).
  • Figure 5 is a bar graph showing quantitative analysis of LUC expression in a murine model (mice) after intramuscularly administration of lipids comprising EA-506 (no fill), EA-506C (vertical lines), EA-2 (horizontal lines), and EA2-C (dots) from left to right. Detailed procedures are described in Example 5.
  • Figure 6 shows the chemical structures of the lipids EA-506, EA-2, EA-519, EA-402, EA-506CN and EA-405.
  • Figure 7A shows GFP mean fluorescence and percentage of GFP + cells analyzed by flow cytometry of LNPs comprising the lipids EA2C (full circles), EA502C (full squares), EA506C (full triangles), EA405C (empty triangles), EA513C (empty circles) and EA524C (empty squares), upon transfected at concentrations 0.25pg/ml, 0.5pg/ml, 1 pg/ml and 2pg/ml.
  • Figure 7B shows the GFP mean fluorescence intensities of LNPs comprising the lipids EA2C, EA502C (large squares), EA506C (horizonal lines), EA405C (vertical lines), EA513C (diagonal lines) and EA524C (small squares), upon transfected at concentrations 0.25pg/ml (left block), 0.5pg/ml (second to left block), 1 pg/ml (second to right block) and 2pg/ml (second to right block).
  • Figure 8 shows the GFP mean fluorescence intensities of LNPs comprising the lipids EA2C, EA502C (large squares), EA506C (horizonal lines), EA405C (vertical lines), EA513C (diagonal lines) and EA524C (small squares), upon transfection of HCC cells at concentrations 0.25pg/ml (left block), 0.5pg/ml (second to left block), Ipg/ml (second to right block) and 2pg/ml (second to right block).
  • Figure 9 shows the GFP mean fluorescence intensities of LNPs comprising the lipids EA2C, EA502C (large squares), EA506C (horizonal lines), EA405C (vertical lines), EA513C (diagonal lines) and EA524C (small squares), upon transfection of 0vcar8 cells at concentrations 0.25pg/ml (left block), 0.5pg/ml (second to left block), Ipg/ml (second to right block) and 2pg/ml (second to right block).
  • Figure 10 shows the GFP mean fluorescence intensities of LNPs comprising the lipids EA2C, EA502C (large squares), EA506C (horizonal lines), EA405C (vertical lines), EA513C (diagonal lines) and EA524C (small squares), upon transfection of Detroit562 pharyngeal cancer cells at concentrations 0.25pg/ml (left block), 0.5pg/ml (second to left block), Ipg/ml (second to right block) and 2pg/ml (second to right block).
  • Figure 11 is a heat map depicting cancer cell lines: ovcar8, Detroid562, HepG2 and Z138 versus LNPs expressed therein, the LNPs comprising the lipids EA2C, EA502C, EA506C, EA405C, EA513C and EA524C. Optimized concentration is shown for each cell line.
  • Figure 12A shows GFP mean fluorescence and percentage of GFP + Primary T cells analyzed by flow cytometry of LNPs comprising the lipids EA2C (full circles), EA502C (full squares), EA506C (full triangles), EA405C (empty triangles), EA513C (empty circles) and EA524C (empty squares), upon transfected at concentrations 0.25pg/ml, 0.5pg/ml, Ipg/ml and 2pg/ml.
  • Figure 12B shows the GFP mean fluorescence intensities of LNPs comprising the lipids EA2C, EA502C (large squares), EA506C (horizonal lines), EA405C (vertical lines), EA513C (diagonal lines) and EA524C (small squares), upon transfection of Primary T cells at concentrations 0.25pg/ml (left block), 0.5pg/ml (second to left block), Ipg/ml (second to right block) and 2pg/ml (second to right block).
  • Figure 13A shows % Indel mutations upon gene editing in HCC cells with sgHPRT-LNPs and sgNCAPG-LNPs.
  • Figure 13B shows cellular viability measured via XTT upon gene editing in HCC cells with sgHPRT-LNPs and sgNCAPG-LNPs.
  • Figure 14A shows fluorescence imaging of femurs 4h after LNPs injection for LNPs comprising the lipids EA506C and EA405C.
  • Figure 14B shows fluorescence imaging of femurs 4h after injection to MCL bearing mice LNPs comprising the lipids EA506C and EA405C.
  • Figure 14C shows Relative Total Flux [p/s] of liver, spleen, kidneys, heart and lungs and femurus normalized to the mock after injection to MCL bearing mice LNPs comprising the lipids EA506C and EA405C.
  • Figure 15A shows Total Flux [p/s] of the femurs normalized to the mock after injection to MCL bearing mice LNPs comprising the lipids EA506C and EA405C.
  • Figure 15B shows Relative Total Flux [p/s] of liver, spleen, kidneys, heart and lungs and femurus normalized to the mock after injection to MCE bearing mice ENPs comprising the lipid EA524C.
  • Figure 15C shows Total Flux [p/s] of the femurs normalized to the mock after injection to MCE bearing mice LNPs comprising the lipid EA524C.
  • Figure 16 is a bar chart of % gene editing using EA524-LNPs and EA524C-LNPs formulations encapsulated with sgSOXl l and with sgGFP.
  • Figures 17A-17D relative decomposition measured as %purity over 6 days at room temperature for lipid EA-524 in ethanol (Figure 17A), lipid EA-524 in ethanol: water 3: 1 v/v ( Figure 17B), lipid EA-524C in ethanol ( Figure 17C), and lipid EA-524C in ethanol: water 3: 1 v/v ( Figure 17D).
  • the present invention based on the discovery of hydrolytically and alcoholytically stable lipids useful in preparing lipid nanoparticles to deliver active agents in vitro and in vivo.
  • the lipids of the present invention are useful in delivery of nucleic acids such as siRNA, miRNA and mRNA etc.
  • the present invention provides carbamate lipids.
  • the lipids are ionizable lipids.
  • the lipid of the present invention is a cationic lipid.
  • the terms “cationic lipid” and “ionizable lipid” are as defined herein below.
  • the present invention relates to carbamate lipid(s) represented by the structure of Formula (V): including salts, hydrates, solvates, polymorphs, optical isomers, geometrical isomers, enantiomers, diastereomers, and mixtures thereof.
  • n is selected from the group consisting of: 0, 1, 2, 3, 4, 5, 6, 7 and 8. Each possibility represents a separate embodiment of the invention. According to some embodiments, n is selected from the group consisting of: 1, 2, 3, 4, 5, 6 and 7. According to some embodiments, n is selected from the group consisting of: 1, 2, 3, 4 and 5. According to some embodiments, n is selected from the group consisting of: 1, 2 and 3. According to some embodiments, n is 2.
  • m is selected from the group consisting of: 0, 1, 2, 3, 4, 5, 6, 7 and 8. Each possibility represents a separate embodiment of the invention. According to some embodiments, m is selected from the group consisting of: 1, 2, 3, 4, 5, 6 and 7. According to some embodiments, m is selected from the group consisting of: 1, 2, 3, 4 and 5. According to some embodiments, m is selected from the group consisting of: 1, 2 and 3. According to some embodiments, m is 2.
  • n equals to m.
  • each one of n and m is individually selected from the group consisting of: 0, 1, 2, 3, 4, 5, 6, 7 and 8.
  • each possibility represents a separate embodiment of the invention.
  • each one of n and m is individually selected from the group consisting of: 1, 2, 3, 4, 5, 6 and 7.
  • each one of n and m is individually selected from the group consisting of: 1, 2, 3, 4 and 5.
  • each one of n and m is individually selected from the group consisting of: 1, 2 and 3.
  • each one of n and m is individually 2.
  • each one of n and m is individually selected from the group consisting of: 1, 2, 3, 4 and 5.
  • each one of n and m is 2, and the lipid is represented by the structure of Formula (III) or a salt thereof:
  • R 1 is selected from the group consisting of: C 3-9 alkylene- CO 2 -C 8-18 alkyl, C 3-9 alkylene-CO 2 -C 8-18 alkenyl, C 3-9 alkenylene-CO 2 -C 8-18 alkyl, C 3-9 alkenylene-CO 2 -C 8-18 alkenyl, C 3-9 alkylene-O 2 C-C 8-18 alkyl, C 3-9 alkylene-O 2 C-C 8-18 alkenyl, C 3-9 alkenylene-O 2 C-C 8-18 alkyl and C 3-9 alkenylene-O 2 C-C 8-18 alkenyl.
  • Each possibility represents a separate embodiment of the invention.
  • alkyl As detailed herein, unless specified otherwise, the terms “alkyl”, “alkylene”, “alkenyl” and “alkenylene” relate to both substituted and unsubstituted groups. Also as detailed herein, unless specified otherwise, the terms “alkyl”, “alkylene”, “alkenyl” and “alkenylene” relate to both linear and branched chains. According to some embodiments, the alkyl is a branched alkyl. According to some embodiments, the alkyl is a straight alkyl. According to some embodiments, the alkyl is an unsubstituted alkyl. According to some embodiments, the alkylene is a straight alkylene.
  • the alkylene is an unsubstituted alkylene.
  • the alkenyl is a straight alkenyl.
  • the terms “alkenyl” and “alkenylene” relate to fragments, which include one or more double carbon- carbon bonds, and thus include dienes, trienes, tetraenes and the like.
  • the alkenyl is an unsubstituted alkenyl.
  • the alkenylene is a straight alkenylene.
  • the alkenyl is a dienyl.
  • the alkenylene is an unsubstituted alkenylene.
  • ester groups within the terminal lipidic substituent do not cause a substantial reduction of hydrolytic or alcoholytic stability of the ionizable lipid.
  • an ester core group of the ionizable lipid results in a substantially less stable lipid than its counterpart that includes a core carbamate group.
  • R 1 is C 3-9 alkylene-CO 2 -C 8-18 alkyl.
  • R 1 is C 3-9 alkylene-CO 2 -C 8-18 alkenyl.
  • R 1 is C 3-9 alkenylene-CO 2 -C 8-18 alkyl.
  • R 1 is C 3-9 alkenylene-CO 2 -C 8-18 alkenyl. According to some embodiments, R 1 is C 3-9 alkylene-O 2 C- C 8-18 alkyl. According to some embodiments, R 1 is C 3-9 alkylene-O 2 C-C 8-18 alkenyl. According to some embodiments, R 1 is C 3-9 alkenylene-O 2 C-C 8-18 alkyl. According to some embodiments, R 1 is C 3-9 alkenylene-O 2 C-C 8-18 alkenyl.
  • R 1 is selected from the group consisting of: C 3-9 alkylene-CO 2 -C 8-18 alkyl, C 3-9 alkylene-CO 2 -C 8-18 alkenyl, C 3-9 alkenylene-CO 2 -C 8-18 alkyl, C 3-9 alkylene-O 2 C-C 8- 18 alkyl, C 3-9 alkylene-O 2 C-C 8-18 alkenyl, C 3-9 alkenylene-O 2 C-C 8-18 alkyl.
  • R 1 is selected from the group consisting of: C 3-9 alkylene- CO 2 -C 8-18 alkyl and C 3-9 alkylene-O 2 C-C 8-18 alkyl. According to some embodiments, R 1 is selected from the group consisting of: C 6-9 alkylene-CO 2 -C 14-18 alkyl, C 6-9 alkylene- O 2 C— C 14-18 alkyl.
  • R 1 is C 6-9 alkylene-CO 2 -C 14-18 alkyl. According to some embodiments, R 1 is C7-8 alkylene-CO 2 -C 15-17 alkyl. According to some embodiments, R 1 is -C7H 14 -CO 2 -CH(C 8 H1 7 )C 8 H 17 .
  • R 1 is C 6-9 alkylene- O 2 C-C 14-18 alkyl. According to some embodiments, R 1 is C 7-8 alkylene- O 2 C-C 15-17 alkyl. According to some embodiments, R 1 is -C 8 H16-O 2 C-CH(C 6 H 13 )C 8 H 17 .
