Cholesterol derivatives and methods of use

Abstract

Disclosed herein are cholesterol derivatives, and methods of making and using the cholesterol derivatives. Cholesterol derivatives can be utilized in lipid nanoparticles for tissue specific (e.g., spleen) delivery of cargo. Methods of spleen specific delivery of a lipid nanoparticle comprising a cholesterol derivative according to the present invention are also provided.

Description

CHOLESTEROL DERIVATIVES AND METHODS OF USERELATED APPLICATION INFORMATION

[0001] This application claims the benefit of U.S. Provisional Application Serial No. 63 / 512,765, filed July 10, 2023, the disclosure of which is incorporated herein by reference in its entirety.FIELD OF INVENTION

[0002] The invention relates to cholesterol derivatives, lipid nanoparticles comprising the cholesterol derivatives, and methods of making the cholesterol derivatives. The invention further relates to methods of using the cholesterol derivatives in lipid nanoparticles for tissue-specific delivery.BACKGROUND

[0003] Even though lipid nanoparticles (LNPs) can deliver messenger RNA (mRNA) payloads into cells, improvement of delivery approaches to specific cells and targets is needed for therapeutic applications. Generation of novel synthetic materials for LNPs, including those that target delivery of therapeutics such as RNA cargoes (including mRNAs) in a tissue specific manner is desirable for clinical applications. Novel materials that result in different tropism (i.e., selectivity), including from clinically approved LNP formulations such as the Moderna and Pfizer / BioNTech COVID-19 LNP formulations, are needed.INVENTION SUMMARY

[0004] The present invention is based, in part, on the development of cholesterol derivatives that can be utilized in lipid nanoparticles for spleen-specific delivery of cargoes.

[0005] In an aspect, a cholesterol derivative according to Formula I is provided:Formula I wherein Ri and R2 are independently H, C1-C8 alkyl, C2-C8 alkenyl, C2-C8 alkynyl, -(CH2)n- NRaRb, -(CH2)nO(CH2)n, -(CH2)nOH, orRi and R2 together with the N atom to which they are bonded form a 4-15 membered ring, each member independently selected from the group consisting of C, N, O, S, CH, CH2, and NH, the ring being saturated, partially unsaturated, or aromatic, and optionally substituted by one to three substituents selected from the group consisting of: C1-C8 alkyl, C2-C8 alkenyl, C2-C8 alkynyl, C1-C8 alkoxyl, (CH2)n(NRaRb), (CH2)nO(CH2)n, -(CH2)nOH, and a 4-8 membered ring, each member independently selected from C, N, O, S, CH, CH2, and NH, the ring being saturated, partially unsaturated, or aromatic, wherein each of the substituents can be further substituted with a 3-8 membered ring, each member independently selected from the group consisting of C, N, O, S, CH, CH2, and NH, the ring being saturated, partially unsaturated, or aromatic; wherein Ra and Rb are each independently selected from H, C1-C8 alkyl, C2-C8 alkenyl, C2-C8 alkynyl, -(CH2)n-NRaRb, -(CH2)nO(CH2)n, -(CH2)nOH; and each n is independently an integer between 1 and 8.

[0006] Another aspect comprises compositions, e.g., LNPs, comprising an ionizable lipid, a phospholipid, a poly(ethylene glycol) (PEG)-lipid and a cholesterol derivative of the present invention. In some embodiments, the ratios of the components in the LNPs are optimized for delivery and / or expression of RNA.

[0007] In another aspect of the invention, a nanoparticle comprising a cholesterol derivative of the present invention, or a composition of the present invention, and a cargo is provided.

[0008] In a further aspect, a method of making a cholesterol derivative according to the present invention is provided, comprising:

[0009] reacting a molecule according towith a secondary amine compound, e.g., at room temperature, to thereby yield the compound according to Formula I.

[0010] Another aspect of the invention comprises a method of delivering a cargo to a target cell, comprising contacting the target cell with a nanoparticle of the present invention.

[0011] In an aspect, a method of modulating expression of a gene in a target cell is provided, comprising contacting the target cell with a nanoparticle of the present invention.

[0012] In an aspect, a method of delivering a cargo to a subject in a tissue specific manner is provided, comprising contacting the tissue with a nanoparticle of the present invention.

[0013] In an aspect, a method of treating a disease or disorder in a subject in need thereof is provided, comprising administering to the subject a nanoparticle of the present invention, wherein the cargo provides a therapeutic effect for the disease or disorder.

[0014] In an aspect, a method of vaccinating a subject in need thereof is provided, comprising administering to the subject a nanoparticle of the present invention, wherein the cargo provides an immunostimulatory effect.

[0015] A further aspect of the invention relates to the nanoparticle of the present invention for use in a method of delivering a cargo to a target cell, a method of modulating expression of a gene in a target cell, a method of delivering a cargo to a subject in a tissue specific manner, a method of treating a disease or disorder in a subject in need thereof, or a method of vaccinating a subject in need thereof.

[0016] Another aspect of the invention relates to use of the nanoparticle of the present invention in the preparation of a medicament for a method of delivering a cargo to a target cell, a method of modulating expression of a gene in a target cell, a method of delivering a cargo to a subject in a tissue specific manner, a method of treating a disease or disorder in a subject in need thereof, or a method of vaccinating a subject in need thereof

[0017] In an aspect, a method of making a cargo-loaded nanoparticle is provided, comprising adding an organic phase comprising a cholesterol derivative of the present invention, a PEG lipid, an ionizable lipid, and a phospholipid to an aqueous phase comprising a cargo to make a mixture, wherein the nanoparticle self-assembles via electrostatic interactions between the cargo and the cholesterol derivative, the PEG lipid, the ionizable lipid, and the phospholipid.

[0018] These and other aspects of the invention are set forth in more detail in the description of the invention below.BRIEF DESCRIPTION OF THE DRAWINGS

[0019] FIG. 1. Firefly luciferase (FLuc) mRNA delivered alone (top), with lipid nanoparticle (LNP) formulation comprising cholesterol (middle), and with nanoparticle formulation comprising example cholesterol derivative (bottom).

[0020] FIG. 2.1H NMR of cholesterol derivative A00 (CDCh 400 MHz).

[0021] FIG. 3.JH NMR of cholesterol derivative Al (CDCh 400 MHz).

[0022] FIG. 4.1H NMR of cholesterol derivative A2 (CDCh 400 MHz).

[0023] FIG. 5.1H NMR of cholesterol derivative A3 (CDCh 400 MHz).

[0024] FIG. 6. ' H NMR of cholesterol derivative A4 (CDCh 400 MHz).

[0025] FIG. 7.1H NMR of cholesterol derivative A5 (CDCh 400 MHz).

[0026] FIG. 8.JH NMR of cholesterol derivative A6 (CDCh 400 MHz).

[0027] FIG. 9. ' H NMR of cholesterol derivative A7 (CDCh 400 MHz).

[0028] FIG. 10.1H NMR of cholesterol derivative A8 (CDCh 400 MHz).

[0029] FIG. 11.rH NMR of cholesterol derivative A9 (CDCh 400 MHz).

[0030] FIG. 12.1H NMR of cholesterol derivative A10 (CDCh 400 MHz).

[0031] FIG. 13.1H NMR of cholesterol derivative Al 1 (CDCh 400 MHz).

[0032] FIG. 14. ' H NMR of cholesterol derivative A12 (CDCh 400 MHz).

[0033] FIG. 15. ' H NMR of cholesterol derivative A13 (CDCh 400 MHz).

[0034] FIG. 16. ‘HNMR of cholesterol derivative Al 4 (CDCh 400 MHz).

[0035] FIG. 17. ‘H NMR of cholesterol derivative Al 5 (CDCh 400 MHz).

[0036] FIG. 18. ‘H NMR of cholesterol derivative Al 6 (CDCh 400 MHz).

[0037] FIG. 19. ‘H NMR of cholesterol derivative A17 (CDCh 400 MHz).

[0038] FIG. 20. ‘H NMR of cholesterol derivative Al 8 (CDCh 400 MHz).

[0039] FIG. 21. ‘HNMR of cholesterol derivative A19 (CDCh 400 MHz).

[0040] FIG. 22. Overall concept for development and evaluation of LNPs comprising the cholesterol derivatives A00-A19.

[0041] FIGS. 23A-23C. Representative in vivo images of tissue specific delivery of exemplary LNPs with their respective size, charge, and encapsulation efficiency for LNPs comprising Al, A2, A3, A8, A16, A17, A18, or A19, naked Flue mRNA, and representative Moderna LNP administered via IV infusion at 0.75 mg / mL; 24 hr; n=3.

[0042] FIG. 24. Microfluidic mixing schematic containing respective ethanol phase and aqueous phase to formulate SE-LNPs.

[0043] FIGS. 25A-25C. (FIG. 25A) Design of Experiment-guided workflow used to identify lead SE-LNP formulations. (FIG. 25B) Size, zeta potential, mRNA encapsulation efficiency, and PDI of SE-LNP libraries. (FIG. 25C) In vitro FLuc expression of J774A.1 macrophages treated with SE-LNP libraries at an overall 50 ng FLuc mRNA doses per well. Cells treated with only cell culture media, naked FLuc mRNA and first-generation SE-LNP A0 formulation as controls (All data presented as mean ± SD, n = 3).

[0044] FIGS. 26A-26C. (FIG. 26A) 24-h viability evaluation of Library A upon dosing on J774A.1 macrophages at 50 ng p / well. (FIG. 26B) 24 h viability of Library B upon dosing on J774A.1 macrophages at 50 ng p / well. (FIG. 26C) 24 h viability of Library C upon dosing on J774A.1 macrophages at 50 ng p / well.

[0045] FIGS. 27A-27C. (FIG. 27A) FLuc expression in the dose responsive scenario (50, 100, 200 ng) and time course scenario (2, 6, 24, 48 h) of J774A.1 macrophages treated with lead SE- LNP libraries (all data presented as mean ± SD, n = 3). (FIG. 27B) Effects of hypoxia (1% O2) on lead SE-LNPs upon increasing dose (50, 100, 200 ng) at 24 hours. (FIG. 27C) Heat map of FLuc expression of the ratio hypoxia to normoxia in J774A.1 macrophages.

[0046] FIG. 28. Size, charge, and encapsulation efficiency of Moderna LNP formulation.

[0047] FIGS. 29A-29C. (FIG. 29A) Cellular association of J774A.1 cells treated with lead SE- LNP A6, B7, Cl compared to the Moderna (M) LNP formulation measured at 2 and 24 h. % Cy- 5 positive cells indicates cellular association of SE-LNPs (measured via flow cytometry). (FIG. 29B) Cellular uptake mechanism studies of J774A.1 cells treated with lead SE-LNP A6, B7, Cl compared to Moderna LNP formulation. Inhibitors of each respective uptake mechanism as follows: Pitstop 2 (Clathrin-Mediated Endocytosis), EIPA (Macropinocytosis), Filipin, (Caveolae Mediated Endocytosis), Cytochalasin D ((Cyto D) Phagocytosis). (FIG. 29C) Endosomal escape studies of J774A.1 cells treated with lead SE-LNP A6, B7, Cl compared to Moderna (M) LNP formulation. Pearson correlation coefficient (PCC) Scale bars are 20 pm. In all experiments cells were treated with 500 ng mL'1of respective LNP at 37°C. Data represented as mean + / - SD, n=3. Significance testing was performed using One-Way Anova (ns p>0.05, *p<0.05).

[0048] FIG. 30. Flow cytometry gating strategy used for cellular association and uptake mechanism studies on J774A.1 macrophages.

[0049] FIGS. 31A-31C. (FIG. 31A) Biodistribution imaging of C57BL / 6 mice organs upon intravenous injection of lead SE-LNPs A6, B7, Cl formulations and PBS, naked mRNA and Moderna as controls (24 hrs). (FIG. 31B) Quantification of organ biodistribution and quantification of FLuc expression within the spleen of treated mice (all data presented as mean ± SD, n = 3). (FIG. 31C) Histological images of spleen, liver, and lungs of treated groups (images 50 nm scale).

[0050] FIG. 32. Viability of lead LNP formulations at respective time and dose.

