EP-4734954-A1 - LIPID NANOPARTICLE FORMULATIONS FOR CELL THERAPY AND RELATED METHODS

EP4734954A1EP 4734954 A1EP4734954 A1EP 4734954A1EP-4734954-A1

Abstract

Provided is a lipid formulation which includes cholesteryl hemisuccinate as a cholesterol substitute and which is mixed with nucleic acid to form lipid nucleic acid nanoparticles which are capable of transfecting cells of the immune system.

Inventors

HARVIE, PIERROT
JEFFS, Lloyd Brian
ZHANG, Ruo Yu
KAZEMIAN, Mohammadreza
CHAKRAPANI, Harish
JAIN, NIKITA
THOMAS, Anitha
KONDRATOWICZ, Andrew

Assignees

Global Life Sciences Solutions Canada ULC

Dates

Publication Date: 20260506
Application Date: 20231222

Claims (1)

Leydig 770158/US DHR P2023-3706-WO01 68 CLAIM(S): 1. A lipid formulation for forming a lipid-based nanoparticle suitable for transfecting nucleic acid cargo into cells of hematopoietic lineage comprising an ionizable lipid, a phospholipid, a sterol, and optionally a stabilizer. 2. A lipid formulation comprising an ionizable lipid, a phospholipid, a sterol, and optionally a stabilizer, for forming a lipid-based nanoparticle suitable for transfecting a nucleic acid cargo into T cells, Jurkat cells, hematopoietic stem cells, natural killer cells, or antigen presenting cells. 3. The lipid formulation of claim 1, further comprising a nucleic acid cargo. 4. The lipid formulation of claim 2, further comprising a nucleic acid cargo. 5. The lipid formulation of claim 4, wherein the nucleic acid cargo comprises chimeric antigen receptor (CAR) encoded mRNA. 6. The lipid formulation of claim 5, wherein the transfected cells are T cells. 7. The lipid formulation of claim 5, wherein the transfected cells are hematopoietic stem cells. 8. The lipid formulation of claim 4, wherein the nucleic acid cargo comprises a gene editing nuclease and a guide RNA to perform permanent gene knockout and/or insertion. 9. The lipid formulation of claim 8, wherein the transfected cells are T cells. 10. The lipid formulation of claim 8, wherein the transfected cells are hematopoietic stem cells. 11. The lipid formulation of claim 3, wherein the nucleic acid cargo comprises an mRNA encoding a protein to correct a genetic deficiency. 12. The lipid formulation of claim 3, wherein the nucleic acid cargo comprises an mRNA encoding a protein to treat disease. Leydig 770158/US DHR P2023-3706-WO01 69 13. The lipid formulation of any of claims 1-12, wherein the stabilizer comprises from about 0.1 to about 10.0 %Mol. 14. The lipid formulation of claim 13, wherein the stabilizer is a PEGylated lipid. 15. The lipid formulation of claim 13, wherein the stabilizer is a polysarcosine. 16. The lipid formulation of claim 13, wherein the stabilizer is vitamin E polyethylene glycol succinate (TPGS). 17. The lipid formulation of any of claims 1-12, wherein the stabilizer is absent. 18. The lipid formulation of any one of claims 1-17, wherein the ionizable lipid comprises from about 0.1 to about 85 %Mol. 19. The lipid formulation of any one of claims 1-18 wherein the ionizable lipid has a jasmonic acid headgroup. 20. The lipid formulation of any one of claims 1-18 wherein the ionizable lipid has a tetrahydrofuran head group. 21. The lipid formulation of any of claims 1-18, wherein the ionizable lipid is selected from the group consisting of (Z)-3-(2-((1,17-bis(2-octylcyclopropyl)heptadecan-9- yl)oxy)-2-oxoethyl)-2-(pent-2-en-1-yl)cyclopentyl 4-(dimethylamino)butanoate (PNI 516), 3-(2-((1,17-bis(2-octylcyclopropyl)heptadecan-9-yl)oxy)-2-oxoethyl)cyclopentyl 4- (dimethylamino)butanoate (PNI 550), Z)-3-(2-((1,17-bis(2-octylcyclopropyl)heptadecan-9- yl)oxy)-2-oxoethyl)-2-(pent-2-en-1-yl)cyclopentyl 1,4-dimethylpiperidine-4-carboxylate (PNI 560), (2R,3S,4S)-2-(((1,4-dimethylpiperidine-4-carbonyl)oxy)methyl)tetrahydrofuran- 3,4-diyl (9Z,9'Z,12Z,12'Z)-bis(octadeca-9,12-dienoate) (PNI 127), (2R,3S,4S)-2-(((4- (dimethylamino)butanoyl)oxy)methyl)tetrahydrofuran-3,4-diyl bis(2-hexyldecanoate) (PNI 580), ((2R,3R,4S)-3,4-bis((2-hexyldecyl)oxy)tetrahydrofuran-2-yl)methyl 4- (dimethylamino)butanoate (PNI 659), (2R,3S,4S)-2-((((2- (dimethylamino)ethyl)carbamoyl)oxy)methyl)tetrahydrofuran-3,4-diyl bis(2- hexyldecanoate) (PNI 721), 2-(((2R,3R,4S)-3,4-bis((2-hexyldecyl)oxy)tetrahydrofuran-2- yl)methoxy)-N,N-dimethylethan-1-amine (PNI 722), ((2R,3R,4S)-3,4-bis((2- hexyldecyl)oxy)tetrahydrofuran-2-yl)methyl 4-(diethylamino)butanoate (PNI 723), (2R,3S,4S)-2-((3-(dimethylamino)propoxy)methyl)tetrahydrofuran-3,4-diyl bis(2- Leydig 770158/US DHR P2023-3706-WO01 70 hexyldecanoate) (PNI 726), ((2R,3R,4S)-3,4-bis((2-hexyldecyl)oxy)tetrahydrofuran-2- yl)methyl (2-(dimethylamino)ethyl)carbamate (PNI 728), and (2R,3S,4S)-2-((2- (dimethylamino)ethoxy)methyl)tetrahydrofuran-3,4-diyl bis(2-hexyldecanoate) (PNI 730). 22. The lipid formulation of any of claims 1-21, wherein the ionizable lipid comprises a mixture of ionizable lipids 23. The lipid formulation of any of claims 1-22, wherein the phospholipid comprises from about 0.1 to about 85 %Mol. 24. The lipid formulation of any of claims 1-23, wherein the phospholipid is distearoylphosphatidylcholine (DSPC). 25. The lipid formulation of any of claims 1-24, wherein the phospholipid is dioleoyl-phosphatidylethanolamine (DOPE). 26. The lipid formulation of any of claims 1-25, wherein the sterol comprises from about 0.1 to about 85 % Mol. 27. The lipid formulation of any of claims 1-26, wherein the sterol is cholesteryl hemisuccinate. 28. The lipid formulations of any of claims 1-27, wherein the formulation is substantially free of cholesterol. 29. The lipid formulation of claim 1 or 2, wherein the ionizable lipid is PNI 516, PNI 659, or PNI 721 at from about 40 to about 60 %Mol (e.g.48 %Mol), the phospholipid is DSPC at from about 5 to about 20 %Mol (e.g.13 %Mol), and wherein the sterol is cholesteryl hemisuccinate at from about 30 to about 50 %Mol (e.g.39 %Mol). 30. The lipid formulation of claim 1 or 2, wherein the ionizable lipid is PNI 516, PNI 659, or PNI 721at from about 45 to about 55 %Mol (e.g.48 %Mol), the phospholipid is DSPC at from about 10 to about 15 %Mol (e.g.13 %Mol), and wherein the sterol is cholesteryl hemisuccinate at from about 35 to about 45 %Mol (e.g.39 %Mol). 31. The lipid formulation of claim 1 or 2, wherein the ionizable lipid is PNI 516, PNI 659, or PNI 721at from about 50 to about 70 %Mol (e.g.61.1 %Mol), the phospholipid Leydig 770158/US DHR P2023-3706-WO01 71 is DSPC at from about 0.1 to about 10 %Mol (e.g.0.5 %Mol), and wherein the sterol is cholesteryl hemisuccinate at from about 30 to about 50 %Mol (e.g.38.4 %Mol). 32. The lipid formulation of claim 1 or 2, wherein the ionizable lipid is PNI 516, PNI 659, or PNI 721 at from about 55 to about 65 %Mol (e.g.61.1 %Mol), the phospholipid is DSPC at from about 0.1 to about 1.0 %Mol (e.g.0.5 %Mol), and wherein the sterol is cholesteryl hemisuccinate at from about 35 to about 45 %Mol (e.g.38.4 %Mol). 33. The lipid formulation of claim 1 or 2, wherein the ionizable lipid is PNI 516, PNI 659, or PNI 721 at from about 1 to about 20 %Mol (e.g.10.5 %Mol), the phospholipid is DSPC at from about 65 to about 85 %Mol (e.g.75.5 %Mol), and wherein the sterol is cholesteryl hemisuccinate at from about 5 to about 25 %Mol (e.g.14 %Mol). 34. The lipid formulation of claim 1 or 2, wherein the ionizable lipid is PNI 516, PNI 659, or PNI 721 at from about 5 to about 15 %Mol (e.g.10.5 %Mol), the phospholipid is DSPC at from about 70 to about 80 %Mol (e.g.75.5 %Mol), and wherein the sterol is cholesteryl hemisuccinate at from about 10 to about 20 %Mol (e.g.14 %Mol). 