  • R 2 is selected from the group consisting of: C 10-20 alkyl, C 10-20 alkenyl, C 10-20 alkynyl, C 3-9 alkylene-CO 2 -C 8-18 alkyl, C 3-9 alkylene-CO 2 -C 8-18 alkenyl, C 3-9 alkenylene-CO 2 -C 8-18 alkyl, C 3-9 alkenylene-CO 2 -C 8-18 alkenyl, C 3-9 alkylene-O 2 C-C 8-18 alkyl, C 3-9 alkylene-O 2 C-C 8-18 alkenyl, C 3-9 alkenylene- O 2 C-C 8-18 alkyl and C 3-9 alkenylene-O 2 C-C 8-18 alkenyl.
  • alkyl refers to both substituted and unsubstituted groups.
  • alkyl refers to both linear and branched chains.
  • the alkyl is a branched alkyl.
  • the alkyl is a straight alkyl.
  • the alkyl is an unsubstituted alkyl.
  • the alkylene is a straight alkylene.
  • the alkylene is an unsubstituted alkylene.
  • the alkenyl is a straight alkenyl.
  • the terms “alkenyl” and “alkenylene” relate to fragments, which include one or more double carbon-carbon bonds, and thus include dienes, trienes, tetraenes and the like.
  • the alkenyl is an unsubstituted alkenyl.
  • the alkenylene is a straight alkenylene.
  • the alkenyl is a dienyl.
  • the alkenylene is an unsubstituted alkenylene.
  • the alkenyl is an unsubstituted alkenyl.
  • the alkenylene is a straight alkenylene.
  • the alkenylene is an unsubstituted alkenylene.
  • R 2 is selected from the group consisting of: C 10-20 alkenyl, C 3-9 alkylene-CO 2 -C 8-18 alkyl and C 3-9 alkylene-O 2 C-C 8-18 alkyl. According to some embodiments, R 2 is selected from the group consisting of: C16-20 alkenyl, C 6-9 alkylene- CO 2 -C 14-18 alkyl and C 6-9 alkylene-O 2 C-C 14-18 alkyl.
  • R 2 is C 6-9 alkylene-CO 2 -C 14-18 alkyl. According to some embodiments, R 2 is C7-8 alkylene-CO 2 -C 15-17 alkyl. According to some embodiments, R 1 is -C7H 14 -CO 2 -CH(C 8 H1 7 )C 8 H 17 . According to some embodiments, R 2 is C 6-9 alkylene- O 2 C-C 14-18 alkyl. According to some embodiments, R 2 is C7-8 alkylene- O 2 C-C15- 17 alkyl. According to some embodiments, R 2 is -C 8 H16-O 2 C-CH(C6H13)C 8 H 17 .
  • each one of R 1 and R 2 is individually selected from the group consisting of: C 3-9 alkylene-CO 2 -C 8-18 alkyl, C 3-9 alkylene-CO 2 -C 8-18 alkenyl, C 3-9 alkenylene-CO 2 -C 8-18 alkyl, C 3-9 alkylene-O 2 C-C 8-18 alkyl, C 3-9 alkylene-O 2 C-C 8-18 alkenyl, C 3-9 alkenylene-O 2 C-C 8-18 alkyl.
  • Each possibility represents a separate embodiment of the invention.
  • each one of R 1 and R 2 is individually selected from the group consisting of: C 3-9 alkylene-CO 2 -C 8-18 alkyl and C 3-9 alkylene-O 2 C-C 8-18 alkyl. According to some embodiments, each one of R 1 and R 2 is C 6-9 alkylene-CO 2 -C 14-18 alkyl.
  • R 3 is a C 1-4 alkyl.
  • alkyl relates to both substituted and unsubstituted alkyls.
  • alkyl relates to both linear and branched alkyls.
  • R 3 is a C1-3 alkyl.
  • R 3 is a C 1-2 alkyl.
  • R 3 is a Ci alkyl.
  • the alkyl is a straight alkyl.
  • the alkyl is an unsubstituted alkyl
  • R 3 is CH 3 or CH2CH 3 .
  • R 3 is CH 3 .
  • R 4 is a C 1-4 alkyl.
  • alkyl relates to both substituted and unsubstituted alkyls.
  • alkyl relates to both linear and branched alkyls.
  • R 4 is a C1-3 alkyl.
  • R 4 is a C 1-2 alkyl.
  • R 4 is a Ci alkyl.
  • the alkyl is a straight alkyl.
  • the alkyl is an unsubstituted alkyl.
  • R 4 is CH 3 or CH2CH 3 .
  • R 4 is CH 3 .
  • R 3 is the same as R 4 .
  • each one of R 3 and R 4 is individually, a C 1-4 alkyl or wherein R 3 and R 4 together with the nitrogen atom to which they are bound form a 5-6 membered ring.
  • R 3 and R 4 together with the nitrogen atom to which they are bound form a 5- 6 membered ring.
  • each one of R 3 and R 4 individually is a C1-3 alkyl.
  • each one of R 3 and R 4 individually is a C 1-2 alkyl.
  • each one of R 3 and R 4 is individually a Ci alkyl.
  • each one of R 3 and R 4 is individually CH 3 or CH2CH 3 . According to some embodiments, each one of R 3 and R 4 is individually CH 3 .
  • each one of n and m is 2, and the lipid is represented by the structure of Formula (IVa) or a salt thereof:
  • each one of n and m is 2, each one of R 3 and R 4 is individually CH 3 , and the lipid is represented by the structure of Formula (IV) or a salt thereof: Carbamate lipids - Formula (I)
  • the lipid is hydrolytically stable for at least 2 hours.
  • Y is NH and Z is O.
  • Z is NH and Y is O.
  • Y is NH
  • Z is O
  • the lipid is represented by the structure of Formula (la) or a salt thereof:
  • n is 2, 3 or 4. Each possibility represents a separate embodiment of the invention. According to some embodiments, n is 2.
  • n is 2, 3 or 4. Each possibility represents a separate embodiment of the invention. According to some embodiments, m is 2.
  • each one of n and m is 2, and the lipid is represented by the structure of Formula (II) or a salt thereof:
  • each one of n and m is 2, Y is NH and Z is O, and the lipid is represented by the structure of Formula (III) or a salt thereof:
  • each one of R 3 and R 4 is individually, a C 1-4 alkyl.
  • R 3 is a C 1-2 alkyl. According to some embodiments, R 3 is CH 3 . According to some embodiments, R 4 is a C 1-2 alkyl. According to some embodiments, R 4 is CH 3 .
  • each one of n and m is 2, Y is NH and Z is O, each one of R 3 and R 4 is individually CH 3 , and the lipid is represented by the structure of Formula (IV) or a salt thereof:
  • R 1 is selected from the group consisting of: C 10-20 alkenyl, C 3-9 alkylene-CO 2 -C 8-18 alkyl, C 3-9 alkylene-O 2 C-C 8-18 alkyl and C 3-9 alkylene-CO 2 -C 8- 18 alkenyl. According to some embodiments, R 1 is selected from the group consisting of: C 14-18 alkenyl, C 6-9 alkylene-CO 2 -C 12-18 alkyl, C 6-9 alkylene-O 2 C-C 9-16 alkyl and C 4-6 alkylene-CO 2 -C 8-10 alkenyl.
  • R 1 contains an ester group.
  • R 1 is selected from the group consisting of: C 3-9 alkylene-CO 2 -C 8-18 alkyl, C 3-9 alkylene-CO 2 -C 8-18 alkenyl, C 3-9 alkenylene-CO 2 -C 8-18 alkyl, C 3-9 alkenylene-CO 2 - C 8-18 alkenyl, C 3-9 alkylene-O 2 C-C 8-18 alkyl, C 3-9 alkylene-O 2 C-C 8-18 alkenyl, C 3-9 alkenylene-O 2 C-C 8-18 alkyl and C 3-9 alkenylene-O 2 C-C 8-18 alkenyl
  • R 2 contains an ester group.
  • R 2 is selected from the group consisting of: C 10-20 alkenyl, C 3-9 alkylene- CO 2 -C 8-18 alkyl, C 3-9 alkylene-O 2 C-C 8-18 alkyl and C 3-9 alkylene-CO 2 -C 8-18 alkenyl.
  • R 2 is selected from the group consisting of: C 14-18 alkenyl, C 6-9 alkylene-CO 2 -C 12-18 alkyl, C 6-9 alkylene-O 2 C-C 9-16 alkyl and C4-6 alkylene-CO 2 -C 8- 10 alkenyl.
  • At least one of R 1 and R 2 contains an ester group. According to some embodiments, each one of R 1 and R 2 contains an ester group. According to some embodiments, each one of R 1 and R 2 is individually selected from the group consisting of: C 10-20 alkenyl, C 3-9 alkylene-CO 2 -C 8-18 alkyl, C 3-9 alkylene-O 2 C-C 8-18 alkyl and C 3-9 alkylene-CO 2 -C 8-18 alkenyl.
  • each one of R 1 and R 2 is individually selected from the group consisting of: C 14-18 alkenyl, C 6-9 alkylene-CO 2 - C 12-18 alkyl, C 6-9 alkylene-O 2 C-C 9-16 alkyl and C4-6 alkylene-CO 2 -C 8-10 alkenyl.
  • each one of R 1 and R 2 is individually selected from the group consisting of: C 18 alkenyl, C 5 alkylene-CO 2 -C 9 alkenyl; C 7-9 alkylene-CO 2 -C 13-17 alkyl and C 7-8 alkylene-O 2 C-C 9-15 alkyl.
  • each one of the alkyl, alkenyl, alkynyl, alkylene and alkenylene groups within Formula 1 is unsubstituted.
  • each one of the alkyl, alkenyl, alkynyl, alkylene and alkenylene groups is straight chain or branched.
  • Each possibility represents a separate embodiment of the invention.
  • each one of the alkyl and alkenyl groups is unsubstituted.
  • each one of the alkyl and alkenyl groups is straight-chain or branched.
  • Y is NH
  • Z is O
  • R 1 is selected from the group consisting of: C 3-9 alkylene-CO 2 -C 8-18 alkyl, C 3-9 alkylene-CO 2 -C 8-18 alkenyl, C 3-9 alkenylene-CO 2 - C 8-18 alkyl, C 3-9 alkenylene-CO 2 -C 8-18 alkenyl, C 3-9 alkylene-O 2 C-C 8-18 alkyl, C 3-9 alkylene-O 2 C-C 8-18 alkenyl, C 3-9 alkenylene-O 2 C-C 8-18 alkyl and C 3-9 alkenylene-O 2 C- C 8-18 alkenyl;
  • R 2 is selected from the group consisting of: C 10-20 alkyl, C 10-20 alkenyl, C10- 20 alkynyl, C 3-9 alkylene-CO 2 -C 8-18 alkyl, C 3-9 alkylene-CO 2 -C 8-18 alkenyl, C 3-9
  • the present invention relates to a lipid represented by the structure of any one of formulas (I), (la), (II), (III), (IV), (IVa) or (V) as described above.
  • a lipid represented by the structure of any one of formulas (I), (la), (II), (III), (IV), (IVa) or (V) as described above.
  • Each possibility represents a separate embodiment of the invention, including salts, hydrates, solvates, polymorphs, optical isomers, geometrical isomers, enantiomers, diastereomers, and mixtures thereof.
  • the lipid of the present invention is selected from the group consisting of:
  • the lipid of Formula (I) is selected from the group consisting of: EA-2C, EA-402C, EA-402C2, EA-404C, EA-405C, EA-406C, EA-408C, EA-502C, EA-505C, EA-506C, EA-513C, EA-516C, EA-521C, EA-522C, EA-524C EA-526C and salts thereof.
  • EA-2C EA-402C, EA-402C2, EA-404C, EA-405C, EA-406C, EA-408C, EA-502C, EA-505C, EA-506C, EA-513C, EA-516C, EA-521C, EA-522C, EA-524C EA-526C and salts thereof.
  • EA-2C EA-402C, EA-402C2, EA-404C, EA-405C, EA-406C, EA-408
  • the lipid of the present invention is selected from the group consisting of: EA-402C, EA-404C, EA-405C, EA-406C, EA-408C, EA-502C, EA- 505C, EA-506C, EA-513C, EA-521C, EA-522C, EA-524C, EA-526C and salts thereof.
  • the lipid of Formula (V) is selected from the group consisting of: EA-402C, EA-404C, EA-405C, EA-406C, EA-408C, EA-502C, EA-505C, EA-506C, EA-513C, EA-521C, EA-522C, EA-524C, EA-526C and salts thereof.