[0051] FIG. 33. Percent weight gain in mice reported as mean ± SD (n = 3) 24 h after respective intravenous dose of FLuc mRNA encapsulated LNPs.

[0052] FIG. 34. Size, charge, and encapsulation efficiency of LNP formulations F00-F18 (Table 9) using compound A8.

[0053] FIGS. 35A-35B. (FIG. 35A) hi vitro FLuc expression of DC 2.4 cells treated with LNP formulations F00-F18 (Table 9) at an overall 50 ng FLuc mRNA doses per well. Cells treated with only cell culture media, naked FLuc mRNA, lipofectamine and Moderna were used as controls. (FIG. 35B) 24 h viability of optimized formulations upon dosing on DC 2.4 cells at 50 ng p / well. (All data presented as mean ± SD, n = 3).

[0054] FIG. 36. Size, charge, and encapsulation efficiency of LIMP formulations F00-F18 (Table 10) using compound A9.

[0055] FIGS. 37A-37B. (FIG. 37A) In vitro FLuc expression of DC 2.4 cells treated with LNP formulations F00-F18 (Table 10) at an overall 50 ng FLuc mRNA doses per well. Cells treated with only cell culture media, naked FLuc mRNA, lipofectamine and Moderna were used as controls. (FIG. 37B) 24 h viability of optimized formulations upon dosing on DC 2.4 cells at 50 ng p / well. (All data presented as mean ± SD, n = 3).

[0056] FIG. 38. Size, charge, and encapsulation efficiency of LNP formulations F00-F18 (Table 11) using compound Al 6.

[0057] FIGS. 39A-37B. (FIG. 39A) In vitro FLuc expression of DC 2.4 cells treated with LNP formulations F00-F18 (Table 11) at an overall 50 ng FLuc mRNA doses per well. Cells treated with only cell culture media, naked FLuc mRNA, lipofectamine and Moderna were used as controls. (FIG. 39B) 24 h viability of optimized formulations upon dosing on DC 2.4 cells at 50 ng p / well. (All data presented as mean ± SD, n = 3).

[0058] FIG. 40. Size, charge, and encapsulation efficiency of LNP formulations F00-F18 (Table 12) using compound Al 8.

[0059] FIGS. 41A-41B. (FIG. 41A) In vitro FLuc expression of J774A.1 macrophages treated with LNP formulations F00-F18 (Table 12) at an overall 50 ng FLuc mRNA doses per well. Cells treated with only cell culture media, naked FLuc mRNA, lipofectamine and Moderna were used as controls. (FIG. 41B) 24 h viability of optimized formulations upon dosing on DC 2.4 cells at 50 ng p / well. (All data presented as mean ± SD, n = 3).

[0060] FIG. 42. Representative in vivo images of tissue specific delivery of exemplary LNPs with their respective size, charge, and encapsulation efficiency for LNPs comprising A8, A9, Al 6, or Al 8 administered via IV infusion at 0.65 mg / mL; 24 hr; n=3.DETAILED DESCRIPTION

[0061] The present invention will now be described in more detail with reference to the accompanying drawings, in which preferred embodiments of the invention are shown. This invention may, however, be embodied in different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the invention tothose skilled in the art. In addition, any references cited herein are incorporated by reference in their entireties.

[0062] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. All publications, patent applications, patents, patent publications and other references cited herein are incorporated by reference in their entireties for the teachings relevant to the sentence and / or paragraph in which the reference is presented.

[0063] Amino acids are represented herein in the manner recommended by the IUPAC-IUB Biochemical Nomenclature Commission, or (for amino acids) by either the one-letter code, or the three-letter code, both in accordance with 37 C.F.R. §1.822 and established usage.

[0064] Except as otherwise indicated, standard methods known to those skilled in the art may be used for cloning genes, amplifying and detecting nucleic acids, and the like. Such techniques are known to those skilled in the art. See, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual 4th Ed. (Cold Spring Harbor, NY, 2012); Ausubel et al. Current Protocols in Molecular Biology: (Green Publishing Associates, Inc. and John Wiley & Sons, Inc., New York).

[0065] Unless the context indicates otherwise, it is specifically intended that the various features of the invention described herein can be used in any combination.

[0066] Moreover, the present invention also contemplates that in some embodiments of the invention, any feature or combination of features set forth herein can be excluded or omitted.

[0067] To illustrate, if the specification states that a complex comprises components A, B and C, it is specifically intended that any of A, B or C, or a combination thereof, can be omitted and disclaimed singularly or in any combination.

[0068] As used in the description of the invention and the appended claims, the singular forms “a,” “an,” and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise.

[0069] Also as used herein, “and / or” refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations when interpreted in the alternative (“or”).

[0070] The term “about,” as used herein when referring to a measurable value such as an amount of polypeptide, dose, time, temperature, enzymatic activity or other biological activity and the like, is meant to encompass variations of ± 10%, ± 5%, ± 1%, ± 0.5%, ± 0.1%, or even 0.01% of the specified amount.

[0071] As used herein, the transitional phrase “consisting essentially of’ (and grammatical variants) is to be interpreted as encompassing the recited materials or steps and those that do not materially affect the basic and novel characteristic(s) of the claimed invention. Thus, the term “consisting essentially of’ as used herein should not be interpreted as equivalent to “comprising.”

[0072] The term “consists essentially of’ (and grammatical variants), as applied to a polypeptide or polynucleotide sequence of this invention, means a polypeptide or polynucleotide that consists of both the recited sequence (e.g., SEQ ID NO) and a total of ten or less (e.g, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) additional amino acids on the N-terminal and / or C-terminal ends of the recited sequence or additional nucleotides on the 5’ and / or 3’ ends of the recited sequence such that the function of the polypeptide or polynucleotide is not materially altered. The total of ten or less additional amino acids or nucleotides includes the total number of additional amino acids or nucleotides on both ends added together. The term “materially altered,” as applied to polypeptides of the invention, refers to an increase or decrease in biological activities / properties (e.g., remodeling activity) of at least about 50% or more as compared to the activity of a polypeptide consisting of the recited sequence.

[0073] As used herein, the term “polypeptide” encompasses both peptides and proteins, unless indicated otherwise.

[0074] The terms “polynucleotide,” “nucleic acid,” “nucleic acid molecule,” and “oligonucleotide” are used interchangeably and refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides or analogs thereof. Polynucleotides can have any three-dimensional structure and may perform any function, known or unknown. The following are non-limiting examples of polynucleotides: a gene or gene fragment (for example, a probe, primer, EST or SAGE tag), exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, genomic DNA, chimeras of RNA and DNA, isolated DNA of any sequence, isolated RNA of any sequence, synthetic DNA of any sequence (e.g., chemically synthesized),synthetic RNA of any sequence (e.g, chemically synthesized), nucleic acid probes and primers. A polynucleotide can comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs or derivatives (e.g, inosine or phosphorothioate nucleotides). Such nucleotides can be used, for example, to prepare nucleic acid molecules that have altered basepairing abilities or increased resistance to nucleases.

[0075] The term “modulate,” “modulates,” or “modulation” refers to enhancement (e.g, an increase) or inhibition (e.g., a decrease) in the specified level or activity.

[0076] The term “enhance” or “increase” refers to an increase in the specified parameter of at least about 1.25-fold, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 8-fold, 10-fold, twelve-fold, or even fifteen-fold and / or can be expressed in the enhancement and / or increase of a specified level and / or activity of at least about 1%, 5%, 10%, 15%, 25%, 35%, 40%, 50%, 60%, 75%, 80%, 90%, 95% or more.

[0077] The term “inhibit” or “reduce” or grammatical variations thereof as used herein refers to a decrease or diminishment in the specified level or activity of at least about 1%, 5%, 10%, 15%, 25%, 35%, 40%, 50%, 60%, 75%, 80%, 90%, 95% or more. In particular embodiments, the inhibition or reduction results in little or essentially no detectible activity (at most, an insignificant amount, e.g., less than about 10% or even 5%).

[0078] The term “contact” or grammatical variations thereof refers to bringing two or more substances in sufficiently close proximity to each other for one to exert a biological effect on the other.

[0079] It will be understood that "substitution" or "substituted with" includes the implicit proviso that such substitution is in accordance with permitted valence of the substituted atom and the substituent, and that the substitution results in a stable compound, e.g., which does not spontaneously undergo transformation such as by rearrangement, cyclization, elimination, etc. In a broad aspect, the permissible substituents include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, aromatic and nonaromatic substituents of organic compounds. The permissible substituents can be one or more and the same or different for appropriate organic compounds. For purposes of this disclosure, the heteroatoms such as nitrogen may have hydrogen substituents and / or any permissible substituents of organic compounds described herein which satisfy the valences of the heteroatoms. Non-limiting examples of optional substituents as referred to herein include halogen, alkyl, aralkyl, heteroalkyl, alkenyl, alkynyl,cycloalkyl, hydroxyl, alkoxy, amino, amido, nitro, cyano, amido, carbonyl, carboxyl, silyl, ether, alkylthio, sulfonyl, sulfonamide, ketone, aldehyde, ester, heterocyclyl, aryl, and heteroaryl.

[0080] As used herein, the term “alkyl”, used either alone or in compound words such as “haloalkyl” includes straight-chain or branched saturated hydrocarbon having 1 to 20 carbon atoms (or in lower alkyl 1 to 8 carbon atoms), such as methyl, ethyl, n-propyl, i-propyl, or the different butyl, pentyl or hexyl isomers, etc. The term “alkyl” is intended to include both substituted and unsubstituted alkyl or lower alkyl unless otherwise indicated.

[0081] As used herein, the term “alkenyl” alone or as part of another group, refers to a straight or branched chain hydrocarbon containing from 2 to 20 carbon atoms (or in lower alkenyl 2 to 8 carbon atoms) that includes 1 to 8 double bonds in the normal chain, and can be referred to as a C2-C20 alkenyl. Representative examples of alkenyl include, but are not limited to, vinyl, 2- propenyl, 3-butenyl, 2-butenyl, 4-pentenyl, 3-pentenyl, 2-hexenyl, 3-hexenyl, 2,4-heptadiene, and the like. The term “alkenyl” or “lower alkenyl” is intended to include both substituted and unsubstituted alkenyl or lower alkenyl unless otherwise indicated.

[0082] As used herein, the term “alkynyl” refers to a straight or branched chain hydrocarbon containing from 2 to 20 carbon atoms (or in lower alkynyl 2 to 8 carbon atoms) which include 1 triple bond in the normal chain, and can be referred to as a C2-C20 alkynyl. Representative examples of alkynyl include, but are not limited to, 2-propynyl, 3-butynyl, 2-butynyl, 4-pentynyl, 3-pentynyl, and the like. The term “alkynyl” is intended to include both substituted and unsubstituted alkynyl or lower alkynyl unless otherwise indicated.

[0083] As used herein, the term “alkoxy” refers to an alkyl or lower alkyl group, as defined herein, appended to the parent molecular moiety through an oxy group, -O-. Representative examples of alkoxy include, but are not limited to, methoxy, ethoxy, propoxy, 2-propoxy, butoxy, tert-butoxy, pentyloxy, hexyloxy and the like.

[0084] As used herein, “unsaturated” refers to compounds or structures having at least one degree of unsaturation (e.g., at least one double or triple bond).

[0085] Substituents around a carbon-carbon double bond alternatively can be referred to as “cis" or “trans,” where “cis” represents substituents on the same side of the double bond and “trans” represents substituents on opposite sides of the double bond. The arrangement of substituents around a carbocyclic ring can also be designated as “cis” or “trans.” The term “cis” represents substituents on the same side of the plane of the ring, and the term “trans” representssubstituents on opposite sides of the plane of the ring. Mixtures of compounds wherein the substituents are disposed on both the same and opposite sides of plane of the ring are designated “cis / trans.”

[0086] All chiral, diastereomeric, racemic, and geometric isomeric forms of a structure are intended, unless specific stereochemistry or isomeric form is specifically indicated. All processes used to prepare compounds and intermediates made therein are encompassed by the present disclosure. All tautomers of shown or described compounds are also encompassed by the present disclosure.