35. The lipid formulation of claim 1 or 2, wherein the ionizable lipid is PNI 516, PNI 659, or PNI 721 at from about 30 to about 50 %Mol (e.g.37.9 %Mol), the phospholipid is DSPC at from about 1 to about 20 %Mol (e.g.10.5 %Mol), and wherein the sterol is cholesteryl hemisuccinate at from about 40 to about 60 %Mol (e.g.51.6 %Mol). 36. The lipid formulation of claim 1 or 2, wherein the ionizable lipid is PNI 516, PNI 659, or PNI 721 at from about 35 to about 45 %Mol (e.g.37.9 %Mol), the phospholipid is DSPC at from about 5 to about 15 %Mol (e.g.10.5 %Mol), and wherein the sterol is cholesteryl hemisuccinate at from about 45 to about 55 %Mol (e.g.51.6 %Mol). 37. The lipid formulation of claim 1 or 2, wherein the ionizable lipid is PNI 516, PNI 659, or PNI 721 at from about 15 to about 35 %Mol (e.g.24 %Mol), the phospholipid is DSPC at from about 0.1 to about 10 %Mol (e.g.0.5 %Mol), and wherein the sterol is cholesteryl hemisuccinate at from about 65 to about 85 %Mol (e.g.75.5 %Mol). 38. The lipid formulation of claim 1 or 2, wherein the ionizable lipid is PNI 516, PNI 659, or PNI 721 at from about 20 to about 30 %Mol (e.g.24 %Mol), the phospholipid is Leydig 770158/US DHR P2023-3706-WO01 72 DSPC at from about 0.1 to about 1.0 %Mol (e.g.0.5 %Mol), and wherein the sterol is cholesteryl hemisuccinate at from about 70 to about 80 %Mol (e.g.75.5 %Mol). 39. The lipid formulation of claim 1 or 2, wherein the ionizable lipid is PNI 516, PNI 659, or PNI 721 at from about 30 to about 50 %Mol (e.g.39.1 %Mol), the phospholipid is DSPC at from about 20 to about 40 %Mol (e.g.30.4 %Mol), and wherein the sterol is cholesteryl hemisuccinate at from about 20 to about 40 %Mol (e.g.30.5 %Mol). 40. The lipid formulation of claim 1 or 2, wherein the ionizable lipid is PNI 516, PNI 659, or PNI 721 at from about 35 to about 45 %Mol (e.g.39.1 %Mol), the phospholipid is DSPC at from about 25 to about 35 %Mol (e.g.30.4 %Mol), and wherein the sterol is cholesteryl hemisuccinate at from about 25 to about 35 %Mol (e.g.30.5 %Mol). 41. A lipid nanoparticle comprising the lipid formulation of any of claims 3-40. 42. A lipid nanoparticle comprising the lipid formulation of claim 1 or 2. 43. The lipid nanoparticle of claim 41 or 42, further comprising a peptide or polypeptide. 44. A method of preferentially transfecting cells of hematopoietic lineage using the lipid nanoparticles of any one of claims 41-43, the method comprising contacting the cells with lipid nanoparticles, wherein the cells maintain at least 50 percent immune cell viability post contact. 45. The method of claim 44, wherein the nucleic acid cargo comprises two or more nucleic acids. 46. The method of claim 45, wherein the two or more nucleic acids are packaged in separate nanoparticles. 47. The method of any of claims 44-46, wherein the transfection is performed in vitro. 48. The method of any of claims 44-46, wherein the transfection is performed in vivo. Leydig 770158/US DHR P2023-3706-WO01 73 49. The method of any of claims 44-46, wherein the transfection is performed ex vivo. 50. A pharmaceutical composition, comprising the lipid nanoparticle of any one of claims 41-43 and a pharmaceutically acceptable excipient. 51. A vaccine comprising the lipid formulation of any of claims 3-40 or the lipid nanoparticle of any of claims 41-43, wherein the nucleic acid cargo is a nucleic acid vaccine element. 52. The vaccine of claim 51, wherein the nucleic acid vaccine element encodes an antigen selected from coronavirus spike protein or influenza hemagglutinin protein. 53. The vaccine of claim 51, wherein the nucleic acid vaccine element is derived from influenza virus. 54. The vaccine of claim 51, wherein the nucleic acid vaccine element is derived from coronavirus. 55. The vaccine of claim 51, wherein the nucleic acid vaccine element encodes an antigen to cancer cells or solid tumors. 