  • EA-402C EA-404C
  • EA-405C EA-406C
  • EA-408C EA-502C
  • EA-505C EA-506C
  • EA-513C EA-521C
  • EA-522C EA-524C
  • EA-526C EA-526C and salts thereof.
  • the lipid of the present invention is selected from the group consisting of: EA-402C, EA-405C, EA-406C, EA-408C, EA-502C, EA-505C, EA- 506C, EA-513C, EA-521C, EA-522C, EA-524C, EA-526C and salts thereof.
  • the lipid of Formula (V) is selected from the group consisting of: EA- 402C, EA-405C, EA-406C, EA-408C, EA-502C, EA-505C, EA-506C, EA-513C, EA- 521C, EA-522C, EA-524C, EA-526C and salts thereof.
  • the lipid of the present invention is selected from the group consisting of: EA-405C, EA-502C, EA-506C, EA-513C, EA-524C and salts thereof.
  • the lipid of Formula (V) is selected from the group consisting of: EA-405C, EA-502C, EA-506C, EA-513C, EA-524C and salts thereof.
  • the lipid of the present invention is selected from the group consisting of: EA-405C, EA-506C, EA-513C, EA-524C and salts thereof.
  • the lipid of Formula (V) is selected from the group consisting of: EA-405C, EA-506C, EA-513C, EA-524C and salts thereof.
  • the lipid of the present invention is selected from the group consisting of: EA-405C, EA-513C, EA-524C and salts thereof.
  • the lipid of Formula (V) is selected from the group consisting of: EA-405C, EA-513C, EA-524C and salts thereof.
  • the lipid of the present invention is selected from the group consisting of: EA-506C, EA-513C, EA-524C and salts thereof.
  • the lipid of Formula (V) is selected from the group consisting of: EA-506C, EA-513C, EA-524C and salts thereof.
  • the lipid of the present invention is selected from the group consisting of: EA-405C, EA-506C, EA-524C and salts thereof.
  • the lipid of Formula (V) is selected from the group consisting of: EA-405C, EA-506C, EA-524C and salts thereof.
  • the lipid of the present invention is selected from the group consisting of: EA-506C, EA-524C and salts thereof. According to some embodiments, the lipid of the present invention is selected from the group consisting of: EA-405C, EA-524C and salts thereof. According to some embodiments, the lipid of the present invention is selected from the group consisting of: EA-513C, EA-524C and salts thereof. According to some embodiments, the lipid of the present invention is EA-524C or a salt thereof.
  • the carbamate lipid of the present invention is hydrolytically stable, alcoholytically stable or both for at least 2 hours, at least 3 hours, at least 4 hours, at least 5 hours, at least 10 hours, at least 12 hours, at least 24 hours, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 10 days, at least 15 days, at least 20 days and/or at least 30 days.
  • Each possibility represents a separate embodiment of the invention.
  • hydrolytic stability refers to the material's ability to resist hydrolysis.
  • a material is considered “hydrolytically stable” if it does not undergo substantial hydrolysis upon exposure to hydrolytic conditions.
  • Hydrolytic conditions include immersion of the material at room temperature in a solution having physiological pH (e.g., upon immersion in PBS containing solutions) for a predetermined time period.
  • the level of hydrolysis can be determined e.g., using HPLC, to monitor the decomposition of the starting material upon exposure to the hydrolytic conditions.
  • substantially hydrolysis refers to hydrolysis of at least 10%, at least 5%, at least 3%, at least 2% or at least 1%. Each possibility represents a separate embodiment of the invention.
  • lipids such as cationic lipids undergo gradual hydrolysis upon exposure to hydrolytic conditions. Without wishing to be bound by any theory of mechanism of action, the gradual hydrolysis results in dissociation of ester and/or carbamate groups contained within the lipid.
  • alcoholysis refers to the material's ability to resist alcoholysis.
  • a material is considered “alcoholytically stable” if it does not undergo substantial alcoholysis upon exposure to alcoholytic conditions.
  • Alcoholytic conditions include immersion of the material at room temperature in an alcohol for a predetermined time period. The level of hydrolysis can be determined e.g., using HPLC, to monitor the decomposition of the starting material upon exposure to the alcoholytic conditions.
  • substantially alcoholysis refers to alcoholysis of at least 10%, at least 5%, at least 3%, at least 2% or at least 1%. Each possibility represents a separate embodiment of the invention.
  • lipids such as ionizable lipids undergo gradual alcoholysis upon exposure to alcoholysis conditions.
  • the gradual alcoholysis results in dissociation of ester and/or carbamate groups contained within the lipid.
  • the present lipids may form lipid nanoparticles formulated with nucleic acids to facilitate their intracellular delivery both in vitro and for in vivo therapeutic applications.
  • the lipids of the present are required to be hydrolytically stable for time frames relevant for such applications.
  • alcoholytic stability entails stability to ethanolysis upon immersion of the material at room temperature in ethanol for a predetermined time period.
  • the lipid is hydrolytically stable for at least 2 hours, at least 3 hours, at least 4 hours, at least 5 hours, at least 10 hours, at least 12 hours, at least 24 hours, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 10 days, at least 15 days, at least 20 days and/or at least 30 days.
  • Each possibility represents a separate embodiment of the invention.
  • the lipid is alcoholytically stable for at least 2 hours, at least 3 hours, at least 4 hours, at least 5 hours, at least 10 hours, at least 12 hours, at least 24 hours, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 10 days, at least 15 days, at least 20 days and/or at least 30 days.
  • Each possibility represents a separate embodiment of the invention.
  • the lipid is hydrolytically stable and alcoholytically stable upon immersion at room temperature in aqueous ethanol for at least 2 hours, at least 3 hours, at least 4 hours, at least 5 hours, at least 10 hours, at least 12 hours, at least 24 hours, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 10 days, at least 15 days, at least 20 days and/or at least 30 days.
  • aqueous ethanol for at least 2 hours, at least 3 hours, at least 4 hours, at least 5 hours, at least 10 hours, at least 12 hours, at least 24 hours, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 10 days, at least 15 days, at least 20 days and/or at least 30 days.
  • the lipid of the present invention is hydrolytically stable for at least 24 hours, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 10 days, at least 15 days, at least 20 days and/or at least 30 days.
  • Each possibility represents a separate embodiment of the invention.
  • the lipid of the present invention is alcoholytically stable for at least 24 hours, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 10 days, at least 15 days, at least 20 days and/or at least 30 days.
  • the lipid of the present invention is hydrolytically stable and alcoholytically stable upon immersion at room temperature in aqueous ethanol for at least 24 hours, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 10 days, at least 15 days, at least 20 days and/or at least 30 days.
  • Each possibility represents a separate embodiment of the invention
  • the present lipids may form lipid nanoparticles formulated with nucleic acids to facilitate their intracellular delivery both in vitro and for in vivo therapeutic applications.
  • the lipids of the present are required to be hydrolytically stable for time frames relevant for such applications.
  • ionizable lipids are prepared in batches and are typically stored in ethanol until use. Therefore, their alcoholytic stability is important.
  • no more than 10% of the lipid of the present invention decomposes in ethanol at RT after 15 days.
  • no more than 15% of the lipid of the present invention decomposes in ethanol at RT after 1 day.
  • no more than 20% of the lipid of the present invention decomposes in ethanol at RT after 15 days.
  • no more than 25% of the lipid of the present invention decomposes in ethanol at RT after 1 day.
  • no more than 30% of the lipid of the present invention decomposes in ethanol at RT after 15 days.
  • no more than 35% of the lipid of the present invention decomposes in ethanol at RT after 1 day.
  • no more than 20% of the lipid of the present invention decomposes in ethanol at RT after 30 days.
  • no more than 25% of the lipid of the present invention decomposes in ethanol at RT after 2 days.
  • no more than 30% of the lipid of the present invention decomposes in ethanol at RT after 30 days.
  • no more than 35% of the lipid of the present invention decomposes in ethanol at RT after 2 days.
  • no more than 40% of the lipid of the present invention decomposes in ethanol at RT after 30 days.
  • no more than 45% of the lipid of the present invention decomposes in ethanol at RT after 2 days.
  • no more than 25% of the lipid of the present invention decomposes in ethanol at RT after 90 days.
  • no more than 30% of the lipid of the present invention decomposes in ethanol at RT after 3 days.
  • no more than 35% of the lipid of the present invention decomposes in ethanol at RT after 90 days.
  • no more than 40% of the lipid of the present invention decomposes in ethanol at RT after 3 days.
  • no more than 45% of the lipid of the present invention decomposes in ethanol at RT after 90 days.
  • no more than 50% of the lipid of the present invention decomposes in ethanol at RT after 3 days.
  • no more than 30% of the lipid of the present invention decomposes in ethanol at RT after 1 year.
  • no more than 35% of the lipid of the present invention decomposes in ethanol at RT after 4 days.
  • no more than 40% of the lipid of the present invention decomposes in ethanol at RT after 1 year.
  • no more than 45% of the lipid of the present invention decomposes in ethanol at RT after 4 days.
  • no more than 50% of the lipid of the present invention decomposes in ethanol at RT after 1 year.
  • no more than 55% of the lipid of the present invention decomposes in ethanol at RT after 4 days.
  • no more than 20% of the lipid of the present invention decomposes in 75% ethanol 25% v/v water solution at RT after 15 days.
  • no more than 25% of the lipid of the present invention decomposes in 75% ethanol 25% v/v water solution at RT after 15 days.
  • no more than 30% of the lipid of the present invention decomposes in 75% ethanol 25% v/v water solution at RT after 15 days.
  • no more than 35% of the lipid of the present invention decomposes in 75% ethanol 25% v/v water solution at RT after 15 days.
  • no more than 40% of the lipid of the present invention decomposes in 75% ethanol 25% v/v water solution at RT after 15 days.
  • no more than 30% of the lipid of the present invention decomposes in 75% ethanol 25% v/v water solution at RT after 30 days.
  • no more than 35% of the lipid of the present invention decomposes in 75% ethanol 25% v/v water solution at RT after 30 days.
  • no more than 40% of the lipid of the present invention decomposes in 75% ethanol 25% v/v water solution at RT after 30 days.
  • no more than 45% of the lipid of the present invention decomposes in 75% ethanol 25% v/v water solution at RT after 30 days.
  • no more than 50% of the lipid of the present invention decomposes in 75% ethanol 25% v/v water solution at RT after 30 days. According to some embodiments, no more than 55% of the lipid of the present invention decomposes in 75% ethanol 25% v/v water solution at RT after 30 days.
  • no more than 50% of the lipid of the present invention decomposes in 75% ethanol 25% v/v water solution at RT after 1 year.
  • no more than 60% of the lipid of the present invention decomposes in 75% ethanol 25% v/v water solution at RT after 1 year.
  • no more than 65% of the lipid of the present invention decomposes in 75% ethanol 25% v/v water solution at RT after 1 year.
  • no more than 70% of the lipid of the present invention decomposes in 75% ethanol 25% v/v water solution at RT after 1 year.
  • no more than 75% of the lipid of the present invention decomposes in 75% ethanol 25% v/v water solution at RT after 1 year. According to some embodiments, no more than 80% of the lipid of the present invention decomposes in 75% ethanol 25% v/v water solution at RT after 1 year.
  • carbamate lipid refers to any lipid, which contains a carbamate moiety.
  • alkyl refers to any saturated aliphatic hydrocarbon, including straight-chain and branched-chain alkyl groups.
  • the alkyl group may be unsubstituted or substituted by one or more groups selected from halogen, hydroxy, alkoxy carbonyl, amido, alkylamido, dialkylamido, nitro, amino, alkylamino, dialkylamino, carboxyl, thio and thioalkyl.
  • C n-m alkyl refers to an alkyl group having n to m carbon atoms.
  • an unsubstituted C 17 alkyl may refer to the straight chain n-CnCss, or to any branched alkyl have the appropriate number of carbon atoms, e.g., -CHICsCi?) .
  • An alkyl group formally corresponds to an alkane with one C-H bond replaced by the point of attachment of the alkyl group to the remainder of the compound.