[0087] When any variable (e.g., Ri) occurs more than one time in any constituent or formula for a compound, its definition at each occurrence is independent of its definition at every other occurrence. Thus, for example, if a group is shown to be substituted with one or more Ri moi eties, then Ri at each occurrence is selected independently from the Markush group recited for Ri. Also, combinations of substituents and / or variables are permissible, but only if such combinations result in stable compounds within a designated atom’s normal valency.

[0088] A “subject” may be any vertebrate organism in various embodiments. A subject may be an individual to whom an agent is administered, e.g., for experimental, diagnostic, and / or therapeutic purposes or from whom a sample is obtained or on whom a procedure is performed. In some embodiments a subject is a mammal, e.g., a human, non-human primate, lagomorph (e.g., rabbit), or rodent (e.g., mouse, rat). In some embodiments a human subject is a neonate, child, adult, or geriatric subject.

[0089] Grammatical variations of “administer,” “administration,” and “administering” to a subject include any route of introducing or delivering to a subject an agent. Administration can be carried out by any suitable route, including oral, topical, intravenous, subcutaneous, transcutaneous, transdermal, intramuscular, intra-joint, parenteral, intra-arteriole, intradermal, intraventricular, intracranial, intraperitoneal, intralesional, intranasal, rectal, vaginal, by inhalation, via an implanted reservoir, parenteral (e.g., subcutaneous, intravenous, intramuscular, intra-articular, intra-synovial, intrasternal, intrathecal, intraperitoneal, intrahepatic, intralesional, and intracranial injections or infusion techniques), and the like. “Concurrent administration,” “administration in combination,” “simultaneous administration,” or “administered simultaneously” as used herein, means that the compounds are administered at the same point in time, overlapping in time, or one following the other. In the latter case, the two compounds areadministered at times sufficiently close that the results observed are indistinguishable from those achieved when the compounds are administered at the same point in time. “Systemic administration” refers to the introducing or delivering to a subject an agent via a route which introduces or delivers the agent to extensive areas of the subject’s body (e.g., greater than 50% of the body), for example through entrance into the circulatory or lymph systems. By contrast, “local administration” refers to the introducing or delivery to a subject an agent via a route which introduces or delivers the agent to the area or area immediately adjacent to the point of administration and does not introduce the agent systemically in a therapeutically significant amount. For example, locally administered agents are easily detectable in the local vicinity of the point of administration but are undetectable or detectable at negligible amounts in distal parts of the subject's body. Administration includes self-administration and the administration by another.

[0090] “ Treat,” “treating,” and similar terms as used herein in the context of treating a subject refer to providing medical and / or surgical management of a subject. Treatment may include, but is not limited to, administering an agent or composition (e.g., a pharmaceutical composition) to a subject. Treatment is typically undertaken in an effort to alter the course of a disease (which term is used to indicate any disease, disorder, syndrome, or undesirable condition warranting or potentially warranting therapy) in a manner beneficial to the subject. The effect of treatment may include reversing, alleviating, reducing severity of, delaying the onset of, curing, inhibiting the progression of, and / or reducing the likelihood of occurrence or recurrence of the disease or one or more symptoms or manifestations of the disease. A therapeutic agent may be administered to a subject who has a disease or is at increased risk of developing a disease relative to a member of the general population. In some embodiments a therapeutic agent may be administered to a subject who has had a disease but no longer shows evidence of the disease. The agent may be administered e.g., to reduce the likelihood of recurrence of evident disease. A therapeutic agent may be administered prophylactically, i.e., before development of any symptom or manifestation of a disease. “Prophylactic treatment” refers to providing medical and / or surgical management to a subject who has not developed a disease or does not show evidence of a disease in order, e.g., to reduce the likelihood that the disease will occur, delay the onset of the disease, or to reduce the severity of the disease should it occur. The subject may have been identified as being at riskof developing the disease (e.g., at increased risk relative to the general population or as having a risk factor that increases the likelihood of developing the disease.

[0091] The present invention relates to cholesterol derivatives, and methods of making and using the cholesterol derivatives. The cholesterol derivatives can advantageously be used in lipid nanoparticles (LNPs), and can be used for tissue-specific (e. ., splenic) delivery of cargoes.

[0092] In some embodiments, the cholesterol derivative is according to Formula I:Formula I wherein Ri and R.2 are independently H, C1-C8 alkyl, C2-C8 alkenyl, C2-C8 alkynyl, -(CH2)n- NRaRb, -(CH2)nO(CH2)n, -(CH2)nOH, orRi and R2 together with the N atom to which they are bonded form a 4-15 membered ring, each member independently selected from the group consisting of C, N, O, S, CH, CH2, and NH, the ring being saturated, partially unsaturated or aromatic, and optionally substituted by one to three substituents selected from the group consisting of: C1-C8 alkyl, C2-C8 alkenyl, C2-C8 alkynyl, C1-C8 alkoxyl, (CH2)n(NRaRb), (CH2)nO(CH2)n, -(CH2)nOH, and a 4-8 membered ring, each member independently selected from C, N, O, S, CH, CH2, and NH, the ring being saturated, partially unsaturated, or aromatic, wherein each of the substituents can be further substituted with a 3-8 membered ring each member independently selected from the group consisting of C, N, O, S, CH, CH2, and NH, the ring being saturated, partially unsaturated or aromatic; wherein Ra and Rb are each independently selected from H, C1-C8 alkyl, C2-C8 alkenyl, C2-C8 alkynyl, -(CH2)n-NRaRb, -(CH2)nO(CH2)n, -(CH2)nOH; and each n is independently an integer between 1 and 8.

[0093] In an embodiment, the Ri and R2 together with the N atom to which they are bonded form a 4-15 membered ring, the ring selected from:wherein the ring is optionally substituted by one to three substituents selected from the group consisting of: C1-C8 alkyl, C2-C8 alkenyl, C2-C8 alkynyl, C1-C8 alkoxyl, (CH2)n(NRaRb), (CH2)nO(CH2)n„ — (CH2)nOH, and a 4-8 membered ring, each member independently selected from C, N, O, S, CH, CH2, and NH, the ring being saturated, partially unsaturated, or aromatic, wherein each of the substituents can be further substituted with a 4-8 membered ring each member independently selected from the group consisting of C, N, O, S, CH, CH2, and NH, the ring being saturated, partially unsaturated or aromatic.

[0094] In an embodiment, the cholesterol derivative is selected from:

[0095] Compositions, e.g., LNPs, comprising the cholesterol derivative are described herein. In an embodiment, a composition comprising an ionizable lipid, a phospholipid, a poly(ethylene glycol) (PEG)-lipid and a cholesterol derivative according to the present invention is provided. In some embodiments, the composition comprises about 5-95% ionizable lipid, about 5-30% phospholipid, about 20-60% of the cholesterol derivative, and about 1-5 mol% PEG-lipid. The compositions may comprise additional elements, including for example, a polyphenol such as tannic acid.

[0096] Another aspect of the invention relates to optimized LNP formulations optimized for delivery of cargo to the spleen, e.g., having enhanced spleen tropism. In some embodiments, the LNPs comprise 45-65 mol% ionizable lipid, 20-50 mol% cholesterol derivative, 5-25 mol% phospholipid, and 0.4-2.0 mol% PEG-lipid.

[0097] In some embodiments, the LNPs comprise 46-48 mol% ionizable lipid, 44-46 mol% cholesterol derivative, 5-25 mol% phospholipid, and 0.5-0.6 mol% PEG-lipid.

[0098] In some embodiments, the LNPs comprise SM-102, a cholesterol derivative, DSPC and / or DOPE, and 14:0 PEG IK.

[0099] Specific examples of optimized LNP formulations include the following (in mol%). Table 1. Optimized LNP formulations.

[0100] In an embodiment, the ionizable lipid of the composition is selected from 9- heptadecanyl 8-{(2-hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino}octanoate (SM-102) and / or [(4-hydroxybutyl)azanediyl]di(hexane-6,l-diyl) bis(2-hexyldecanoate)(ALC-0315).

[0101] In an embodiment, the composition comprises a phospholipid selected from dioleoylphosphatidylethanolamine (DOPE), dioleoylphosphatidylcholine (DOPC), di stearoylphosphatidylcholine (DSPC), or any combination thereof.

[0102] In an embodiment, the composition comprises a PEG-lipid, for example, DMG- PEG(2000) or 14:0 PEG IK.

[0103] Nanoparticles are provided herein. In an embodiment, a nanoparticle comprising the cholesterol derivative of the present invention and a cargo is provided. In an embodiment, a nanoparticle comprising a composition of the present invention and a cargo is provided.

[0104] The composition comprising the cholesterol derivative may comprise a cargo. In an aspect, the cargo is a biologically active agent, imaging agent, or therapeutic agent. The cargo may be, for example, a nucleic acid, a protein, a complex of a nucleic acid and a protein, a carbohydrate, a lipid, or a small molecule, and salts thereof. In an aspect, the cargo nucleic acid is an RNA, for example, an mRNA, guide RNA, antisense oligonucleotide, DNA / RNA hybrid, or siRNA.

[0105] The cargo can be a gene modulating agent. In an aspect, the gene modulating agent is a gene editing system comprising a CRISPR-Cas system, a zinc finger nuclease, a TALEN, a base editor or a meganuclease.

[0106] In an embodiment, example LNPs of the present invention are about 75 nm to about 1000 nm average particle size, e.g., about 90 nm to about 800 nm, about 95 nm to about 800 nm, about 100 nm to about 600 nm, about 125 nm to about 500 nm, about 150 nm to about 400 nm, or about 125 nm to about 350 nm average particle size.

[0107] In an embodiment, example LNPs of the present invention have a charge of about -1.75 to about 1.75 eV. In an embodiment, example LNPs of the present invention have apoly dispersity index (PDI) of between about 0 and 1, between about 0 and 0.8, between about 0 and 0.6, between about 0 and 0.5, between about 0 and 0.4, or between about 0 and 0.35.

[0108] The LNPs comprising a cholesterol derivative of the present invention can be provided with cargo for delivery to a cell, tissue, or subject. In an embodiment, the LNPs comprising a cholesterol derivative have a high affinity to interact with biomolecules such as proteins, polysaccharides, and nucleic acids, allowing for strong interaction between a cargo and the LNP. In some embodiments, the LNPs deliver cargo in a tissue specific manner.Ionizable lipid

[0109] The ionizable lipid can be provided at about 5-95 mol%, e.g. about 5 mol%, about 10 mol%, about 15 mol%, about 20 mol%, about 21 mol%, about 22 mol%, about 23 mol%, about 24 mol%, about 25 mol%, about 26 mol%, about 27 mol%, about 28 mol%, about 29 mol%, about 30 mol%, about 31 mol%, about 32 mol%, about 33 mol%, about 34 mol%, about 35 mol%, about 36 mol%, about 37 mol%, about 38 mol%, about 39 mol%, about 40 mol%, about 41 mol%, about 42 mol%, about 43 mol%, about 44 mol%, about 45 mol%, about 46 mol%, about 47 mol%, about 48 mol%, about 49 mol%, or about 50 mol%, about 41 mol%, about 42 mol%, about 43 mol%, about 44 mol%, about 45 mol%, about 46 mol%, about 47 mol%, about 48 mol%, about 49 mol%, about 50 mol%, about 51 mol%, about 52 mol%, about 53 mol%, about 54 mol%, about 55 mol%, about 56 mol%, about 57 mol%, about 58 mol%, about 59 mol%, about 60 mol%, about 61 mol%, about 62 mol%, about 63 mol%, about 64 mol%, about 65 mol%, about 66 mol%, about 67 mol%, about 68 mol%, about 69 mol%, about 70 mol%, about 71 mol%, about 72 mol%, about 73 mol%, about 74 mol%, about 75 mol%, about 76 mol%, about 77 mol%, about 78 mol%, about 79 mol%, about 80 mol%, about 81 mol%, about 82 mol%, about 83 mol%, about 84 mol%, about 85 mol%, about 86 mol%, about 87 mol%, about 88 mol%, about 89 mol%, about 90 mol%, about 91 mol%, about 92 mol%, about 93 mol%, about 94 mol%, or about 95 mol% or any range therein in the composition. The ionizable lipid can be provided at about 15-95 mol%, about 15-85 mol%, about 15-75 mol%, about 15-65 mol%, about 30-65 mol%, about 35-50 mol%, about 40-50 mol%, about 42-48 mol%, or about 46-48 mol% in the composition. In an aspect, the ionizable lipid can be provided at about 20-50 mol%, at about 25-50 mol%, at about 30-50 mol%, or at about 30-45 mol% in the compositions. In an embodiment, the composition (e.g., LNP) comprises one or more ionizable lipids, e.g., 1, 2, 3, 4, or 5 or more ionizable lipids. In an aspect, the ionizable lipid is an unsaturated, multi-tail,polymeric, biodegradable, or branched-tail ionizable lipid. See, Han et al. (2021) Nature Communications 12:7233 7233, Figures 1 and 2, incorporated herein by reference. Example ionizable lipids include l,2-dilinoleyl-N,N-dimethyl-3 -aminopropane (DLinDMA), 2,2- dilinoleyl- 4-dimethylaminoethyl-[l,3]-dioxolane (DLin-KC2-DMA), and dilinoleylmethyl-4- dimethylaminobutyrate (DLin-MC3-DMA). The ionizable lipid can preferably be selected from 9-heptadecanyl 8- { (2-hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino } octanoate ( SM- 102) and / or [(4-hydroxybutyl)azanediyl]di(hexane-6,l-diyl) bis(2-hexyldecanoate)(ALC-0315).Phospholipid