56. A method of treating or preventing a disease in a subject, the method comprising administering to the subject an effective amount of the lipid nanoparticle of any of claims 41-43 or the pharmaceutical composition of claim 50 or the vaccine of any of claims 51-55 to transfect cells of hematopoietic lineage. 57. The method of claim 56, wherein the cells of hematopoietic lineage are T cells, Jurkat cells, hematopoietic stem cells, natural killer cells, or antigen presenting cells. 58. The method of claim 56 or 57, wherein the administration is in vivo. 59. The method of claim 58, wherein the administration is intravenous, intramuscular, intrathecal, or intraperitoneal by bolus injection. 60. The method of claim 58, wherein the method comprises administering to the subject an effective amount of the vaccine of any of claims 50-54 and the administration is intramuscular. Leydig 770158/US DHR P2023-3706-WO01 74 61. The method of claim 58, wherein the administration is intravenous and wherein the cells of hematopoietic lineage are in the liver of a subject. 62. The method of any of claims 56-61, wherein an effective amount of a lipid nanoparticle is from about 0.1 to about 2.5 mg/kg of a subject. 63. The method of claim 56 or 57, wherein the administration is ex vivo. 64. The method of claim 63, wherein cells of hematopoietic lineage are (i) removed from the subject, (ii) transfected, (iii) washed, and (iv) returned to the subject. 65. The method of claim 63 or 64, wherein the nucleic acid cargo is a RNA encoding a chimeric antigen receptor. 66. The method of any of claims 63-65, wherein an effective amount of a lipid nanoparticle is from about 0.1 to about 2.5 µg per million cells of hematopoietic lineage. 67. The method of any of claims 56-66, wherein the disease is a genetic disease. 68. The method of any of claims 56-66, wherein the disease is a cancer. 69. The method of any of claims 56-68, wherein the subject is a mammal. 70. The method of any of claims 56-69, wherein the subject is human. 71. A method for screening lipid composition of lipid nanoparticles for preferential delivery of RNA to T cells, comprising: (a) preparing a plurality of lipid nanoparticles with different lipid compositions and a reporter RNA, (b) administering the lipid nanoparticles to Jurkat cells, (c) measuring the relative abundance of reporter RNA, (d) comparing the relative abundance of reporter RNA to a threshold, and (e) identifying lipid compositions above threshold as candidates for preferential delivery of RNA to T cells. Leydig 770158/US DHR P2023-3706-WO01 75 72. The method of claim 71, wherein the different lipid compositions of step (a) comprise an ionizable lipid and a phospholipid. 73. The method of claim 72, wherein the different lipid compositions of step (a) further comprise at least one of a sterol or a stabilizing agent. 74. The method of any of claims 71-73, wherein the different lipid compositions of step (a) vary in at least one of (i) types of components, (ii) ratios of components, or (iii) the ratio of the total lipid components. 75. The method of any of claims 71-74, wherein the reporter RNA is an mRNA encoding a bioluminescent or biofluorescent protein. 76. The method of any of claims 71-74, wherein the reporter RNA is an mRNA encoding a gene editing nuclease and a guide RNA targeting a reporter gene. 77. The method of any of claims 71-74, wherein the reporter RNA is an siRNA targeting a reporter gene. 78. The method of any of claims 71-74, wherein the reporter RNA is labelled with a contrast agent. 79. The method of claim 78, wherein the contrast agent is a radionuclide, an iodine agent, a luminescent dye, or a fluorescent dye. 80. The method of any of claims 71-79, wherein the relative abundance of reporter RNA in step (c) is measured by optical imaging.