  • alkyl moieties include, but are not limited to, chemical groups such as methyl, ethyl, n-propyl, isopropyl, n-butyl, tert-butyl, isobutyl, sec-butyl; higher homologs such as 2- methyl-1 -butyl, 3-pentyl, hexyl, 1,2,2- trimethylpropyl and the like.
  • alkylene employed alone or in combination with other terms, refers to a divalent alkyl linking group.
  • An alkylene group formally corresponds to an alkane with two C-H bonds replaced by points of attachment of the alkylene group to the remainder of the compound.
  • C n-m alkylene refers to an alkylene group having n to m carbon atoms.
  • alkylene groups include, but are not limited to, ethan-1,2- diyl, ethan-l,l-diyl, propan-1, 3-diyl, propan-1, 2-diyl, propan-1, 1-diyl, butan-l,4-diyl, butan- 1, 3-diyl, butan-1,2- diyl, 2-methyl-propan-l, 3-diyl and the like.
  • Cn-m ranges include any sub range thereof, for example, C4- 14 alkyl may include and/or be directed to: C4-8 alkyl, C 8-14 alkyl, C6-12 alkyl, C9 alkyl etc.
  • alkenyl refers to an aliphatic hydrocarbon group containing at least one carboncarbon double bond including straight-chain, branched-chain and cyclic alkenyl groups.
  • alkenyl groups include ethenyl, propenyl, n-butenyl, i-butenyl, 3-methylbut-2- enyl, n-pentenyl, heptenyl, octenyl, cyclohexyl-butenyl and decenyl.
  • the alkenyl group can be unsubstituted or substituted through available carbon atoms with one or more groups defined hereinabove for alkyl.
  • Alkenyls according to the present invention may include more than one carbon-carbon double bond.
  • dienes see e.g., compound EA-513C, substituent R 2
  • trienes are within the definition of alkenyl.
  • the alkenyl is a dienyl.
  • C n-m alkenyl refers to an alkyl group having n to m carbon atoms.
  • C n-m alkenyl refers to an alkenyl group having n to m carbon atoms.
  • An alkenyl group formally corresponds to an alkene with one C-H bond replaced by the point of attachment of the alkenyl group to the remainder of the compound.
  • alkenyl moieties include, but are not limited to, chemical groups such as ethenyl, propenyl, isopropenyl, n- butenyl, sec-butenyl the like.
  • alkenylene employed alone or in combination with other terms, refers to a divalent alkenyl linking group.
  • An alkenylene group formally corresponds to an alkane with two C- H bonds replaced by points of attachment of the alkenylene group to the remainder of the compound.
  • C n-m alkenylene refers to an alkenylene group having n to m carbon atoms.
  • alkenyl chains are presented, e.g., C 2 -8 alkenyl, C 4.2 o alkenyl etc. It is to be understood that such ranges include any sub range thereof, for example, C4- 14 alkenyl may include and/or be directed to: C 4 -8 alkenyl, C 8- i 4 alkenyl, Ce-i 2 alkenyl, C9 alkenyl etc.
  • salt encompasses both basic and acid addition salts, including but not limited to, carboxylate salts or salts with amine nitrogen atoms, and include salts formed with the organic and inorganic anions and cations discussed below. Furthermore, the term includes salts that form by standard acid-base reactions with basic groups (such as amino groups) and organic or inorganic acids.
  • Such acids include hydrochloric, hydrofluoric, trifluoroacetic, sulfuric, phosphoric, acetic, succinic, citric, lactic, maleic, fumaric, palmitic, cholic, pamoic, mucic, D-glutamic, D-camphoric, glutaric, phthalic, tartaric, lauric, stearic, salicylic, methanesulfonic, benzenesulfonic, sorbic, picric, benzoic, cinnamic, and like acids.
  • organic or inorganic cation refers to counter-ions for the anion of a salt.
  • the counter-ions include, but are not limited to, alkali and alkaline earth metals (such as lithium, sodium, potassium, barium, aluminum and calcium); ammonium and mono-, di- and trialkyl amines such as trimethylamine, cyclohexylamine; and the organic cations, such as dibenzylammonium, benzylammonium, 2-hydroxyethylammonium, bis(2- hydroxyethyl)ammonium, phenylethylbenzylammonium, dibenzylethylenediammonium, and like cations. See, for example, Berge et al., J. Pharm. Sci. (1977), 66:1-19, which is incorporated herein by reference.
  • the present invention provides a particle comprising the lipid according to the present invention.
  • the present invention provides a particle comprising the lipid according to the present invention and at least one membrane stabilizing lipid.
  • the present invention provides a composition comprising a lipid according to any one of formulae (I), (la), (II), (III), (IV), (IVa) and (V), e.g., any one of lipids EA-402C, EA-404C, EA-405C, EA-406C, EA-408C, EA-502C, EA-505C, EA-506C, EA-513C, EA-521C, EA-522C, EA- 524C, EA-526C or a salt thereof and a pharmaceutically acceptable excipient.
  • compositions comprising a plurality of particles as discloses herein and a pharmaceutically acceptable carrier, diluent or excipient.
  • the composition is a liposomal composition.
  • the particles of the present invention are in the form of liposomes.
  • the ratio between the various lipids in the particle may vary. In some embodiments, the ratio is a molar ratio. In some embodiments, the ratio is a weight ratio. In some embodiments, each of the lipid groups may be at molar ratio/a weight ratio of about l%-99%, including each value and sub-range within the specified range.
  • the particle comprises 10% to 70% mol% of the lipid according to the present invention. According to some embodiments, the particle comprises 20% to 70% mol% of the lipid according to the present invention. According to some embodiments, the particle comprises 30% to 65% mol% of the lipid according to the present invention. According to some embodiments, the particle comprises 40% to 60% mol% of the lipid according to the present invention. According to some embodiments, the particle comprises 45% to 55% mol% of the lipid according to the present invention. According to some embodiments, the particle comprises about 50 mol% of the lipid according to the present invention.
  • the molar percentage of the lipid is at least 15 mol% of the particle. According to some embodiments, the molar percentage of the lipid is at least 25 mol% of the particle. According to some embodiments, the molar percentage of the lipid is at least 35 mol% of the particle. According to some embodiments, the molar percentage of the lipid is at least 45 mol% of the particle. According to some embodiments, the molar percentage of the lipid is no more than 75 mol% of the particle. According to some embodiments, the molar percentage of the lipid is no more than 65 mol% of the particle. According to some embodiments, the molar percentage of the lipid is no more than 60 mol% of the particle. According to some embodiments, the molar percentage of the lipid is no more than 55 mol% of the particle.
  • the molar percentage of the lipid is at least x mol% of the particle” it is meant that at least x% of the particle molecules are of the lipid.
  • the same terminology is reflected with other components of the present particle.
  • the unit “mol%” is also sometimes referred as “mokmol” or “% mokmol”.
  • the composition further comprises one or more components selected from the group consisting of a neutral lipid, a charged lipid, a steroid, and a polymer-conjugated lipid.
  • a neutral lipid a charged lipid
  • a steroid a polymer-conjugated lipid
  • the membrane stabilizing lipid is selected from the group consisting of a sterol, a phospholipid, a cephalin, a sphingolipid and a glycoglycerolipid. Each possibility represents a separate embodiment of the invention.
  • the membrane stabilizing lipid comprises a sterol.
  • the membrane stabilizing lipid comprises cholesterol.
  • the lipid nanoparticle formulation comprises 15 to 65 mol% of the sterol.
  • the lipid nanoparticle formulation comprises 20 to 60 mol% of the sterol.
  • the lipid nanoparticle formulation comprises 25 to 55 mol% of the sterol.
  • the lipid nanoparticle formulation comprises 30 to 50 mol% of the sterol. According to some embodiments, the lipid nanoparticle formulation comprises 35 to 45 mol% of the sterol. According to some embodiments, the lipid nanoparticle formulation comprises about 38 mol% of the sterol. According to some embodiments, the lipid nanoparticle formulation comprises at least 20 mol% of the sterol. According to some embodiments, the lipid nanoparticle formulation comprises at least 30 mol% of the sterol. According to some embodiments, the lipid nanoparticle formulation comprises at least 35 mol% of the sterol. According to some embodiments, the lipid nanoparticle formulation comprises no more than 80 mol% of the sterol.
  • the lipid nanoparticle formulation comprises no more than 70 mol% of the sterol. According to some embodiments, the lipid nanoparticle formulation comprises no more than 60 mol% of the sterol. According to some embodiments, the lipid nanoparticle formulation comprises no more than 50 mol% of the sterol.
  • the lipid nanoparticle formulation comprises 15 to 65 mol% cholesterol. According to some embodiments, the lipid nanoparticle formulation comprises 20 to 60 mol% cholesterol. According to some embodiments, the lipid nanoparticle formulation comprises 25 to 55 mol% cholesterol. According to some embodiments, the lipid nanoparticle formulation comprises 30 to 50 mol% cholesterol. According to some embodiments, the lipid nanoparticle formulation comprises 35 to 45 mol% cholesterol. According to some embodiments, the lipid nanoparticle formulation comprises about 38 mol% cholesterol. According to some embodiments, the lipid nanoparticle formulation comprises at least 20 mol% cholesterol. According to some embodiments, the lipid nanoparticle formulation comprises at least 30 mol% cholesterol.
  • the lipid nanoparticle formulation comprises at least 35 mol% cholesterol. According to some embodiments, the lipid nanoparticle formulation comprises no more than 80 mol% cholesterol. According to some embodiments, the lipid nanoparticle formulation comprises no more than 70 mol% cholesterol. According to some embodiments, the lipid nanoparticle formulation comprises no more than 60 mol% cholesterol. According to some embodiments, the lipid nanoparticle formulation comprises no more than 50 mol% cholesterol.
  • the membrane stabilizing lipids may be selected from, but not limited to: cholesterol, phospholipids (such as, for example, phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, phosphatidylserine, phosphatidylglycerol, diphosphatidylglycerols), cephalins, sphingolipids (sphingomyelins and glycosphingolipids), glycoglycerolipids, and combinations thereof.
  • phospholipids such as, for example, phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, phosphatidylserine, phosphatidylglycerol, diphosphatidylglycerols
  • cephalins such as, for example, phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, phosphatidy
  • the phosphatidylethanolamines may be selected from, but not limited to: 1,2-dilauroyl-L-phosphatidyl-ethanolamine (DLPE), l,2-Dioleoyl-sn-glycero-3- phosphoethanolamine (DOPE), l,2-Diphytanoyl-sn-glycero-3 -phosphoethanolamine (DPhPE) l,3-Dipalmitoyl-sn-glycero-2-phosphoethanolamine (1,3-DPPE), l-Palmitoyl-3- oleoyl-sn-glycero-2 -phosphoethanolamine ( 1 ,3-POPE), Biotin-Phosphatidylethanolamine, 1 ,2-Dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE),
  • DLPE 1,2-dilauroyl-L-phosphatidyl-ethanolamine
  • DOPE 1,2-Dilauroyl-L-phosphat
  • DPPE Dipalmitoylphosphatidylethanolamine
  • DSPE l,2-Distearoyl-sn-glycero-3- phosphoethanolamine
  • the Phosphatidylethanolamines may be conjugated to a PEG- Amine derivative.
  • the particle further comprising one or more additional components selected from the group consisting of a PEG-lipid conjugate, a neutral lipid and a charged lipid.
  • the additional component comprises 1,2- Distearoyl-sn-glycero-3-phosphocholine (DSPC).
  • the additional component comprises 1 ,2-Dimyristoyl-sn-glyceryl-methoxy polyethylene glycol (DMG-PEG).
  • the particle comprises 4 to 35 mol% DSPC. According to some embodiments, the particle comprises 5 to 30 mol% DSPC. According to some embodiments, the particle comprises 5 to 20 mol% DSPC. According to some embodiments, the particle comprises 6 to 15 mol% DSPC. According to some embodiments, the particle comprises 8 to 12 mol% DSPC. According to some embodiments, the particle comprises about 10 mol% DSPC.
  • the particle comprises at least 2 mol% DSPC. According to some embodiments, the particle comprises at least 3 mol% DSPC. According to some embodiments, the particle comprises at least 5 mol% DSPC. According to some embodiments, the particle comprises at least 7 mol% DSPC. According to some embodiments, the particle comprises at least 9 mol% DSPC.