[0110] The phospholipid can be provided at about 5-30 mol%, about 8-30 mol%, about 8-25 mol%, about 8-20 mol%, or about 8-15 mol% in the LNP comprising a cholesterol derivative. In an aspect, phospholipid can be provided at about 8-30 mol%, about 8-25 mol%, about 8-20 mol%, or about 8-15 mol% in the composition. The phospholipid can be provided at about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%. about 19%, about 20 mol%, about 21 mol%, about 22 mol%, about 23 mol%, about 24 mol%, about 25 mol%, about 26 mol%, about 27 mol%, about 28 mol%, about 29 mol%, or about 30 mol% or any range therein in the composition. In an embodiment, the composition comprises one or more phospholipids, e.g., 1, 2, 3, 4, or 5 or more phospholipids. In an aspect, the phospholipid is dioleoylphosphatidylethanolamine (DOPE), dioleoylphosphatidylcholine (DOPC), di stearoylphosphatidylcholine (DSPC), or any combination thereof.Cholesterol derivative

[0111] The cholesterol derivative of the present invention can be provided at about 30-60 mol% in the composition. In an aspect the cholesterol derivative of the present invention can be provided at about 30 mol% to about 45 mol%, about 30 mol% to about 50 mol%, about 35 mol% to about 55 mol%, about 35 mol% to about 45 mol%, about 40 mol% to about 50 mol%, about 42 mol% to about 48 mol%, or about 44 mol% to about 46 mol% in the LNP. The cholesterol derivative of the present invention can be provided at about 30 mol%, about 31 mol%, about 32 mol%, about 33 mol%, about 34 mol%, about 35 mol%, about 36 mol%, about 37 mol%, about 38 mol%, about 39 mol%, about 40 mol%, about 41 mol%, about 42 mol%, about 43 mol%, about 44 mol%, about 45 mol%, about 46 mol%, about 47 mol%, about 48 mol%, about 49 mol%, about 50 mol%, about 51 mol%, about 52 mol%, about 53 mol%, about 54 mol%, about55 mol%, about 56 mol%, about 57 mol%, about 58 mol%, about 59 mol%, or about 60 mol% or any range therein in the composition. In an embodiment, the composition comprises one or more cholesterol derivatives of the present invention, e.g., 1, 2, 3, 4, or 5 or more cholesterol derivatives.PEG lipid

[0112] In an aspect, the PEG lipid is provided at about 0.1-5 mol% in the composition. In an aspect, the PEG lipid is provided at about 2 to about 5 mol%, about 2 to about 4 mol%, about 2 to about 3 mol%, about 1 to about 3 mol%, about 0.1 to about 2 mol%, about 0.2 to about 1.5 mol%, about 0.3 to about 0.8 mol%, or about 0.5 to about 0.6 mol% in the composition. In an aspect, the PEG lipid is provided at about 0.1 mol%, 0.5 mol%, 1 mol%, about 2 mol%, about 3 mol%, about 4 mol%, or about 5 mol% or any range therein in the composition. In an embodiment, the composition comprises one or more PEG lipids, e.g., 1, 2, 3, 4, or 5 or more PEG lipids. The amount of PEG lipid can be tuned to adjust particle size, particle stability and aggregation, and zeta potential. Exemplary PEG lipids may comprise varying PEG lengths. In an aspect, the PEG is a l,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy- (poly ethylene glycol)] of varying lengths, for example, l,2-dimyristoyl-sn-glycero-3- phosphoethanolamine-N-[methoxy-(polyethylene glycol)-350] (ammonium salt) (C14-PEG350), C14-PEG1000 (14:0 PEG IK), C14-PEG2000, C18-PEG2000, C14-PEG3000, or any combination thereof. In an aspect, the PEG lipid is C14-PEG-2000, i.e., 1,2-dimyristoyl-sn- glycero-3-phosphoethanolamine-N-[methoxy-(polyethylene glycol)-2000] (ammonium salt).

[0113] In an embodiment, a method of making the cholesterol derivative according to the present invention is provided, comprising reacting a molecule according to:with a secondary amine compound, e.g., at room temperature, to thereby yield the compound according to Formula I. In an embodiment, the secondary amine is selected from the group consisting of

[0114] In an embodiment, the reaction is performed at about 18°C, 19°C, 20°C, 21 °C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C, 40°C, 41°C, 42°C, 43°C, 44°C, 45°C, 46°C, 47°C, 48°C, 49°C, or 50°C.

[0115] Methods of making a cargo-loaded nanoparticle are provided, comprising adding an organic phase comprising a cholesterol derivative of the present invention, a PEG lipid, an ionizable lipid and a phospholipid to an aqueous phase comprising a cargo to make a mixture. The cargo and nanoparticle components can be selected to optimize electrostatic interactions between the cargo and the nanoparticle composition. The ratios of the nanoparticle components can be further tuned to optimize interactions with the cargo. In an aspect, one or more of the amounts or compositions of the cholesterol derivative, ionizable lipid, phospholipid, and / or PEG lipid components can be adjusted for optimization.

[0116] In an embodiment, a method of making a nanoparticle that targets the spleen is provided, comprising using a cholesterol derivative of the present invention in a nanoparticle formulation. In some embodiments, the nanoparticle formulation comprises the cholesterol derivative, a PEG lipid, an ionizable lipid and a phospholipid. “Targets the spleen”, as used herein means that at least 50% of the cargo administered to a subject is delivered to the spleen relative to other tissues in the subject, e.g., at least 60%, 70%, 80%, or 90%.Cargo

[0117] The compositions disclosed herein may be provided with cargo molecules as nanoparticles for delivery. Agents including biologically active agents, imaging agents and therapeutic agents may be utilized as cargo molecules in the present invention. Example cargos may be a nucleic acid, a protein, a complex of a nucleic acid and a protein, a carbohydrate, a lipid, or a small molecule. The cargo may be formulated as a salt. One or two or more different cargos may be delivered by the delivery particles described herein. The cargo can be selected for the treatment of a disease or disorder, for the prevention of a disease or disorder, for the detection of a disease or disorder, or for monitoring of a disease or disorder. In an example embodiment, the cargo is an mRNA for delivery to a cell to increase expression of one or more proteins. In an embodiment, the cargo is an antisense oligonucleotide or siRNA for delivery to a cell to decrease expression or achieve gene silencing of one or more target sequences. The cargo may be an imaging agent or detectable label or may be a molecule comprising an imaging agent or detectable label, that can be delivered to a cell or tissue. In an aspect, the cargo is a genetic modulating agent such as a CRISPR-Cas system comprising a CRISPR-Cas protein, or a polynucleotide encoding the CRISPR-Cas protein, and a guide sequence specific for a target sequence that can be utilized for a variety of gene editing applications. One or both of the CRISPR-Cas system components can be provided in the same or in different particles. Accordingly, the particles comprising cargo will find use in therapeutic, prophylactic, detection, diagnostic, and other applications.Imaging Agents

[0118] Imaging agents, including reporter probes, can be delivered in the compositions, nanoparticles, and methods of the present invention. The imaging agents and reporter probes may be associated with another molecule, covalently or otherwise, for delivery in the nanoparticle. In an aspect, a reporter probe may be associated with a nucleic acid specific for a target sequence in a cell, that can be delivered to a cell, tissue, or a subject in need thereof.

[0119] Imaging agents can include molecules suitable for any imaging modality, including positron emission tomography (PET) and single-photon emission computed tomography (SPECT), magnetic resonance imaging (MRI), ultrasound (US), and Computed Tomography (CT) imaging. Example agents can include magnetic agents including paramagnetic agents, such as gadolinium, and superparamagnetic iron-based agents. See, e.g., Xiao et al. (2016) Int’L J.Mol. Med. 38: 1319-1326; doi:10.3892 / ijmm.2016.2744. Other contrast media may include iodinated, e.g., tri-iodinated, benzene rings, or colloidal or micronized barium sulfate.

[0120] Reporter probes, including fluorescent, radioactive, and other photo-emitting compounds, may also be delivered, including synthetic dye families such as tetramethylindo(di)- carbocyanines, fluorescein, and rhodamines. Biosensors may also be used, including in in vitro detection applications for bacterial, viral, and other clinical applications. See, e.g., Castillo- Henriquez et al. (2020) Sensors (Basel) 20(23):6926; doi: 10.3390 / s20236926.Biologically Active Agents

[0121] A biologically active agent, i.e., an agent that modulates an effect or activity in a cell, tissue, organ, or other biological media such as biological fluid, includes nucleic acids, proteins, small molecules, carbohydrates, lipids, and complexes, salts thereof, and combinations thereof. The biologically active agent can be a genetic modifying agent. Representative nucleic acids include DNA, RNA, transposon DNA, antisense nucleic acids, ribozymes, plasmids, expression constructs, and RNA, such as mRNA, guide RNA, tRNA, ribosomal RNA, small nucleolar RNA, antisense oligonucleotides, or RNAi, such as siRNA, shRNA, and miRNA.

[0122] RNAi therapeutic agents comprise a polynucleotide that is complementary to a portion of the target sequence mRNA. In an example embodiment, the siRNA is a nucleic acid that can form a double stranded RNA with the ability to reduce or inhibit expression of a gene or target gene: each complementary sequence of the double stranded siRNA is about 15-50 nucleotides in length, and the double stranded siRNA is about 15-50 base pairs in length. A small hairpin RNA (shRNA) is also contemplated for use. The shRNA is an antisense strand of about 19 to about 25 nucleotides followed by a short nucleotide loop (approximately 5 to 9 nt) followed by the analogous sense strand. In an embodiment, an RNAi is a microRNA or miRNA, endogenous RNAs, some of which are known to regulate the expression of protein-coding genes at the posttranscriptional level. See, e.g., Lim et al. (2003) Science 299: 1540; Lee & Ambros (2001) Science 294:862; Lau et al. (2001) Science 294:858-861; Lagos-Quintana et al. (2003) RNA 9: 175-179.