Description

Leydig 770158/US DHR P2023-3706-WO01 1 LIPID NANOPARTICLE FORMULATIONS FOR CELL THERAPY AND RELATED METHODS CROSS-REFERENCE TO RELATED APPLICATIONS [0001] This patent application claims priority to International Application No. PCT/US2023/069303, filed June 28, 2023, which claims the benefit of U.S. Provisional Application 63/357,094, filed June 30, 2022, which are incorporated by reference in their entireties herein. BACKGROUND [0002] Nucleic-acid-based cell therapy reagents offer advantages over electroporation and traditional oncological treatments in terms of safety and efficacy. In particular, RNA cell therapies are subject to degradation by exonucleases and endonucleases in vivo without a delivery system, so they need a carrier. [0003] Lipid nanoparticles (also referred to herein as “LNP”) generally consist of different lipids, each serving distinct functions. LNP can have a lipidic or aqueous core and may contain bilayer structures depending on the abundance of each type of lipids. [0004] Generally, the components of LNP formulations include: ionizable cationic lipids, which spontaneously encapsulate negatively-charged nucleic acids by a combination of attractive electrostatic interactions with and hydrophobic interactions; neutral phospholipids to reduce charge-related toxicity and to maintain structure of the LNP; and cholesterol to stabilize the LNP and help with cell entry, and a lipid-conjugated polyethylene glycol (PEG). [0005] Recognized challenges to creating successful LNP cell therapy are safety, manufacturability, stability, and efficacy. There is a continued need in the art to provide additional solutions to a better cell therapy delivery agent with both high efficiency and high live cell yield. The present disclosure provides for ameliorating at least some of the disadvantages of the prior art. These and other advantages of the present disclosure will be apparent from the description as set forth below. [0006] It will be appreciated that this background description has been created to aid the reader and is not to be taken as an indication that any of the indicated problems were themselves appreciated in the art. While the described principles can, in some aspects and embodiments, alleviate the problems inherent in other systems, it will be appreciated that the Leydig 770158/US DHR P2023-3706-WO01 2 scope of the protected innovation is defined by the attached claims and not by the ability of any disclosed feature to solve any specific problem noted herein. BRIEF SUMMARY [0007] The disclosure provides, e.g., lipid formulations and related methods suitable for forming nucleic acid-based cell therapy reagents in lipid nanoparticles. In an aspect, the disclosure provides a lipid formulation for forming a lipid-based nanoparticle suitable for transfecting nucleic acid cargo into cells of hematopoietic lineage comprising an ionizable lipid, a phospholipid, a sterol, and optionally a stabilizer. [0008] In another aspect, the disclosure provides a lipid formulation comprising an ionizable lipid, a phospholipid, a sterol, and optionally a stabilizer, for forming a lipid-based nanoparticle suitable for transfecting a nucleic acid cargo into T cells, Jurkat cells, hematopoietic stem cells, natural killer cells, or antigen presenting cells. [0009] In a further aspect, the disclosure provides a method for screening lipid composition of lipid nanoparticles for preferential delivery of RNA to T cells. The method comprises: (a) preparing a plurality of lipid nanoparticles with different lipid compositions and a reporter RNA, (b) administering the lipid nanoparticles to Jurkat cells, (c) measuring the relative abundance of reporter RNA, (d) comparing the relative abundance of reporter RNA to a threshold, and (e) identifying lipid compositions above threshold as candidates for preferential delivery of RNA to T cells. [0010] Further and alternative aspects and features of the disclosed principles will be appreciated from the following detailed description and the accompanying drawings. Other aspects and features of the present invention will become apparent to those ordinarily skilled in the art upon review of the following description of specific embodiments of the invention in conjunction with the accompanying figures. Accordingly, it is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and do not restrict the scope of the appended claims. BRIEF DESCRIPTION OF THE DRAWINGS [0011] The figure is graphical representation of the half maximal effective concentration (EC50) dose for transfection in BHK cells of lipid formulations containing cholesterol (gray bars) and cholesteryl hemisuccinate (black bars) as discussed in Example 4. Leydig 770158/US DHR P2023-3706-WO01 3 [0012] It should be understood that the drawings are not necessarily to scale and that the disclosed embodiments are sometimes illustrated diagrammatically