  • the particle comprises no more than 40 mol% DSPC.
  • the particle comprises no more than 30 mol% DSPC.
  • the particle comprises no more than 25 mol% DSPC.
  • the particle comprises no more than 20 mol% DSPC.
  • the particle comprises no more than 15 mol% DSPC.
  • the particle further comprises a PEGylated lipid.
  • PEG refers to polyethylene glycol.
  • PEGylated lipid means a lipid that is bonded to PEG.
  • the PEGylated lipid comprises a PEG moiety having a molecular weight in the range of 1000 gr/mol to 3000 gr/mol, including each value and subrange within the specified range. According to some embodiments, the PEGylated lipid comprises a PEG moiety having a molecular weight in the range of 1000 gr/mol to 2000 gr/mol. According to some embodiments, the PEG moiety has a molecular weight of about 2000 gr/mol.
  • the PEGylated lipid comprises DMG-PEG. According to some embodiments, the PEGylated lipid comprises DMG-PEG-2000.
  • DMG-PEG means 1 ,2-Dimyristoyl-sn-glycero-3 -methoxypolyethylene glycol, or ,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol.
  • DMG-PEG-2000 means l,2-Dimyristoyl-sn-glycero-3-methoxypoly ethylene glycol, or ,2-dimyristoyl-rac- glycero-3-methoxypolyethylene glycol, wherein the polyethylene glycol has a molecular weight of about 2000 gr/mol.
  • the particle comprises 1 to 5 mol% PEGylated lipid.
  • the particle comprises 1 to 4 mol% PEGylated lipid.
  • the particle comprises 1 to 3 mol% PEGylated lipid.
  • the particle comprises 1.5 to 2.5 mol% PEGylated lipid.
  • the particle comprises about 2 mol% PEGylated lipid.
  • the particle comprises at least 1 mol% PEGylated lipid.
  • the particle comprises no more than 10 mol% PEGylated lipid. According to some embodiments, the particle comprises no more than 5 mol% PEGylated lipid.
  • the particle comprises the lipid of the present invention, cholesterol, 1 ,2-Distearoyl-sn-glycero-3 -phosphocholine (DSPC) and 1,2-Dimyristoyl-sn- glyceryl-methoxy polyethylene glycol (DMG-PEG).
  • particle comprises the lipid according to the present invention, a membrane stabilizing lipid, an additional phospholipid and PEG-lipid conjugate.
  • the particle comprises 40%-60% of the present lipid, 30- 50% mol% cholesterol, 5-15% DSPC and 0.5-5% of the PEG-lipid conjugate.
  • the particles of the present intention are nanoparticles.
  • the lipidic particles of the present intention are lipid nanoparticles.
  • the particle has average nanoparticle size (Z average) in the range of 10 to 500 nanometers. According to some embodiments, the particle has average nanoparticle size in the range of 25 to 400 nanometers. According to some embodiments, the particle has nanoparticle size (Z average) in the range of 50 to 200 nanometers. According to some embodiments, the particle has nanoparticle size (Z average) in the range of 50 to 100 nanometers. According to some embodiments, the particle has nanoparticle size (Z average) in the range of 60 to 95 nanometers. According to some embodiments, the particle has nanoparticle size (Z average) in the range of 65 to 90 nanometers.
  • the particle has nanoparticle size (Z average) in the range of 70 to 85 nanometers.
  • the particles (including any nucleic acid, therapeutic agent and the like encapsulated within and any targeting moiety conjugated thereto) have a particle size (diameter) in the range of about 10 to about 500 nm. In some embodiments, the particles have a particle size (diameter) in the range of about 10 to about 350 nm. In some embodiments, the particles have a particle size (diameter) in the range of about 40 to about 270 nm. In some embodiments, the particles have a particle size (diameter) in the range of over about 10 nm.
  • the particles have a particle size (diameter) of over about 20 nm. In some embodiments, the particles have a particle size (diameter) of over about 30 nm. In some embodiments, the particles have a particle size (diameter) of over about 40 nm. In some embodiments, the particles have a particle size (diameter) of over about 45 nm. In some embodiments, the particles have a particle size (diameter) of over about 50 nm. In some embodiments, the particles have a particle size (diameter) of over about 60 nm. In some embodiments, the particles have a particle size (diameter) of over about 70 nm.
  • the particles have a particle size (diameter) of not more than about 500 nm. In some embodiments, the particles have a particle size (diameter) of not more than about 250 nm. In some embodiments, the particles have a particle size (diameter) of not more than about 150 nm. In some embodiments, the particles have a particle size (diameter) of not more than about 100 nm. In some embodiments, the particles have a particle size (diameter) of not more than about 90 nm. In some embodiments, the size is a hydrodynamic diameter.
  • the particle has Zeta potential of at least ImV. According to some embodiments, the particle has Zeta potential of at least 1.5mV. According to some embodiments, the particle has Zeta potential of at least 2mV. According to some embodiments, the particle has Zeta potential of at least 2.5mV. According to some embodiments, the particle has Zeta potential of at least 2.9mV.
  • the particle has Zeta potential of no more than 25mV.
  • the particle has Zeta potential of no more than 15mV.
  • the particle has Zeta potential of no more than 12mV.
  • the particle has Zeta potential of no more than lOmV.
  • the particle has Zeta potential of no more than 8mV. According to some embodiments, the particle has Zeta potential in the range of 2 to 14 mV, including each value and sub-range within the specified range. According to some embodiments, the particle has Zeta potential in the range of 2.8 to 7.2 mV. According to some embodiments, the particle has Zeta potential in the range of 2.8 to 6.5 mV.
  • zeta potential refers to a physical measurement of a colloidal system by electrophoresis. It gives the value of the potential (in mV) of a colloid in a suspension at the boundary between the Stern layer and the diffuse layer.
  • the zeta potential in a colloidal system is the difference in potential between the immovable layer attached to the surface of the dispersed phase and the dispersion medium.
  • the zeta potential is related to stability of suspensions of particles. Zeta potential may be adjusted, in part, for example, by adjusting the concentration of an electrolyte in the buffer system.
  • the particle has polydispersity index (PDI) of no more than 0.75. According to some embodiments, the particle has PDI of no more than 0.5. According to some embodiments, the particle has PDI of no more than 0.25. According to some embodiments, the particle has PDI of no more than 0.2. According to some embodiments, the particle has PDI of no more than 0.1.
  • PDI polydispersity index
  • the particle further comprises a nucleic acid.
  • the nucleic acid is encapsulated within a particle comprising the lipid.
  • the nucleic acid is selected from the group consisting of small interfering RNA (siRNA), micro RNA (miRNA), antisense oligo nucleotides, messenger RNA (mRNA), ribozymes, pDNA, CRISPR mRNA, gRNA, circular RNA and immune stimulating nucleic acids.
  • the composition may further comprise a nucleic acid.
  • nucleic acids examples include small interfering RNA (siRNA), micro RNA (miRNA), antisense oligo nucleotides, messenger RNA (mRNA), ribozymes, pDNA, CRISPR mRNA, gRNA, circular RNA and immune stimulating nucleic acids.
  • siRNA small interfering RNA
  • miRNA micro RNA
  • antisense oligo nucleotides messenger RNA (mRNA)
  • mRNA messenger RNA
  • ribozymes pDNA
  • CRISPR mRNA CRISPR mRNA
  • gRNA gRNA
  • immune stimulating nucleic acids include a separate embodiment of the present invention.
  • the weight ratio between the nucleic acid and the lipid mixture may be adjusted so as to achieve maximal biological effect by the nucleic acid on the target site.
  • the ratio between the nucleic acid and the lipid phase may be 1: 1.
  • the weight ratio between the nucleic acid and the lipid phase may be 1 :2.
  • the weight ratio between the nucleic acid and the lipid phase may be 1:5.
  • the weight ratio between the nucleic acid and the lipid phase may be 1:10.
  • the weight ratio between the nucleic acid and the lipids phase may be 1:16.
  • the weight ratio between the nucleic acid and the lipid phase may be 1:20.
  • the weight ratio between the nucleic acid and the lipid phase is about 1: 1 to 1:20 (w:w).
  • the particle further comprises a therapeutic agent.
  • the therapeutic agent is encapsulated within a particle comprising the lipid.
  • the therapeutic agent is RNA comprising an open reading frame encoding a polypeptide that comprises a SARS-CoV-2 spike protein or an immunogenic fragment or variant thereof.
  • the therapeutic agent is RNA comprising an open reading frame encoding a polypeptide that comprises a SARS- CoV-2 spike protein, an immunogenic fragment of SARS-CoV-2 or a SARS-CoV-2 variant.
  • the therapeutic agent is RNA comprising an open reading frame encoding a polypeptide that comprises a SARS-CoV-2 spike protein.
  • a method of gene editing comprising the step of contacting a cell with a composition comprising a plurality of particles according to the present invention and a pharmaceutically acceptable carrier, diluent or excipient.
  • the present invention provides a method of gene editing, comprising the step of contacting a cell with a composition comprising a lipid of the present invention.
  • the cell is a cancer cell.
  • a method of gene silencing comprising the step of contacting a cell with a composition comprising a plurality of particles according to the present invention and a pharmaceutically acceptable carrier, diluent or excipient.
  • the present invention provides a method of gene silencing, comprising the step of contacting a cell with a composition comprising a lipid of the present invention.
  • the cell is a cancer cell.
  • the compositions of the present invention may be used as a delivery system to administer a therapeutic agent to its target location in the body.
  • the present invention relates to a method for administering a therapeutic agent, by preparing a composition comprising a lipid as described herein and a therapeutic agent, and administering the composition to a subject in need thereof.
  • the present invention relates to a method for administering a therapeutic agent, by preparing a particle as described herein comprising a therapeutic agent, and administering the composition to a subject in need thereof.
  • the method further comprises encapsulating the therapeutic agent within a particle comprising the lipid.
  • the therapeutic agent is RNA comprising an open reading frame encoding a polypeptide that comprises a SARS- CoV-2 spike protein, an immunogenic fragment of SARS-CoV-2 or a SARS-CoV-2 variant.
  • RNA comprising an open reading frame encoding a polypeptide that comprises a SARS- CoV-2 spike protein, an immunogenic fragment of SARS-CoV-2 or a SARS-CoV-2 variant.
  • the present invention provides novel lipids that enable the formulation of improved compositions for the in vitro and in vivo delivery of IVT- mRNA and/or other oligonucleotides.
  • these lipid nanoparticle compositions are useful for expression of protein encoded by mRNA.
  • these improved lipid nanoparticles compositions are useful for upregulation of endogenous protein expression by delivering miRNA inhibitors targeting one specific miRNA or a group of miRNA regulating one target mRNA or several mRNA.
  • these improved lipid nanoparticle compositions are useful for downregulating (e.g., silencing) the protein levels and/or mRNA levels of target genes.
  • the lipid nanoparticles are also useful for delivery of mRNA and plasmids for expression of transgenes.
  • the lipid nanoparticle compositions are useful for inducing a pharmacological effect resulting from expression of a protein, e.g., increased production of red blood cells through the delivery of a suitable erythropoietin mRNA, or protection against infection through delivery of mRNA encoding for a suitable antibody.
  • the lipid may be in the form of nanoparticles and administered as is.
  • the nanoparticles may be administered in a solution.
  • the nanoparticles may be formulated to a suitable pharmaceutical composition to be administered by any desired route of administration. Exemplary routes of administration include such routes as, but not limited to: topical, oral or parenteral.
  • compositions used may be in the form of solid, semi-solid or liquid dosage forms, such, as for example, tablets, suppositories, pills, capsules, powders, liquids, suspensions, or the like, preferably in unit dosage forms suitable for single administration of precise dosages.
  • the pharmaceutical compositions may include the particles, a pharmaceutical acceptable excipient, and, optionally, may include other medicinal agents, pharmaceutical agents, carriers, adjuvants, and the like. It is preferred that the pharmaceutically acceptable carrier be one which is inert to the nucleic acid encapsulated within the particles and which has no detrimental side effects or toxicity under the conditions of use.
  • the administration is localized. In some embodiments, the administration is systemic.