[0123] Different criteria are available for selecting the nucleic acid for use and may comprise scanning the mRNA sequence of the target, and may include empiric determination in accordance with, for example, Sui et al. (2002) Proc. Natl. Acad. Sci. USA 99:5515-20 and may include confirmation the sequence lacks significant sequence homology with other genes asanalyzed by BLAST search. Additional approaches may comprise any accessible site in endogenous mRNA that can be targeted for degradation by synthetic oligodeoxyribonucleotide / RNase H method (see, e.g., Lee et al. (2002) Nature Biotechnol. 20:500-05). RNAi treatment may comprise miRNA or siRNA, or a pre-miRNA which is processed by Dicer to form a miRNA. The RNAi may also comprise a dsRNA or shRNA which is processed by Dicer to form a siRNA. The polynucleotides may comprise one or more modifications to suppress innate immune activation, enhance activity and specificity, and reduce off-target induced toxicity. Example teachings can be found, for example, in Provost et al. (2002) EMBO J. 21 (21 ):5864- 5874; Tabara et al. (2002) Cell 109(7):861-71 ; Martinez et al. (2002) Cell 110(5):563; Hutvagner & Zamore(2002) Science 297:2056. In certain embodiments, a single-stranded RNAi agent disclosed herein can comprise substitutions, or modifications, including chemically modified nucleotides, and non-nucleotides which may include incorporation in the backbone, sugars, bases, or nucleosides. In an example, siRNA may comprise dual ribose modifications, including 2', 4'- and 2',5'-modifications, 5'-E / 'Z-vinylphosphonate, and northern methanocarbacyclic (NMC) modifications. See, Gangopadhyay (2022) RNA Biol. 19(l):452-467; doi:10. 1080 / 15476286.2022.2052641. The use of substituted or modified single-stranded RNAi agents can be designed to have an increased half-life in a subject. Furthermore, certain substitutions or modifications can be used to improve the bioavailability of single-stranded RNAi agents by targeting particular cells or tissues or improving cellular uptake of the single-stranded RNAi agents. Exemplary modifications and locations within a RNAi polynucleotide are described in Hu et al. (2020) Signal Transduction and Targeted Therapy 5:100, incorporated herein by reference, see, e.g., Figures 2 and 3, specifically for its teachings of modifications.

[0124] The RNAi molecule may decrease the mRNA level in a cell for a target gene by at least about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 99%, or about 100% of the mRNA level found in the cell without the presence of the miRNA or RNA interference molecule.

[0125] The cargo may include a gene modulating agent, including gene editing systems, or a component thereof. Gene editing systems may comprise a CRISPR system, a zinc finger nuclease system, a meganuclease, or a TALE system. A CRISPR-Cas system can comprise a Class 1 or Class 2 CRISPR-Cas system, which may comprise a guide sequence engineered to specifically bind a polynucleotide of interest. A polynucleotide encoding the CRISPR-Caspolypeptide, a guide sequence designed to complex with the CRISPR-Cas polypeptide at a target of interest, or both components, can be utilized as cargo, delivered in the same nanoparticle or in different nanoparticles. As such, a ribonucleoprotein comprised of a Cas protein and a guide polynucleotide can be delivered. The CRISPR-Cas system that can be used to modify a target polynucleotide of the present invention described herein can be a Class 1 CRISPR-Cas system. Class 1 CRISPR-Cas systems are divided into types I, II, and IV. Makarova et al. (2020) Nat. Rev. 18:67-83, particularly as described in Figure 1. Type I CRISPR-Cas systems include Types I-A, I-B, I-C, I-D, I-E, I-Fl, I-F2, 1-F3, and IG; Type III CRISPR-Cas systems can be Types III- A, III-B, III-C, III-D, III-E, and III-F; which can contain a CaslO that can include an RNA recognition motif called Palm and a cyclase domain that can cleave polynucleotides; Type IV CRISPR-Cas systems include Types IV-A, IV-B, and IV-C. Class 2 systems comprise a single, large, multi-domain effector protein and can be a Type II, Type V, or Type VI system, which are described in Makarova et al. (2020) Nature Rev. Microbiol. 18:67-81, incorporated herein by reference. Class 2, Type II systems include II- A, II-B, II-C1, and II-C2; Type V systems includeV-A, V-Bl, V-B2, V-C, V-D, V-E, V-Fl, V-F1(V-U3), V-F2, V-F3, V-G, V-H, V-I, V-K (V- U5), V-Ul, V-U2, and V-U4. Class 2, Type VI systems include VI-A, VI-B1, VI-B2, VI-C, andVI-D. Design of guides for targeting a nucleic acid for modification is known in the art, see, e.g., IDTdna.com and Synthego.com for guidance on custom guide RNAs. Reduction of off-target effects can be tailored using programs such as GUIDE-seq for the design of guide sequences for a desired target. See, e.g., Malinin et al. (2012) Nature Protocols 16:5592-5615.

[0126] TALEN based gene editing is also contemplated and can be used in in vivo applications. See, Becker & Boch (2021) Gene and Genome Editing. Zinc finger nuclease editing can also be utilized, and further modified to ensure high-precision gene editing. See, e.g., Conway et al. (2019) Molecular Therapy 27(4):866-877; Paschon et al. (2019) Nature Comm. 10: 1133.Similarly, editing can be made by meganucleases, characterized by a large recognition site of 12 to 40 based pairs of a double-stranded DNA sequence. See, e.g., U.S. Patent Nos. 8,119,381, 10,273,524. Gene editing tools are well known in the art, with advantages and comparison of the tools that can be considered for the desired application. Rahim et al. (2021) Int’l. J. Innov. Sci. Res. Tech. 6:8, incorporated herein by reference.

[0127] Transposases may be used with the methods of the present invention. Transposases include those comprising RNase H-like nuclease domains, such as Tn5, MuA, Mosl, Hermes,Serine and Tyrosine recombinases, including CTnDOT, Tn916, IS607 and TnpX, transposases comprising an HUH domain, including TnpA of IS91 or ISHp608, and helitron transposases, which can be as detailed in International Patent Publication WO 2022 / 056309, page 26, line 26 - page 27, line 17, specifically incorporated by reference. See also nuclease guided transposase as described in WO 2022 / 150651 (DNA nuclease guided Transposases systems, Tn7-like transposition proteins with a Casl2k protein), WO 2022 / 147321 (Type I-B CRISPR Associated Transposase systems), WO 2022 / 076830 (Type I CRISPR Associated transposase systems), WO 2021 / 257997 (CAST); Li et al. (2020) Int. J. Mol. Sci. 21(21):8329; doi: 10.3390 / ijms21218329 (Tn5 transposase in applied genomic research).

[0128] DNA base editors and RNA base editors may be used as cargo in the present invention. In some embodiments, the DNA base editor is a cytosine base editor and mediates a C to T change. Exemplary base editors include BE3, AID, CDA1, APOBEC3G, BE4, BE4max and AncBEm4max. See, e.g., Koblan et al. (2018) Nat. Biotechnol. 36:843-846; Komor et al. (2017) Sci. Adv. 3(8):eaao4774. In an embodiment, the DNA base editor is an adenine base editor and mediates an A to G change. Exemplary adenine base editors include ABE8e and its variants, See, Gaudelli et al. (2020) Nat. Biotechnol. 38:892-900. Dual deaminase editors, for example, SPACE (synchronous programmable adenine and cytosine editor)(Grunewald et al. (2020) Nat.Biotechnol. 38:861-864), and A&C-BEmax (Zhang et al. (2020) Nat. Biotechnol. 38:856-860). The deaminase base editors can be used with CRIPSR-Cas proteins, including engineered nickase Cas9 or Cpfl. In some embodiments, the base editor is an RNA editor, and may comprise a Casl3 base editor. See, Cox et al. (2017) Science 358(6366): 1019-1027. Exemplary RNA editors can comprise, for example. AD ARI, ADAR2, ADAR2DD(E488Q), and can be used in systems such as REPAIR (RNA editing for programmable A to I (G) replacement as described in Cox, 2017) and RESCUE (C to U base editing as described in Abudayyeh et al. (2019) Science 365(6451):382-386.Therapeutic Agents

[0129] Therapeutic agents that can be used as cargo with the nanoparticles can comprise modulating agents, for example, small molecules such as chemotherapeutic agents, anti- oncogenic agents, anti-microbial agents, peptides, proteins (enzymes, antibodies, peptidic hormones), non-peptidic hormones, other pharmaceutically active substances, and the like.

[0130] Examples of the therapeutic agents can include hormones such as growth hormones, growth factors, cytokines, tumor necrosis factors, growth hormone releasing factors, steroid sparing agents such as cyclosporine, thyroid hormones, adrenaline, insulin, cortisol, estrogen and progesterone. Example enzymes can include transferases, hydrolases, lyases, isomerases, ligases, oxidoreductases, and translocases.

[0131] Anti-microbials such as antibiotics, antivirals, antifungals, and antiparasitics can be delivered in the nanoparticles of the present invention. Accordingly, the antimicrobials can be used to prevent and / or treat infections in a subject.

[0132] Examples of a chemotherapeutic agent include without limitation: alkylating agents (e. ., which may include doxorubicin, cyclophosphamide, estramustine, carmustine, mitomycin, bleomycin and the like); antimetabolites e.g., which may include 5-fluoro-uracil, capecitabine, gemcitabine, nelarabine, fludarabine, methotrexate and the like); platinating agents (e.g., which may include cisplatin, oxaliplatin, carboplatin and the like); topoisomerase inhibitors (e.g., which may include topotecan, irinotecan, etoposide and the like); tubulin agents (e.g, which may include paclitaxel, docetaxel, vinorelbine, vinblastine, vincristine, other taxanes, epothilones, and the like); signaling inhibitors (e.g, kinase inhibitors, antibodies, farnesyltransferase inhibitors, and the like); and other chemotherapeutic agents (e.g, tamoxifen, anti-mitotic agents such as polo-like kinase inhibitors or aurora kinase inhibitors, and the like).

[0133] Antibodies can be utilized in the methods of the invention. An antibody, or antigen binding fragment thereof, including polyclonal and monoclonal antibodies, can be utilized as cargo. The term "antibody fragment" refers to a portion of an immunoglobulin, often the hypervariable region and portions of the surrounding heavy and light chains, that displays specific binding affinity for a particular target, typically a molecule. A hypervariable region is a portion of an immunoglobulin that physically binds to the polypeptide target. An antibody fragment thus includes or consists of one or more portions of a full-length immunoglobulin retaining the targeting specificity of the immunoglobulin. Such antibody fragment may for instance lack at least partially the constant region (Fc region) of the full-length immunoglobulin. In some embodiments, an antibody fragment is produced by digestion of the full-length immunoglobulin. An antibody fragment may also be a synthetic or recombinant construct that contains one or more parts of the immunoglobulin or immunoglobulin chains (see e.g., Holliger & Hudson (2005) Nat. Biotechnol. 23(9): 1126-1136). Examples of an antibody fragment include,but are not limited to, an scFv, a Fab, a Fv, a Fab', a F(ab')2 fragment, a dAb, a VHH, a nanobody, a V(NAR) or a so-called minimal recognition unit.

[0134] In some embodiments of the invention, a method of modulating expression of a gene in a target cell is provided, comprising contacting the target cell with a nanoparticle of the present invention comprising a cargo.

[0135] Methods of delivering a cargo to a target cell can comprise contacting the target cell with a LNP comprising a cholesterol derivative as described herein. The contacting may occur in vivo, ex vivo, or in vitro.

[0136] Exemplary target cells include hematopoietic cells, including myeloid and lymphoid cells. The cell may be, e.g., a prostate cell, breast cell, kidney cell, lung cell, cardiac cell, muscle cell, liver cell, nerve cell, neuron, esophageal cell, brain cell, or epithelial cell. In embodiments the cell can be one or more cells from tissues or organs or part of a tissue or organ (e.g., in vivo applications) which include, e.g., brain, retina, genitals, hypothalamus, lung, liver, large intestine, skeletal muscle, smooth muscle, skin, bones, adipose tissues, hairs, thyroid, trachea, gall bladder, kidney, ureter, bladder, pancreas, aorta, vein, esophagus, diaphragm, stomach, heart, spleen, pancreas, small intestine, rectum, adrenal glands, bronchi, ears, eyes, larynx, nose, tongue, spinal cord, ureters, uterus, ovary, and testis. The cell may be a tumor cell or a nontumor cell. In an embodiment, the target cell is a spleen cell.

[0137] In some embodiments, a method of delivering a cargo to a subject in a tissue specific manner is provided, comprising contacting the tissue with a LNP of the present invention. In an embodiment, the tissue is spleen. In an embodiment, contacting comprises systemic or local administration. In an embodiment, an LNP of the present invention is administered intravenously, intramuscularly, subcutaneously, or topically.

[0138] Another aspect of the invention is a method of treating or preventing a disease in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of one or more nanoparticles comprising a cargo, thereby treating the disease.