  • injectable formulations for parenteral administration can be prepared as liquid solutions or suspensions, solid forms suitable for solution or suspension in liquid prior to injection, or as emulsions.
  • Suitable excipients are, for example, water, saline, dextrose, glycerol, ethanol or the like.
  • the pharmaceutical compositions to be administered may also contain minor amounts of non-toxic auxiliary substances such as wetting or emulsifying agents, pH buffering agents and the like, such as for example, sodium acetate, sorbitan monolaurate, triethanolamine oleate, and the like.
  • Aqueous injection suspensions may also contain substances that increase the viscosity of the suspension, including, for example, sodium carboxymethylcellulose, sorbitol, and/or dextran.
  • the suspension may also contain stabilizers.
  • the parenteral formulations can be present in unit dose or multiple dose sealed containers, such as ampules and vials, and can be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, such as, for example, water, for injections immediately prior to use.
  • parenteral administration includes intravenous administration.
  • a pharmaceutically acceptable, non-toxic composition may be formed by the incorporation of any of the normally employed excipients, such as, for example, mannitol, lactose, starch, magnesium stearate, sodium saccharine, talcum, cellulose, sodium crosscarmellose, glucose, gelatin, sucrose, magnesium carbonate, and the like.
  • excipients such as, for example, mannitol, lactose, starch, magnesium stearate, sodium saccharine, talcum, cellulose, sodium crosscarmellose, glucose, gelatin, sucrose, magnesium carbonate, and the like.
  • Such compositions include solutions, suspensions, tablets, dispersible tablets, pills, capsules, powders, sustained release formulations and the like.
  • Formulations suitable for oral administration can consist of liquid solutions such as effective amounts of the compound(s) dissolved in diluents such as water, saline, or orange juice; sachets, lozenges, and troches, each containing a predetermined amount of the active ingredient as solids or granules; powders, suspensions in an appropriate liquid; and suitable emulsions.
  • Liquid formulations may include diluents such as water and alcohols, (such as, for example ethanol, benzyl alcohol, and the polyethylene alcohols), either with or without the addition of a pharmaceutically acceptable surfactant, suspending agents, or emulsifying agents.
  • the dosage and frequency of administration may be selected in relation to the pharmacological properties of the specific nucleic acids encapsulated within the particles.
  • the lipids of the present invention can be used alone or in combination with other lipid components such as neutral lipids, charged lipids, steroids (including, for example, sterols) and/or their analogs, and/or polymer conjugated lipids to form lipid nanoparticles for the delivery of therapeutic agents.
  • the lipid nanoparticles are used to deliver nucleic acids for the treatment of various diseases or conditions, in particular leukocyte associated conditions such as cancer.
  • the present invention relates to a method of treating a leukocyte associated condition, the method comprising the step of administering to a subject in need thereof a composition according to the present invention.
  • the leukocyte associated condition may be selected from the group consisting of cancer, infection, autoimmune diseases, neurodegenerative diseases and inflammation.
  • a method of treating a leukocyte associated condition comprising the step of administering to a subject in need thereof a composition comprising a plurality of particles according to the present invention and a pharmaceutically acceptable carrier, diluent or excipient.
  • the particle comprises a nucleic acid, such as, for example, siRNA, miRNA, shRNA, anti-sense RNA, and the like, which may be used in the treatment of various leukocyte-associated conditions, depending on the identity of the nucleic acid, the specific target leukocyte, and the like.
  • the nucleic acid encapsulated within the particles may be a nucleic acid capable of inducing silencing of a target gene.
  • the target gene may be any gene, the expression of which is related to the condition to be treated.
  • the target gene may be a gene selected from, but not limited to: growth factors (such as EGFR, PDGFR), genes related to angiogenesis pathways (such as VEGF, Integrins), genes involved in intracellular signaling pathways and cell cycle regulation (such as PI3K/AKT/mT0R, Ras/Raf/MAPK, PDK1, CHK1, PLK1, Cyclins).
  • growth factors such as EGFR, PDGFR
  • genes related to angiogenesis pathways such as VEGF, Integrins
  • genes involved in intracellular signaling pathways and cell cycle regulation such as PI3K/AKT/mT0R, Ras/Raf/MAPK, PDK1, CHK1, PLK1, Cyclins.
  • a combination of nucleic acids, each having one or more targets may be encapsulated within the particles.
  • exemplary leukocyte-associated conditions that may be treated by the targeted particles may be selected from, but not limited to: various types of cancer, various infections (such as, for example, viral infection, bacterial infection, fungal infection, and the like), autoimmune diseases, neurodegenerative diseases, inflammations, and the like.
  • various infections such as, for example, viral infection, bacterial infection, fungal infection, and the like
  • autoimmune diseases such as, for example, neurodegenerative diseases, inflammations, and the like.
  • the targeted particles comprising a nucleic acid may be used for the treatment of cancer.
  • cancer is a disorder in which a population of cells has become, in varying degrees, unresponsive to the control mechanisms that normally govern proliferation and differentiation.
  • the cancer is a blood cancer.
  • blood cancers are lymphoma, leukemia and myloma. Lymphomas may be divided into two categories: Hodgkin lymphoma and non-Hodgkin lymphoma. Most nonHodgkin lymphomas are B-cell lymphomas, that grow quickly (high-grade) or slowly (low- grade). There are 14 types of B-cell non-Hodgkin lymphomas, such as mantle cell lymphoma (MCL). The others are T-cell lymphomas.
  • MCL mantle cell lymphoma
  • the nucleic acid that may be used for the treatment of cancer is directed against a target gene, which is involved in the regulation of cell cycle.
  • the target gene may be Polo-like Kinase 1 (PLK), Cyclin DI, CHK1, Notch pathway genes.
  • the plurality of lipids of the lipid particles may be of natural or synthetic source and may be selected from, but not limited to: cationic lipids, phosphatidylethanolamines, ionized lipids, membrane stabilizing lipids, phospholipids, and the like, or combinations thereof. Each possibility represents a separate embodiment of the present invention.
  • the particle further comprises a targeting moiety connected to a component of the composition.
  • the particle is conjugated to a targeting moiety.
  • the targeting moiety may by conjugated to any one of the lipids included in the present particle.
  • the particles may be comprised of any one or more of the lipids of the present invention, a phospholipid (e.g. DSPC), a membrane stabilizing lipid (e.g. cholesterol), a PEG-lipid conjugate (e.g. DMG-PEG); at various mokmol ratios, and further conjugated to a targeting moiety, wherein the targeting moiety is conjugated, linked or attached to any one of the particle’s components.
  • a phospholipid e.g. DSPC
  • a membrane stabilizing lipid e.g. cholesterol
  • PEG-lipid conjugate e.g. DMG-PEG
  • nucleic acid As referred to herein, the terms “nucleic acid”, “nucleic acid molecules” “oligonucleotide”, “polynucleotide”, and “nucleotide” may interchangeably be used herein.
  • the terms are directed to polymers of deoxy ribonucleotides (DNA), ribonucleotides (RNA), and modified forms thereof in the form of a separate fragment or as a component of a larger construct, linear or branched, single stranded, double stranded, triple stranded, or hybrids thereof.
  • the term also encompasses RNA/DNA hybrids.
  • the polynucleotides may include sense and antisense oligonucleotide or polynucleotide sequences of DNA or RNA.
  • the DNA or RNA molecules may be, for example, but not limited to: complementary DNA (cDNA), genomic DNA, synthesized DNA, recombinant DNA, or a hybrid thereof or an RNA molecule such as, for example, mRNA, shRNA, siRNA, miRNA, Antisense RNA, and the like. Each possibility represents a separate embodiment of the present invention.
  • the terms further include oligonucleotides composed of naturally occurring bases, sugars, and covalent inter nucleoside linkages, as well as oligonucleotides having non-naturally occurring portions, which function similarly to respective naturally occurring portions.
  • polypeptide “peptide” and “protein” are used interchangeably herein to refer to a polymer of amino acid residues.
  • the terms apply to amino acid polymers in which one or more amino acid residue is an artificial chemical analogue of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers.
  • construct refers to an artificially assembled or isolated nucleic acid molecule which may include one or more nucleic acid sequences, wherein the nucleic acid sequences may include coding sequences (that is, sequence which encodes an end product), regulatory sequences, non-coding sequences, or any combination thereof.
  • construct includes, for example, vector but should not be seen as being limited thereto.
  • “Expression vector” refers to constructs that have the ability to incorporate and express heterologous nucleic acid fragments (such as, for example, DNA), in a foreign cell.
  • an expression vector comprises nucleic acid sequences/fragments (such as DNA, mRNA, tRNA, rRNA), capable of being transcribed.
  • nucleic acid sequences/fragments such as DNA, mRNA, tRNA, rRNA
  • Many prokaryotic and eukaryotic expression vectors are known and/or commercially available. Selection of appropriate expression vectors is within the knowledge of those having skill in the art.
  • the expression vector may encode for a double stranded RNA molecule in the target site.
  • the term "expression”, as used herein, refers to the production of a desired end-product molecule in a target cell.
  • the end-product molecule may include, for example an RNA molecule; a peptide or a protein; and the like; or combinations thereof.
  • the terms “introducing” and “transfection” may interchangeably be used and refer to the transfer of molecules, such as, for example, nucleic acids, polynucleotide molecules, vectors, and the like into a target cell(s), and more specifically into the interior of a membrane -enclosed space of a target cell(s).
  • the molecules can be “introduced” into the target cell(s) by any means known to those of skill in the art, for example as taught by Sambrook et al.
  • Means of “introducing” molecules into a cell include, for example, but are not limited to: heat shock, calcium phosphate transfection, PEI transfection, electroporation, lipofection, transfection reagent(s), viral-mediated transfer, and the like, or combinations thereof.
  • the transfection of the cell may be performed on any type of cell, of any origin, such as, for example, human cells, animal cells, plant cells, virus cell, and the like.
  • the cells may be selected from isolated cells, tissue cultured cells, cell lines, cells present within an organism body, and the like.
  • gene editing refers to genetic engineering in which one or more nucleotides are inserted, replaced, or removed from a genome. Gene editing can be performed using a nuclease (e.g., a natural-existing nuclease or an artificially engineered nuclease), such as Cas9 and the like. Gene editing includes, inter alia, gene silencing.
  • the term "silencing” as used herein refers to suppression of expression of a (target) gene. Gene silencing may be achieved via reduction of transcription, and may additionally or alternatively include post-transcriptional suppression of gene expression.
  • the degree of gene silencing can be complete, i.e., substantially abolishing production of the encoded gene product (yielding a null phenotype), although in general partial silencing of the gene expression is achieved, with some degree of expression remaining (yielding an intermediate phenotype). The term should not therefore be taken to require complete "silencing" of expression.
  • treating and “treatment” as used herein refers to abrogating, inhibiting, slowing or reversing the progression of a disease or condition, ameliorating clinical symptoms of a disease or condition or preventing the appearance of clinical symptoms of a disease or condition.
  • preventing is defined herein as barring a subject from acquiring a disorder or disease or condition.
  • treatment of cancer is directed to include one or more of the following: a decrease in the rate of growth of the cancer (i.e. the cancer still grows but at a slower rate); cessation of growth of the cancerous growth, i.e., stasis of the tumor growth, and, the tumor diminishes or is reduced in size.
  • the term also includes reduction in the number of metastases, reduction in the number of new metastases formed, slowing of the progression of cancer from one stage to the other and a decrease in the angiogenesis induced by the cancer. In most preferred cases, the tumor is totally eliminated. Additionally included in this term is lengthening of the survival period of the subject undergoing treatment, improving the well-being of the subject undergoing treatment, lengthening the time of diseases progression, tumor regression, and the like.
  • the cancer is a blood cancer.
  • the term “Leukocytes” is directed to white blood cells (WBCs), produced and derived from a multipotent, hematopoietic stem cell in the bone marrow.
  • the white blood cells have nuclei, and types of white blood cells can be classified in into five main types, including, neutrophils, eosinophils, basophils, lymphocytes, and monocytes, based on functional or physical characteristics.
  • the main types may be classified into subtypes.
  • lymphocytes include B cells, T cells, and NK cells.
  • B-cells for example, release antibodies and assist activation of T cells.
  • T cells for example, can be classified to several subtypes, including: T-helper cells (CD4+ Th) which activate and regulate T and B cells; cytotoxic T cells (CD8+) that can target and kill virus-infected cells and tumor cells; Gamma-delta T cells (y5 T cells) which can bridge between innate and adaptive immune responses and be involved in phagocytosis; and Regulatory (suppressor) T cells which modulate the immune system, maintain tolerance to self-antigens, and abrogate autoimmune conditions.
  • T-helper cells CD4+ Th
  • cytotoxic T cells CD8+
  • y5 T cells Gamma-delta T cells
  • Regulatory (suppressor) T cells which modulate the immune system, maintain tolerance to self-antigens, and abrogate autoimmune conditions.
  • Precursor 1 was synthesized according to previously described procedures [Ramishetti S, Hazan-Halevy I, Palakuri R, Chatterjee S, Naidu Gonna S, Dammes N, Freilich I, Kolik Shmuel L, Danino D, and Peer D “A Combinatorial Library of Lipid Nanoparticles for RNA Delivery to Leukocytes”. Adv Mater. (2020) 32, 1906128].
  • the carbamate moiety was introduced via p-Nitrophenyl chloroformate (PNPCF) activation of the alcohol and concomitant reaction with a nucleophile, namely N,N- dimethylethylenediamine.
  • PNPCF p-Nitrophenyl chloroformate
  • N, N- dimethyl ethylene diamine (O.lmL, 0.896 mmol, 2.0 equiv.) and followed by DMAP (10 mg, 0.089 mmol, 0.2 equiv.) were added and stirred for 16 h at room temperature. Then the reaction was quenched with water followed by extracted with EtOAc (3x15 mL) and washed with brine solution and dried over with anhydrous Na2SC>4. The solvent was evaporated, and the residue was purified by column chromatography using 0-10% IPA in CHCh to bestow EA-2C (273 mg, 90.69%) as pale-yellow oil.
  • N, N-dimethyl ethylene diamine (0.103 mL, 0.940 mmol, 2.0 equiv.) and followed by DMAP (11 mg, 0.094 mmol, 0.2 equiv.) were added and stirred for 16 h at room temperature. Then the reaction was quenched with water followed by extracted with EtOAc (3x15 mL) and washed with brine solution and dried over with anhydrous Na2SO4. The solvent was evaporated, and the residue was purified by column chromatography using 0-10% IPA in CHC13 to bestow EA-502C (304 mg, 81.94%) as pale -yellow oil.
  • N, N-dimethyl ethylene diamine (0.14 mL, 1.318 mmol, 2.0 equiv.) and followed by DMAP (16 mg, 0.1318 mmol, 0.2 equiv.) were added and stirred for 16 h at room temperature. Then the reaction was quenched with water followed by extracted with EtOAc (3 x 20 mL) and washed with brine solution and dried over with anhydrous Na2SO4. The solvent was evaporated, and the residue was purified by column chromatography using 0-10% IPA in CHC13 to bestow EA-513C (482mg, 91%) as pale -brown oil.
  • N, N-dimethyl ethylene diamine (0.10 mL, 0.985 mmol, 2.0 equiv.) and followed by DMAP (12 mg, 0.098 mmol, 0.2 equiv.) were added and stirred for 16 h at room temperature. Then the reaction was quenched with water followed by extracted with EtOAc (3 x 15 mL) and washed with brine solution and dried over with anhydrous Na2SO4. The solvent was evaporated, and the residue was purified by column chromatography using 0-10% IPA in CHC13 to bestow EA-516C (263 mg, 79.21%) as colorless oil.
  • N, N-dimethyl ethylene diamine (0.06 mL, 0.547 mmol, 2.0 equiv.) and followed by DMAP (6.5 mg, 0.0547mmol, 0.2 equiv.) were added and stirred for 16 h at room temperature. Then the reaction was quenched with water followed by extracted with EtOAc (3 x 10 mL) and washed with brine solution and dried over with anhydrous Na2SO4. The solvent was evaporated, and the residue was purified by column chromatography using 0-10% IPA in CH2C12 to bestow EA-505C (150 mg, 82.87%) as colorless oil.
  • Lipids were synthesized as described in Example 1. Cholesterol, DSPC (1,2- distearoyl-sn- glycero-3-phosphocholine), polyethylene glycol (PEG)-DMG ( 1 ,2-dimyristoyl-rac- glycerol), were purchased from Avanti Polar Lipids Inc.
  • lipid mixture ionizable cationic lipid, Cholesterol, DSPC and PEG-DMG at 50:38.5: 10:1.5 molar ratio
  • LUC mRNA 1:9 mole N to P ratio RNA to ionizable lipid
  • the particles were dialyzed against PBS (pH 7.4) for 24 hours with two buffer exchanges to remove ethanol.
  • the prepared LNPs comprising different lipids of the present invention were characterized using dynamic light scattering (DLS) and zeta potential measurements.
  • Table 1 represents the measured hydrodynamic diameter (Z-ave; d.nm), polydispersity index (PDI) and zeta potential (mV) of various LNPs (structures of comparative lipids are show in Ligure 7).
  • the resultant LNPs were spherical and uniformly distributed with a hydrodynamic diameter size of -50 - 260 nm, depending on the lipid in use. Additionally, LNP surface potentials were negative for some of the measured NLPs.
  • LNPs Physicochemical characterization of LNPs made of different lipids
  • the LNPs consist of an ionizable lipid, DSPC, cholesterol, and PEG-DMG (50: 10:38:2 molar ratio).
  • Fig. l.B a novel ionizable amino lipid library
  • the LNPs were -70-80 nm, were highly uniform with low polydispersity index (PDI) of 0.04 to 0.13 and had potential of 2.9 to 15.4 mV, as measured by DLS. All LNP formulations showed high RNA encapsulation efficiency (>96%) as measured by RiboGreen (Fig l.C).
  • EXAMPLE 3 GFP expression by mRNA-LNP in Z138 cells
  • Z138 cells were seeded in a 24-well plate, at a concentration of 200,000 cells/mL. The cells were transfected with different amounts of 6 LNP formulations, which were added to the 1.5mL media already in the plate. 48 hours post transfection, 750uL cells of each treatment were washed and taken for GFP measurement via flow cytometry. 300uL cells of each treatment were re-seeded in a 96-well plate in 3 wells of lOOuL each and were measured for cellular viability via XTT at 72 hours post transfection.
  • LNPs comprising EA-519, EA-402, EA-506C, EA-506CN, EA-405 lipids were prepared according to Example 2 and were encapsulated with mRNA encoding and were used to transfect Z138 cells
  • the level of GFP expression was measured by flow cytometry, showing improved efficiency for GFP expression by the use of EA-506C compared to the other mRNA-LNPs evaluated in this disclosure ( Figures 1A-1B).
  • LNPs comprising EA-2C, EA502-C, EA-506C and EA-405C lipids were prepared according to Example 2 and were encapsulated with mRNA encoding GFP and were used to transfect cells. The level of GFP expression was measured showing improved efficiency for GFP expression by the use of EA-506C compared to the other mRNA-LNPs evaluated ( Figures 2A-2B).
  • EXAMPLE 6 GFP expression by mRNA-LNPs in primary T-cells
  • Human PBMCs were activated with anti-CD3 and anti-CD28 antibodies together with IL2, IL15 and IL7.
  • primary T-cells which are the only cells remaining in the culture, were collected, and re-seeded without the addition of anti-CD3 and anti- CD28.
  • the day after, the cells were collected and reseeded in a 24-well plate, at a concentration of 200,000 cells/mL.
  • the cells were transfected with different amounts of 6 LNP formulations, which were added to the 1.5mL media already in the plate.
  • 48 hours post transfection 750uL cells of each treatment were washed and taken for GFP measurement via flow cytometry.
  • EXAMPLE 7 In vivo transfection in a murine model
  • mice All animal protocols were approved by Tel Aviv University Institutional Animal Care and Usage Committee and in accordance with current regulations and standards of the Israel Ministry of Health. The mice were housed and maintained in laminar flow cabinets under specific pathogen-free conditions. To evaluate LNPs activity. Six- to eight-week-old female C57BLj6 mice, (Envigo, Rehovot, Israel) mice were injected with either LNPs at Img per kg body weight to the retroorbital sinus or at Img per kg body weight to the quadriceps.
  • mice were injected intraperitonially with 15mg of XenoLight D-Luciferin (122799, PerkinElmer Inc.) and imaged by IVIS bioluminescence imaging system (IVIS SpectrumCT. PerkinElmer Inc.) under isoflurane anesthesia.
  • IVIS bioluminescence imaging system IVIS SpectrumCT. PerkinElmer Inc.
  • LNPs comprising EA-506, EA-506C, EA-2, EA-2C lipids were administered intramuscularly to mice, and the organs were excised and imaged to test their transfection rate in-vivo.
  • Figure 5 shows that the lipid EA-506C has an improved transfection efficiency compared to the other tested LNPs.
  • LNPs lipid nanoparticle
  • GPP mRNA Green Pluorescent Protein messenger ribonucleic acid
  • the LNPs include an ionizable lipid, DSPC, cholesterol, and PEG-DMG (50:10:38:2 molar ratio). Lor the screening, six different ionizable lipids were chosen (EA2C, EA502C, EA405C, EA506C, EA513C and EA524C, the structures of which are shown herein).
  • the LNP sizes were -70-80 nm, and highly uniform with low polydispersity index (PDI) of 0.04 to 0.13.
  • Table 2 represents the measured hydrodynamic diameter (Z-ave; d.nm), polydispersity index (PDI) and zeta potential (mV) of the LNPs.
  • Table 2 Physicochemical characterization of LNPs made of different lipids
  • EXAMPLE 8B In-Vitro Screening for mRNA Delivery to Different Cell Types
  • Example 8A the in vitro transfection efficiency of the different LNP formulations of Example 8A was evaluated, following transfection of five different cell types; Z138 Mantle Cell Lymphoma cell line, HepG2 Hepatocellular Carcinoma cell line, 0vcar8 ovarian cancer cell line, Detroit562 pharyngeal cancer cell line, and Primary T cells (Examples 8C-F and Example 8H).
  • EXAMPLE 8C Mantle Cell Lymphoma cell line
  • Z138 cells were seeded in a 24-well plate, at a concentration of 200,000 cells/mL.
  • the cells were transfected with increasing doses (0.25-2pg/mL) of each LNP formulation, which were added to the 1.5mL media already in the plate.
  • 48 hours post transfection 750pL cells of each treatment were washed and taken for GFP measurement via flow cytometry.
  • 300pL cells of each treatment were re-seeded in a 96-well plate in 3 wells of lOOpL each and were measured for cellular viability via XTT at 72 hours post transfection.
  • Figure 7A shows GFP mean fluorescence and percentage of GFP + cells analyzed by flow cytometry of LNPs comprising the lipids EA2C (full circles), EA502C (full squares), EA506C (full triangles), EA405C (empty triangles), EA513C (empty circles) and EA524C (empty squares), upon transfected at concentrations 0.25pg/ml, 0.5pg/ml, Ipg/ml and 2pg/ml.
  • Figure 7B shows the GFP mean fluorescence intensities of LNPs comprising the lipids EA2C, EA502C (large squares), EA506C (horizonal lines), EA405C (vertical lines), EA513C (diagonal lines) and EA524C (small squares), upon transfection of MCL cells at concentrations 0.25pg/ml (left block), 0.5pg/ml (second to left block), Ipg/ml (second to right block) and 2pg/ml (second to right block).
  • EXAMPLE 8D HepG2 Hepatocellular Carcinoma cell line
  • HepG2 cells were pre-seeded in a 24-well plate, at a concentration of 150,000 cells/well. 24 hours post seeding, the cells were transfected with increasing doses (0.05-0.5pg/mL) of each LNP formulation, which were added to the ImL media already in the plate. 48 hours post transfection, cells were collected with Trypsin and neutralized by media at a final volume of ImL. 300pL of the cells were taken and re-seeded in a 96-well plate in 3 wells of lOOpL each, and the rest were washed and taken for GFP measurement via flow cytometry. Cellular viability was measured in the 96-well plate via XTT at 72 hours post transfection.