[0139] In some embodiments, the methods treat or prevent a disease, disorder, or condition by introducing a cargo, e.g., thereby modulating the expression of a gene, stimulating an immune response, etc. Example applications include vaccine therapies, cancer treatments, pathogen (e.g., virus) infection disease therapies, and gene editing. In some embodiments, the disease or disorder is associated with the spleen, e.g., an autoimmune disease, for example, type I diabetes,systemic lupus erythematosus, autoimmune encephalitis, or multiple sclerosis. The administration of a nanoparticle comprising a cargo may increase or enhance expression or reduce or silence gene expression.

[0140] In an aspect, a method of vaccinating a subject in need thereof is provided, comprising administering to the subject a nanoparticle of the present invention, wherein the cargo provides an immunostimulatory effect. In some embodiments, the cargo is an antigen (e.g., a protein) or a nucleic acid encoding the antigen (e.g., an mRNA). The antigen may be from an organism for which protection by vaccination is desired.

[0141] In another aspect, methods to detect, diagnose, or monitor a disease, disorder, or condition are provided. Methods of detection can be in vitro and can comprise obtaining a sample from the subject. Suitable samples may comprise blood, serum, plasma, brain homogenate, interstitial fluid, cerebral spinal fluid, and / or exocrine gland secretion, and enriched forms thereof. According to one embodiment, the sample is a biological sample from a subject in need thereof. The biological sample can be from a subject that requires diagnosis of disease, and / or monitoring of the effectiveness of a treatment.

[0142] Methods of detection can be in vivo and can comprise administering a nanoparticle comprising an imaging agent or detectable label to the subject. The nanoparticle may comprise a labeled biomarker, which includes nucleic acids, proteins, metabolites, and reaction products thereof. Such biomarkers also encompass the mutations, variants, modifications, fragments, and polymorphisms of said biomarkers. In some embodiments, the subject requires diagnosis of, or is suspected of having, a disease or disorder, and optionally monitoring of the effectiveness of a treatment or progression of a disease. Accordingly, the nanoparticles comprising a cargo such as a biomarker, label, or imaging agent are useful in methods of diagnosing, prognosing and / or staging a disease or disorder in a subject by detecting a first level of expression, activity and / or function of one or more biomarker and comparing the detected level to a control level wherein a difference in the detected level and the control level indicates a diagnosis, prognosis, or stage of a disease or disorder, or a factor indicative of a diagnosis, prognosis or staging of a disease or disorder.

[0143] An aspect of the present invention is a method of transferring or delivering a cargo to a cell in vitro. The nanoparticle comprising a cargo may be introduced, e.g., by contact, into the cells at the appropriate cargo dosage suitable for the particular target cells. The amount ofnanoparticle to administer can vary, depending upon the target cell type and number, and the particular nanoparticle and cargo, and can be determined by those of skill in the art without undue experimentation.

[0144] A further aspect of the invention is a method of treating subjects in vivo, comprising administering to a subject a nanoparticle comprising one or more cargo, which may be further comprised in a pharmaceutically acceptable carrier, wherein the pharmaceutical composition is administered in a therapeutically effective amount. Administration of the nanoparticles of the present invention to a human subject or an animal in need thereof can be by any means known in the art for administering compounds.

[0145] Administration may preferably be by intravenous or intramuscular administration. In an example embodiment, the method comprises administering a nanoparticle comprising a therapeutic as a vaccine, wherein the administration is performed intramuscularly. In an example embodiment, the method comprises administering a nanoparticle comprising a therapeutic for treatment or prevention of a disease of disorder comprising administering the nanoparticle intravenously.

[0146] In an aspect, the delivery dose of the nanoparticle is about 0.01 to 10.00 mg ml;1, 0.05 to 5 mg mL-1, or between about 0.1 and 1.0 mg ml;1. In certain embodiments, disclosed nanoparticles are administered at dosage levels greater than about 0.001 mg / kg, such as greater than about 0.01 mg / kg or greater than about 0.1 mg / kg. For example, the dosage level may be from about 0.001 mg / kg to about 50 mg / kg such as from about 0.01 mg / kg to about 25 mg / kg, from about 0.1 mg / kg to about 10 mg / kg, or from about 1 mg / kg to about 5 mg / kg of subject body weight per day, one or more times a day, to obtain the desired therapeutic effect. It will also be appreciated that dosages smaller than about 0.001 mg / kg or greater than about 50 mg / kg (for example about 50-100 mg / kg) can also be administered to a subject.

[0147] Non-limiting examples of formulations of the invention include those suitable for intravenous administration of the nanoparticles. Oral, rectal, buccal (e.g., sub-lingual), vaginal, parenteral (e.g, subcutaneous, intramuscular including skeletal muscle, cardiac muscle, diaphragm muscle and smooth muscle, intradermal, intravenous, intraperitoneal), topical (z.e., both skin and mucosal surfaces, including airway surfaces), intranasal, transdermal, intraarticular, intracranial, intrathecal, and inhalation administration, administration to the liver by intraportal delivery, as well as direct organ injection (e.g., into the liver, into a limb, into thebrain or spinal cord for delivery to the central nervous system, into the pancreas, or into a tumor or the tissue surrounding a tumor) are also envisioned. The most suitable route in any given case will depend on the nature and severity of the condition being treated and on the nature of the particular compound which is being used. In some embodiments, it may be desirable to deliver the formulation locally to avoid any side effects associated with systemic administration. For example, local administration can be accomplished by direct injection at the desired treatment site, by introduction intravenously at a site near a desired treatment site (e.g., into a vessel that feeds a treatment site, or intramuscular administration with muscle specific promoters). In some embodiments, the formulation can be delivered locally to ischemic tissue.

[0148] For injection, the carrier will typically be a liquid, such as sterile pyrogen-free water, pyrogen-free phosphate-buffered saline solution, bacteriostatic water, or Cremophor EL® (BASF, Parsippany, NJ). For other methods of administration, the carrier can be either solid or liquid.

[0149] For oral administration, the compound can be administered in solid dosage forms, such as capsules, tablets, and powders, or in liquid dosage forms, such as elixirs, syrups, and suspensions. Examples of additional inactive ingredients that can be added to provide desirable color, taste, stability, buffering capacity, dispersion or other known desirable features are red iron oxide, silica gel, sodium lauryl sulfate, titanium dioxide, edible white ink and the like. Similar diluents can be used to make compressed tablets. Both tablets and capsules can be manufactured as sustained release products to provide for continuous release of medication over a period of hours. Compressed tablets can be sugar coated or fdm coated to mask any unpleasant taste and protect the tablet from the atmosphere, or enteric-coated for selective disintegration in the gastrointestinal tract. Liquid dosage forms for oral administration can contain coloring and flavoring to increase patient acceptance.

[0150] Formulations suitable for buccal (sub-lingual) administration include lozenges comprising the compound in a flavored base, usually sucrose and acacia or tragacanth; and pastilles comprising the compound in an inert base such as gelatin and glycerin or sucrose and acacia.

[0151] Formulations of the present invention suitable for parenteral administration comprise sterile aqueous and non-aqueous injection solutions of the compound, which preparations are preferably isotonic with the blood of the intended recipient. These preparations can contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient. Aqueous and non-aqueous sterile suspensions can include suspending agents and thickening agents. The formulations can be presented in unit / dose or multi-dose containers, for example sealed ampoules and vials, and can be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example, saline or water-for-inj ection immediately prior to use.

[0152] In one aspect of the present invention, there is provided an injectable, stable, sterile composition comprising a compound of the invention, in a unit dosage form in a sealed container. The compound or salt is provided in the form of a lyophilizate which is capable of being reconstituted with a suitable pharmaceutically acceptable carrier to form a liquid composition suitable for injection thereof into a subject. The unit dosage form typically comprises from about 10 mg to about 10 grams of the compound or salt. When the compound or salt is substantially water-insoluble, a sufficient amount of emulsifying agent which is pharmaceutically acceptable can be employed in sufficient quantity to emulsify the compound or salt in an aqueous carrier. One such useful emulsifying agent is phosphatidyl choline.

[0153] Formulations suitable for rectal administration are preferably presented as unit dose suppositories. These can be prepared by admixing the compound with one or more conventional solid carriers, for example, cocoa butter, and then shaping the resulting mixture.

[0154] Formulations suitable for topical application to the skin preferably take the form of an ointment, cream, lotion, paste, gel, spray, aerosol, or oil. Carriers which can be used include petroleum jelly, lanoline, polyethylene glycols, alcohols, transdermal enhancers, and combinations of two or more thereof.

[0155] Formulations suitable for transdermal administration can be presented as discrete patches adapted to remain in intimate contact with the epidermis of the recipient for a prolonged period of time. Formulations suitable for transdermal administration can also be delivered by iontophoresis (see, for example, Tyle (1986) Pharm. Res. 3:318) and typically take the form of an optionally buffered aqueous solution of the compound. Suitable formulations comprise citrate or bis\tris buffer (pH 6) or ethanol / water and contain from 0.1M to 0.2M of the compound.

[0156] The compound can alternatively be formulated for nasal administration or otherwise administered to the lungs of a subject by any suitable means, e.g., administered by an aerosol suspension of respirable particles comprising the compound, which the subject inhales. Therespirable particles can be liquid or solid. The term “aerosol” includes any gas-borne suspended phase, which is capable of being inhaled into the bronchioles or nasal passages. Specifically, aerosol includes a gas-borne suspension of droplets, as can be produced in a metered dose inhaler or nebulizer, or in a mist sprayer. Aerosol also includes a dry powder composition suspended in air or other carrier gas, which can be delivered by insufflation from an inhaler device, for example. See Ganderton & Jones (1987) Drug Delivery to the Respiratory Tract, Ellis Horwood; Gonda (1990) Crit. Rev. Therap. Drug Carrier Systems 6:273-313; and Raeburn et al. ( 1992) . / . Pharmacol. Toxicol. Meth. 27: 143. Aerosols of liquid particles comprising the compound can be produced by any suitable means, such as with a pressure-driven aerosol nebulizer or an ultrasonic nebulizer, as is known to those of skill in the art. See, e.g., U.S. Patent No. 4,501,729. Aerosols of the particles of the invention can likewise be produced with any solid particulate medicament aerosol generator, by techniques known in the pharmaceutical art.

[0157] Having described the present invention, the same will be explained in greater detail in the following examples, which are included herein for illustration purposes only, and which are not intended to be limiting to the invention.EXAMPLESExample 1. Cholesterol Derivatives and Their Characterization in Lipid Nanoparticles

[0158] An overview of a synthetic reaction for cholesterol derivatives of the present invention is exemplified in Scheme 1. Example cholesterol derivatives of the present invention were synthesized and characterized via1NMR as described below.

[0160] 5 g of Cholesterol (12.93 mmol, 1 eq, Sigma Aldrich C8667) was added to a reaction flask containing 125 mL of toluene and left to stir. 2.42 mL of propiolic acid (32.33 mmol, 2.5 equivalents Sigma Aldrich P51400) was added to the reaction, immediately followed by 0.222 g of / ?ara-toluene sulfonic acid (1.29 mmol, 0.1 equivalents Sigma Aldrich 8. 14725). The reaction refluxed at 165°C using a Dean Stark Trap for 24 hours. The next day the reaction was cooled and the solvent was evaporated through rotary evaporation to yield crude product. A silica column was then run using a gradient column using hexane and diethyl ether (100:0 — 96:4 — 94:6) as the mobile phase to afford the A00 with a yield of 64% (FIG. 2).

[0161] Synthesis of Al

[0162] 9.3 mg of N,N'-dimethyl-l,3-propanediamine (0.091 mmol, 1.0 equivalents Sigma Aldrich 308110) was added into 0.5 mL of chloroform and left to stir. A 0.5 mL solution of chloroform containing A00 (40 mg, 0.091 mmol, 1.0 equivalents) was then added into the reaction flask and left to stir overnight at room temperature. The solvent was then removed through rotary evaporation to yield Al (FIG. 3).

[0164] 20.8 mg of l,8-dimethyl-l,4,8,l 1-tetraazacyclotetradecane (0.091 mmol, 1.0 equivalents Sigma Aldrich 526169) was added into 0.5 mL of chloroform and left to stir. A 0.5 mL solution of chloroform containing A00 (40 mg, 0.091 mmol, 1.0 equivalents) was then added into the reaction flask and left to stir overnight at room temperature. The solvent was then removed through rotary evaporation to yield A2 (FIG. 4).

[0166] 15.71 mg ofN,N'-dimethyl-l ,8-octanediamine (0.091 mmol, 1.0 equivalents Sigma Aldrich 493775) was added into 0.5 mL of chloroform and left to stir. A 0.5 mL solution of chloroform containing A00 (40 mg, 0.091 mmol, 1.0 equivalents) was then added into the reaction flask and left to stir overnight at room temperature. The solvent was then removed through rotary evaporation to yield A3 (FIG. 5).

[0168] 13.15 mg ofN,N'-dimethyl-l,6-hexanediamine (0.091 mmol, 1.0 equivalents Sigma Aldrich DI 61101) was added into 0.5 mL of chloroform and left to stir. A 0.5 mL solution of chloroform containing A00 (40 mg, 0.091 mmol, 1.0 equivalents) was then added into the reaction flask and left to stir overnight at room temperature. The solvent was then removed through rotary evaporation to yield A4 (FIG. 6).

[0170] 11.87 mg ofN,N'-diethyl-l,3-propanediamine (0.091 mmol, 1.0 equivalents Sigma Aldrich 138428) was added into 0.5mL of chloroform and left to stir. A 0.5 mL solution of chloroform containing A00 (40 mg, 0.091 mmol, 1.0 equivalents) was then added into the reaction flask and left to stir overnight at room temperature. The solvent was then removed through rotary evaporation to yield A5 (FIG. 7).

[0171] Synthesis of A6

[0172] 9.13 mg of Homopiperazine (0.091 mmol, 1.0 equivalents Sigma Aldrich Hl 6604) was added into 0.5 mL of chloroform and left to stir. A 0.5 mL solution of chloroform containing A00 (40 mg, 0.091 mmol, 1.0 equivalents) was then added into the reaction flask and left to stir overnight at room temperature. The solvent was then removed through rotary evaporation to yield A6 (FIG. 8).

[0173] Synthesis of A7

[0174] 19.18 mg of 4,4'-Trimethylylenedipiperidine (0.091 mmol, 1.0 equivalents Sigma Aldrich 121207) was added into 0.5 mL of chloroform and left to stir. A 0.5 mL solution of chloroform containing A00 (40 mg, 0.091 mmol, 1.0 equivalents) was then added into the reaction flask and left to stir overnight at room temperature. The solvent was then removed through rotary evaporation to yield A7 (FIG. 9).

[0175] Synthesis of A8

[0176] 5.88 mg of Azetidine (0.103 mmol, 1.0 equivalents Sigma Aldrich 281069) was added into 0.5 mL of chloroform and left to stir. A 0.5 mL solution of chloroform containing A00 (45 mg, 0.103 mmol, 1.0 equivalents) was then added into the reaction flask and left to stir overnight at 45°C. The solvent was then removed through rotary evaporation to yield A8 (FIG. 10).

[0178] 7.33 mg of Pyrrolidine (0.103 mmol, 1.0 equivalents Sigma Aldrich P73803) was added into 0.5 mL of chloroform and left to stir. A 0.5 mL solution of chloroform containing A00 (45 mg, 0.103 mmol, 1.0 equivalents) was then added into the reaction flask and left to stir overnight at 45°C. The solvent was then removed through rotary evaporation to yield A9 (FIG. 11).

[0180] 8.77 mg of Piperidine (0.103 mmol, 1.0 equivalents Sigma Aldrich 104094) was added into 0.5 mL of chloroform and left to stir. A 0.5 mL solution of chloroform containing A00 (45 mg, 0.103 mmol, 1.0 equivalents) was then added into the reaction flask and left to stir overnight at 45°C. The solvent was then removed through rotary evaporation to yield A10 (FIG. 12).

[0182] 10.21 mg of Hexamethyleneimine (0.103 mmol, 1.0 equivalents Sigma AldrichHl 0401) was added into 0.5 mL of chloroform and left to stir. A 0.5 mL solution of chloroform containing A00 (45 mg, 0.103 mmol, 1.0 equivalents) was then added into the reaction flask and left to stir overnight at 45°C. The solvent was then removed through rotary evaporation to yield Al 1 (FIG. 13).

[0184] 11.65 mg of Heptamethyleneimine (0.103 mmol, 1.0 equivalents Sigma Aldrich H2155) was added into 0.5 mL of chloroform and left to stir. A 0.5 mL solution of chloroform containing A00 (45 mg, 0.103 mmol, 1.0 equivalents) was then added into the reaction flask and left to stir overnight at 45°C. The solvent was then removed through rotary evaporation to yield A12 (FIG.14).

[0185] Synthesis of M3

[0186] 10.21 mg of 3 -Methylpiperidine (0.103 mmol, 1.0 equivalents Sigma Aldrich M73001) was added into 0.5 mL of chloroform and left to stir. A 0.5 mL solution of chloroform containing A00 (45 mg, 0.103 mmol, 1.0 equivalents) was then added into the reaction flask and left to stir overnight at 45°C. The solvent was then removed through rotary evaporation to yield Al 3 (FIG.15).

[0188] 13.41 mg ofN,N-diethyl-N'-methylethylenediamine (0.103 mmol, 1.0 equivalentsSigma Aldrich 308099) was added into 0.5 mL of chloroform and left to stir. A 0.5 mL solution of chloroform containing A00 (45 mg, 0.103 mmol, 1.0 equivalents) was then added into the reaction flask and left to stir overnight at 45°C. The solvent was then removed through rotary evaporation to yield A14 (FIG. 16).

[0190] 7.74 mg of 2-(Methylamino)ethanol (0.103 mmol, 1.0 equivalents Sigma Aldrich471445) was added into 0.5 mL of chloroform and left to stir. A 0.5 mL solution of chloroform containing A00 (45 mg, 0.103 mmol, 1.0 equivalents) was then added into the reaction flask andleft to stir overnight at 45°C. The solvent was then removed through rotary evaporation to yieldAl 5 (FIG. 17).

[0192] 8.9 mg of Morpholine (0.103 mmol, 1.0 equivalents Sigma Aldrich 134236) was added into 0.5 mL of chloroform and left to stir. A 0.5 mL solution of chloroform containing A00 (45 mg, 0.103 mmol, 1.0 equivalents) was then added into the reaction flask and left to stir overnight at 45°C. The solvent was then removed through rotary evaporation to yield Al 6 (FIG. 18).

[0194] 13.71 mg of Bis-(2-methoxyethyl)amine (0.103 mmol, 1.0 equivalents Sigma Aldrich B48207) was added into 0.5 mL of chloroform and left to stir. A 0.5 mL solution of chloroform containing A00 (45 mg, (0.103 mmol, 1.0 equivalents) was then added into the reaction flask and left to stir overnight at 45°C. The solvent was then removed through rotary evaporation to yield Al 7 (FIG. 19).

[0195] Synthesis o / A18

[0196] 9.18 mg of (2-Methoxyethyl)methylamine (0.103 mmol, 1.0 equivalents Sigma Aldrich 708917) was added into 0.5 ml of chloroform and left to stir. A 0.5 mL solution of chloroform containing A00 (45 mg, (0.103 mmol, 1.0 equivalents) was then added into the reaction flask and left to stir overnight at 45°C. The solvent was then removed through rotary evaporation to yield Al 8 (FIG. 20).

[0198] 16.60 mg of A, A-Bis-(2-ethoxyethyl)amine (0.103 mmol, 1.0 equivalents SigmaAldrich CDS000629) was added into 0.5 mL of chloroform and left to stir. A 0.5 mL solution of chloroform containing A00 (45 mg, (0.103 mmol, 1.0 equivalents) was then added into the reaction flask and left to stir overnight at 45°C. The solvent was then removed through rotary evaporation to yield A19 (FIG. 21).

[0199] Characterization of Lipid Nanoparticles Formulated with Cholesterol Derivatives. The overall concept for the development and evaluation of LNPs comprising the cholesterol derivatives A00-A19 is provided in FIG. 22. The novel cholesterol derivative was formulated into a lipid nanoparticle at 38.5 mol% cholesterol derivative, 50 mol% SM-102, 10 mol% DSPC, and 2.5 mol% DMG-PEG2000. Lipid nanoparticle and mRNA were administered to mice at 0.75 mg / mL by IV infusion to monitor in vivo activity. Nanoparticle characteristics such as encapsulation efficiency, charge, and size also were determined. Representative in vivo imagesof tissue specific delivery of exemplary LNPs comprising cholesterol derivatives Al , A2, A3, A8, Al 6, Al 7, Al 8, or Al 9 with their respective size, charge, and encapsulation efficiency for LNPs are shown in FIGS. 23A-23C. A control of naked Flue mRNA, and representative Modema LNP with formulation 38.5% Cholesterol, 50% SM-102, 10% DSPC, 2.5% DMG- PEG2000 (% by mol fraction) are also shown. As shown, spleen specific delivery is achieved by in vivo delivery of LNPs comprising cholesterol derivatives and delivering mRNA. See, FIG. 1.Example 2. Optimized Spleen-Expressing mRNA LNP Platform

[0200] Genetic medicines such as mRNA have the capacity to regulate gene expression, an incredibly powerful tool in the treatment of disease. However, its large size, immunogenicity, and strong negative charge make it a largely ineffective drug when administered alone. Lipid nanoparticles (LNPs) have been demonstrated to be effective carriers of mRNA for the treatment of several diseases such as cancer, infectious diseases and autoimmune disorders. However, one central limitation of clinically validated mRNA LNP formulations is that their activity is primarily localized to the liver when administered intravenously. Therefore, expanding the therapeutic impact of this powerful class of drug will require the development of an mRNA LNP formulation that can target extrahepatic organs - such as the spleen.

[0201] The spleen is the largest secondary lymphoid organ in the body and is a site for antigen presentation, filtration of blood borne pathogens and activation of cellular immune responses within the bloodstream, making it a prime target for genetic medicines for diseases involving these pathways. One cell type within the spleen of particular interest is macrophages. Macrophages have a broad role in maintaining homeostasis. Indeed, dysregulation of macrophages can contribute to the generation of immunosuppressive tumor microenvironment in cancer, chronic inflammation in autoimmune disorders, and antigen presentation in cancer vaccine applications. Therefore, developing a novel LNP platform with effective targeting in macrophages can benefit a range of diseases such as cancer, and autoimmune disorders.