  • Figure 8 shows the GFP mean fluorescence intensities of LNPs comprising the lipids EA2C, EA502C (large squares), EA506C (horizonal lines), EA405C (vertical lines), EA513C (diagonal lines) and EA524C (small squares), upon transfection of HCC cells at concentrations 0.25pg/ml (left block), 0.5pg/ml (second to left block), Ipg/ml (second to right block) and 2pg/ml (second to right block).
  • EXAMPLE 8E Ovcar8 ovarian cancer cell line
  • Ovcar8 cells were pre-seeded in a 24-well plate, at a concentration of 40,000 cells/well. 24 hours post seeding, the cells were transfected with increasing doses (0.025-0.5pg/mL) of each LNP formulation, which were added to the ImL media already in the plate. 48 hours post transfection, cells were collected with Trypsin and neutralized by media at a final volume of ImL. 300pL of the cells were taken and re-seeded in a 96-well plate in 3 wells of lOOpL each, and the rest was washed and taken for GFP measurement via flow cytometry. Cellular viability was measured in the 96-well plate via XTT at 72 hours post transfection.
  • Figure 9 shows the GFP mean fluorescence intensities of LNPs comprising the lipids EA2C, EA502C (large squares), EA506C (horizonal lines), EA405C (vertical lines), EA513C (diagonal lines) and EA524C (small squares), upon transfection of Ovcar8 cells at concentrations 0.25pg/ml (left block), 0.5pg/ml (second to left block), Ipg/ml (second to right block) and 2pg/ml (second to right block).
  • EXAMPLE 8F Detroit562 pharyngeal cancer cell line
  • Detroit562 cells were pre-seeded in a 24-well plate, at a concentration of 50,000 cells/well. 24 hours post seeding, the cells were transfected with increasing doses (0.025-0.5pg/mL) of each LNP formulation, which were added to the ImL media already in the plate. 48 hours post transfection, cells were collected with Trypsin and neutralized by media at a final volume of ImL. 300pL of the cells were taken and re-seeded in a 96-well plate in 3 wells of lOOpL each, and the rest was washed and taken for GFP measurement via flow cytometry. Cellular viability was measured in the 96-well plate via XTT at 72 hours post transfection.
  • Figure 10 shows the GFP mean fluorescence intensities of LNPs comprising the lipids EA2C, EA502C (large squares), EA506C (horizonal lines), EA405C (vertical lines), EA513C (diagonal lines) and EA524C (small squares), upon transfection of Detroit562 pharyngeal cancer cells at concentrations 0.25pg/ml (left block), 0.5pg/ml (second to left block), Ipg/ml (second to right block) and 2pg/ml (second to right block).
  • EXAMPLE 8G mRNA Delivery to Different Cell Types - summary
  • Figure 11 is a heat map depicting cancer cell lines: ovcar8, Detroid562, HepG2 and Z138 versus LNPs expressed therein, the LNPs comprising the lipids EA2C, EA502C, EA506C, EA405C, EA513C and EA524C. Optimized concentration is shown for each cell line.
  • human PBMCs were activated with anti-CD3 and anti-CD28 antibodies together with IL2, IL 15 and IL7.
  • primary T-cells which are the only cells remaining in the culture, were collected, and re-seeded without the addition of anti-CD3 and anti-CD28.
  • the cells were transfected with increasing doses (0.25-4pg/mL) of four out of the six LNP formulations, which were added to the 1.5mL media already in the plate.
  • 48 hours post transfection 750pL cells of each treatment were washed and taken for GPP measurement via flow cytometry. 300pL cells of each treatment were re-seeded in a 96-well plate in 3 wells of lOOpL each and were measured for cellular viability via XTT at 72 hours post transfection.
  • Figure 12A shows GFP mean fluorescence and percentage of GFP + Primary T cells analyzed by flow cytometry of LNPs comprising the lipids EA2C (full circles), EA502C (full squares), EA506C (full triangles), EA405C (empty triangles), EA513C (empty circles) and EA524C (empty squares), upon transfected at concentrations 0.25pg/ml, 0.5pg/ml, Ipg/ml and 2pg/ml.
  • Figure 12B shows the GFP mean fluorescence intensities of LNPs comprising the lipids EA2C, EA502C (large squares), EA506C (horizonal lines), EA405C (vertical lines), EA513C (diagonal lines) and EA524C (small squares), upon transfection of Primary T cells at concentrations 0.25pg/ml (left block), 0.5pg/ml (second to left block), Ipg/ml (second to right block) and 2pg/ml (second to right block).
  • LNPs were formulation with Cas9 mRNA and sgRNAs targeting either HPRT or NCAPG.
  • the LNPs consist of the EA513C ionizable lipid, DSPC, cholesterol, and PEG-DMG (50: 10.5:38:1.5 molar ratio).
  • the LNPs were -104-115 nm and were highly uniform with low poly dispersity index (PDI) of 0.04 to 0.07 as measured by DLS. Both LNP formulations showed high RNA encapsulation efficiency (>96%) as measured by RiboGreen.
  • Table 2 represents the measured hydrodynamic diameter (Z-ave; d.nm), polydispersity index (PDI) and zeta potential (mV) of the LNPs.
  • HepG2 cells were seeded at 150,000 cells per well in 1ml media. 24 hours post seeding, cells were transfected with 0.25pg/mL of LNP (Example 9 A) added directly to the media. 72 hours post transfection, cells were collected with trypsin and media in a final volume of ImL, 300ul of which was reseeded as previously described for XTT measurement, while the rest of the cells were taken to DNA extraction. DNA was extracted and the HPRT and NCAPG loci were amplified in PCR.
  • Figure 13A shows % Indel mutations upon gene editing in HCC cells with sgHPRT-LNPs and sgNCAPG-LNPs.
  • Figure 13B shows cellular viability measured via XTT upon gene editing in HCC cells with sgHPRT-LNPs and sgNCAPG-LNPs.
  • LNPs comprising lipids EA405C and EA506C were designed to encapsulate Cas9 mRNA and a Cy 5 -fluorescently labeled siRNA in 1 : 1 weight ratio.
  • R2G2 mice were injected with 1.5xl0 6 Z138 cells that constitutively express luciferase (Z138-Luc), through the caudal artery of immunodeficient mice. The mice were monitored twice a week by weight measurement and luciferase expression measurement by IVIS, indicating the engraftment levels of the cancer cells. As determined by the intensity of luciferase signal, on day 24, mice were mock-treated or injected retro-orbitally with either EA506C or EA405C CRISPR-Cy5 LNPs.
  • liver, spleen, kidneys, heart, lungs, and femurs were extracted 4h post injection and analyzed by IVIS live imaging. Localization of Cy5 signal in the femurs of the treated mice was evident ( Figures 14A-C).
  • Figure 14A shows fluorescence imaging of femurs 4h after injection to MCL bearing mice LNPs comprising the lipids EA506C and EA405C.
  • Figure 14B shows Relative Total Flux [p/s] of liver, spleen, kidneys, heart and lungs and femurus normalized to the mock after injection to MCL bearing mice LNPs comprising the lipids EA506C and EA405C.
  • Figure 14C shows Total Flux [p/s] of the femurs normalized to the mock after injection to MCL bearing mice LNPs comprising the lipids EA506C and EA405C.
  • mice were injected with 1.5xl0 6 Z138-Luc cells, through the caudal artery.
  • the mice were monitored twice a week by weight measurement and luciferase expression measurement by IVIS.
  • mice were mock-treated or injected retro-orbitally with EA524C CRISPR-Cy5 LNPs.
  • the liver, spleen, kidneys, heart, lungs, and femurs were extracted 4h post injection and analyzed by IVIS live imaging. Localization of Cy5 signal in the femurs of the treated mice was evident ( Figures 15A-C).
  • Figure 15A shows fluorescence imaging of femurs 4h after injection to MCL bearing mice LNPs comprising the lipid EA524C.
  • Figure 15B shows Relative Total Flux [p/s] of liver, spleen, kidneys, heart and lungs and femurus normalized to the mock after injection to MCL bearing mice LNPs comprising the lipid EA524C.
  • Figure 15C shows Total Flux [p/s] of the femurs normalized to the mock after injection to MCL bearing mice LNPs comprising the lipid EA524C.
  • EXAMPLE 11 In vivo gene editing in MCL R2G2 mouse model - comparison study EA524 vs. EA524C.
  • LNPs were formulation with Cas9 mRNA and sgRNAs targeting SOX11.
  • the LNPs included the EA524C ionizable lipid, DSPC, cholesterol, and PEG-DMG (50: 10.5:38: 1.5 molar ratio) or with EA524 (which has a similar structure to the EA524C, but with a core ester group instead of carbamate, see Figure 6).
  • the LNPs were ⁇ 89-96nm and were highly uniform with low poly dispersity index (PDI) of 0.04 as measured by DLS. BothLNP formulations showed high RNA encapsulation efficiency (>96%) as measured by RiboGreen.
  • Table 3 represents the measured hydrodynamic diameter (Z-ave; d.nm), polydispersity index (PDI) and zeta potential (mV) of the LNPs.
  • LNPs formulations (EA524 or EA524C) with sgGFP (as a negative control) and with sgSOXl l were injected i.v. into R2G2 MCL-Bearing Mice (as detailed above) at day 33 from tumor implantation.
  • MCL cells were isolated from the BM and sorted via FACSOrting (CD38+, CD138+) and subjected to NGS. All doses were at 0.5mg/Kg as a single dose.
  • Figure 16 is a bar chart of % gene editing using EA524-LNPs and EA524C-LNPs formulations encapsulated with sgSOXl l and with sgGFP.
  • EXAMPLE 12 Hydrolysis stability of hydrolytically stable lipids

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Abstract

La présente invention concerne des lipides et des formulations de nanoparticules lipidiques comprenant ces lipides, seuls ou en combinaison avec d'autres lipides. Ces nanoparticules lipidiques peuvent être formulées avec des acides nucléiques pour faciliter leur administration intracellulaire à la fois in vitro et pour des applications thérapeutiques in vivo. Les lipides selon la présente invention sont caractérisés en ce qu'ils sont particulièrement hydrolytiquement et alcoolytiquement stables, tout en conservant leur activité biologique.
PCT/IL2024/050567 2023-06-13 2024-06-09 Lipides de carbamate biodégradables présentant une stabilité accrue Ceased WO2024257088A1 (fr)

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IL325271A IL325271A (en) 2023-06-13 2024-06-09 Biodegradable carbamate lipids with increased stability
AU2024302998A AU2024302998A1 (en) 2023-06-13 2024-06-09 Biodegradable carbamate lipids with increased stability
CN202480051966.XA CN121729406A (zh) 2023-06-13 2024-06-09 具有提高的稳定性的可生物降解的氨基甲酸酯脂质
KR1020267000768A KR20260048536A (ko) 2023-06-13 2024-06-09 증가된 안정성을 갖는 생분해성 카바메이트 지질
EP24822968.4A EP4727917A1 (fr) 2023-06-13 2024-06-09 Lipides de carbamate biodégradables présentant une stabilité accrue

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KR (1) KR20260048536A (fr)
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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2014007398A1 (fr) * 2012-07-06 2014-01-09 協和発酵キリン株式会社 Lipide cationique
WO2018087753A1 (fr) * 2016-11-08 2018-05-17 Technology Innovation Momentum Fund (Israel) Limited Partnership Lipides cationiques pour l'administration d'acides nucléiques et leur préparation
WO2022168085A1 (fr) * 2021-02-02 2022-08-11 Ramot At Tel-Aviv University Ltd. Lipides appropriés pour l'administration d'acides nucléiques

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2014007398A1 (fr) * 2012-07-06 2014-01-09 協和発酵キリン株式会社 Lipide cationique
WO2018087753A1 (fr) * 2016-11-08 2018-05-17 Technology Innovation Momentum Fund (Israel) Limited Partnership Lipides cationiques pour l'administration d'acides nucléiques et leur préparation
WO2022168085A1 (fr) * 2021-02-02 2022-08-11 Ramot At Tel-Aviv University Ltd. Lipides appropriés pour l'administration d'acides nucléiques

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AU2024302998A1 (en) 2026-01-22
CN121729406A (zh) 2026-03-24
IL325271A (en) 2026-02-01
KR20260048536A (ko) 2026-04-10

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