[0202] Herein, a novel spleen expressing lipid nanoparticle (SE-LNPs) platform is presented through the combination of chemical synthesis and microfluidic formulation. Then, the activity of the SE-LNP library was optimized and screened through the use of design of experiment (DOE) to identify lead SE-LNP formulations with the best mRNA expression performance on macrophages. Upon in vitro screening and identification of three lead SE-LNP formulations,mechanistic studies were performed to elucidate the cellular association, uptake pathways, and endosomal escape process to understand how these formulations behave in macrophages in comparison to the LNP formulation used in the Moderna COVID-19 vaccine. Additionally, how hypoxia affects the overall efficacy of the SE-LNP platform was studied as it has been shown that hypoxia can affect the overall efficacy of mRNA activity. Following mechanistic evaluation, the lead SE-LNPs were administered in vivo and demonstrated nearly 100% expression within the spleen compared to the 30% FLuc expression of Moderna LNP formulation in the spleen. Taken in tandem, it is anticipated that the SE-LNPs can be used as a versatile mRNA delivery platform for spleen cancer immunotherapy and other disorders and conditions.Results

[0203] To create the SE-LNP platform, a novel cholesterol derivative was synthesized in two steps beginning with esterification of cholesterol with propiolic acid via Dean Starks Esterification to form an intermediate. This intermediate was then reacted with (2-Methoxyethyl) methylamine to generate “N-Sterol” (Scheme 2; A00, FIG. 2; Al 8, FIG. 20).N-Sterol40Scheme 2

[0204] We hypothesized that the introduction of a methoxy group and tertiary amine to cholesterol would alter the biodistribution of the formulated LNPs, due to the role cholesterol may have in dictating liver tropism. Then, our first-generation SE-LNP (A0) was formulated by micro fluidic mixing an organic phase containing, ionizable lipid SM-102, phospholipid DOPE N-sterol and PEG lipid with the 14:0 PEG IK with the molar ratio of 50 / 10 / 38.5 / 1.5, and an aqueous phase consisting of mRNA encoding for firefly luciferase (FLuc) (FIG. 24).N-Sterol (Cholesterol Derivative)14:0 PEG 1 KTable 2. (A.) Formulation parameters used to generate Library A (B.) Formulation parameters used to generate Library B (C.) Formulation parameters used to generate Library C.A.B.C

[0205] It was next sought to optimize the first-generation SE-LNP formulation AO through the use of Design of Experiment (DOE) which enabled rapid generation and screening of three libraries, thus enabling refinement of a large formulation design space in an efficient and cost- effective manner. (FIG. 25A) To begin the DOE optimization, an initial formulation space wasconstructed specifically by establishing identities and ranges of the molar ratios of ionizable lipid, phospholipid, N-sterol and PEG lipid building off of the initial formulation, AO. To decrease the number of potential formulations to evaluate, a definitive screening design (DSD) was used to generate an initial library consisting of 18 discrete formulations, which were based on the initial formulation space created (Tables 2A and 3). A DSD was chosen to generate the initial library as this statistical method is meant for initial range-finding and enabled screening of a large number of factors without increasing the number of SE-LNP formulations, while still being able to observe main effects. Upon evaluating the protein expression on J774A.1 macrophages of each formulation in Library A, a lead SE-LNP formulation (termed A6) was identified. Building upon A6 as a lead formulation, the initial design space used in Library A was refined and generated a second library consisting of 8 discrete formulations termed Library B (Tables 2B and 4). Library B was generated using a definitive factorial design leading to identification of a second lead (termed B7). Using A6 and B7 as lead formulations, the design space was further refined and generated a final library, termed Library C, (Tables 2C and 5) consisting of 5 discrete formulations once again by using a definitive factorial design, which enabled identification of a third lead formulation CL After collective analysis of this data, several findings emerged. First, various types of SE-LNPs were successfully formulated with reproducible size, zeta potential, mRNA encapsulation efficiency and PDI. The sizes ranging from ~200 nm to 350 nm; zeta potentials ranging from —40 mV to ~0 mV, mRNA encapsulation efficiency ranging from -20% to -60%; and the PDI ranging from 0.05 to 0.3 (FIG. 25B, Table 6). Second, all three lead SE-LNP formulations (A6, B7, Cl) resulted in a greater than 20-fold increase in FLuc expression in J774A.1 library optimization through the use of design of experiment (FIG. 25C). Third, each formulation within each library was well tolerated, as evidenced by the observed cell viability being similar to untreated cells (FIGS. 26A-26C). Table 3. Formulation Ratios of each LNP in Library A.Table 4. Formulation Ratios of each LNP in Library B.Table 5. Formulation Ratios of each LNP in Library C.Table 6. Size, zeta, EE, PDI of Lead LNPs encapsulating FLuc mRNA in vitro (Data shown as the mean ± SEM, n = 3).

[0206] Within these three libraries, three lead SE-LNP formulations (A6, B7, Cl) were identified which were used for further evaluation. To begin, the molar ratio of each formulation - A6, B7, and Cl - was examined and it was found that all three formulations had similar molar ratios of SM-102, N-Sterol and 14:0 PEG IK ranging from 46-48%, 44-46%, and 0.5-0.6%, respectively (Table 7). It is worth noting that the first-generation SE-LNP formulation A0 consisted of 50% SM-102, 38.5% N-Sterol, and 1.5% 14:0 PEG IK, suggesting the molar ratio of each SE-LNP component plays an important role in mRNA translation. Interestingly, it was found that DSPC, as compared to DOPE (used in SE-LNP A0), was the phospholipid comprising all three lead formulations (Table 7).Table 7. Formulation ratios of lead SE-LNPs.

[0207] Next, lead SE-LNP formulations were evaluated in a dose-response and time-dependent fashion. As a clinical benchmark, the Moderna LNP formulation was chosen as a side-by-side control (Table 8 and FIG. 32). In collectively analyzing these data, several trends were observed. First, it was found all three SE-LNP formulations responded in a dose-responsive fashion with 200 ng mRNA being the highest FLuc expression at 2, 6, and 24 h. However, at 48 h expression, an increase in dose did not correlate to increased expression, with all doses being well tolerated as the cell viability for each was similar to untreated cells (FIG. 27A, FIG. 30). Second, it was found that FLuc expression increased from 2 hours to 6 hours and 24 hours for all LNP formulations, but decreased at 48 hours (FIG. 27A). Finally, Cl had the highest expression at 200 ng at 24 hours as compared to A6 and B7, however, B7 had the highest expression at 200 ng at 2 and 6 h (FIG. 27A). Taken together, these results suggest that the protein expression of SE-LNPs in a dose-responsive and time-dependent fashion.Table 8. Moderna LNP formulation

[0208] Finally, the effects of hypoxia on the SE-LNP platform were evaluated. Previous studies have demonstrated that hypoxia affects LNP-mediated mRNA expression. Consequently, the SE- LNPs were evaluated in normoxia and hypoxia at three respective doses and protein expression quantified, as well as the percent change in expression, between the two conditions. Upon analyzing this data, a complex relationship between hypoxia and LNP-mediated mRNA expression in macrophages was found. At 50 ng, SE-LNP A6 had an increase in expression whereas Moderna, B7, and Cl expression was decreased (FIGS. 27B-27C). At 100 ng, all four formulations had a significant decrease in protein expression; however, comparing previous studies using MDCK, U251, and DC 2.4 cell lines, the percent decrease in expression between normoxia and hypoxia was less (FIGS. 27B-27C). Finally, at 200 ng, expression decrease was non-significant across all four formulations, with Moderna LNP control having an increase in protein expression. Taken together, these findings indicate that LNP-mediated mRNA expression in macrophages under various oxygenation conditions in complex.

[0209] Next, the mechanism of mRNA delivery of our SE-LNPs was explored including the cellular association, cellular uptake pathway, and endosomal escape in macrophages. In the cellular association study, Cy-5 labeled FLuc mRNA was encapsulated in the lead SE-LNPs and Cy-5 positive cells quantified through the use of flow cytometry on J774A.1 cell lines (FIG. 29A). To elucidate the cellular uptake pathway in which macrophages take up our lead SE-LNPs, four main cellular uptake pathways were individually inhibited using Pitstop 2 (Clathrin- Mediated Endocytosis), EIPA (Macropinocytosis), Filipin, (Caveolae Mediated Endocytosis), and Cytochalasin D (Phagocytosis). Upon inhibition, the level of Cy-5 positive cells was observed after each inhibitor and the percent inhibition of uptake quantified. Finally, to quantify the level of endosomal escape of SE-LNPs, confocal microscopy was used and the colocalization (yellow) between Cy-5 LNPs (red) and endo / lysosomes (green) quantified through the Pearson correlation coefficient (PCC). In these three studies, the Moderna LNP formulation was used as a clinically relevant benchmark. Upon analyzing this mechanistic data, several findings emerged. First, the cellular association of SE-LNP A6, and Cl were statistically similar to the Moderna LNP at both 2 and 24 h time points, whereas SE-LNP B7 showed significantly lower cellular association at 2 h but similar association at 24 h. (FIG. 29A, FIG. 30). Second, SE-LNP A6 and Cl were taken up by cells with a combination of macropinocytosis and phagocytosis as both SE- LNPs were inhibited primarily by EIPA and Cytochalasin D. However, SE-LNP B7 wasinhibited most by Pitstop, Filipin, and Cytochalasin D, indicating that it is taken up through a combination of clathrin-mediated endocytosis, caveolin-mediated endocytosis, and phagocytosis (FIG. 29B). Finally, the level of endosomal escape was quantified using confocal microscopy. In this assay, cells were stained with Hoechst Blue and LysoTracker Green to stain for the nucleus (blue), and the lysosome (green) respectively, and mRNA was labeled with Cy-5 fluorophore. Overlap of the green lysosome and red mRNA gives rise to a yellow signal, suggesting less endosomal escape, while separation of the two indicates more endosomal escape (FIG. 29C). To quantify the level of endosomal escape in each image, the Pearson Correlation Coefficient (PCC) was quantified, where 1 indicates no endosomal escape and 0 indicate complete endosomal escape. Upon confocal imaging and PCC analysis, a significantly decreased PCC value was found in all the SE-LNP formulations when compared to the Modema LNP formulation, suggesting better endosomal escape (FIG. 29C). Taken together, these results reveal that SE- LNPs are internalized through a combination of uptake pathways, depending on the molar ratio of the formulation and that SE-LNPs have increased endosomal escape capability when compared to the Modema clinical benchmark.

[0210] After mechanistic analysis, the study was completed by evaluating the in vivo activity of the lead SE-LNPs. To do so, FLuc mRNA was encapsulated with lead SE-LNP A6, B7 and Cl formulations and intravenously injected into C57BL / 6 mice at a dose of 0.65 mg / kg of FLuc mRNA. Mice injected with naked FLuc mRNA and PBS were used as controls. After 24 h, the organs were resected and imaged for bioluminescence. Furthermore, the tolerability of SE-LNPs was evaluated by the histological analysis on the spleen, liver, and kidneys and weight retention data (FIG. 33). Upon analyzing these data, several trends emerged. First, the three lead SE-LNP formulations exhibited -100% mRNA expression within the spleen, whereas the Moderna LNP formulation showed main FLuc expression in the liver (80%) as indicated by luminescence signal (FIGS. 31A-31B) Second, SE-LNP A6 had the highest mRNA expression within the spleen as compared to B7 and Cl, though all three were significantly higher than the Modema LNP formulation. Third, each SE-LNP formulation was well-tolerated, as indicated by the analysis of the histology (FIG. 31C) and weight retention (FIG. 33) where there was no difference observed among the SE-LNP treatment groups with PBS control group. Taken in tandem, these results suggest that the SE-LNPs, especially the SE-LNP A6, can be used as a versatile platform for delivering mRNA to the spleen.Conclusion

[0211] In this example, a novel cholesterol derivative - termed N-sterol - was successfully synthesized and a safe and effective SE-LNP platform formulated for effective mRNA delivery. Specifically, this was accomplished by utilizing DOE to screen the activity of SE-LNP on macrophages, followed by mechanism evaluation, including the cellular association, uptake pathways and endosome escape. The therapeutic understanding of this platform and macrophages in general was further built by evaluating the differences in LNP-mediated mRNA- expression in hypoxia versus normoxia and it was found that each formulation was generally unaffected by oxygenation levels, which is a finding unique to macrophages thus far and will have significant impacts on the therapeutic relevance of LNP-mediated mRNA therapies for the treatment of hypoxia-associated diseases such as cancer and development of cancer vaccines targeting the spleen. Moreover, the results suggest that the SE-LNP formulation facilitates the expression of mRNA cargo almost exclusively in the spleen in vivo. The ability of the SE-LNP platform to target the spleen in vivo and express mRNA cargo in macrophages is especially relevant for cancer immunotherapies and autoimmune disorders, as splenic macrophages play an important role in the pathology of these diseases.Experimental Section

[0212] Materials'. Propiolic Acid, / / ra-Toluenesulfonic acid, (2-Methoxyethyl)methylamine, Cholesterol, Dulbecco’s phosphate-buffered saline (DPBS), phosphate-buffered saline (PBS, pH 7.4), sodium citrate, citrate acid, penicillin-streptomycin, Triton X-100, NaNi, filipin from S. filipinensis, cytochalasin D, (N-ethyl-N-isopropyl) amiloride (EIP A), Tween 20, and were all purchased from Sigma-Aldrich. l,2-dioleoyl- / ?-glycero-3 -phosphoethanolamine (DOPE), 1,2- dimyristoyl-w-glycero-3-phosphoethanolamine-N-[methoxy-(polyethyleneglycol)-l 000] (C14- PEG 1000), l,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG 2000), and l,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) were purchased from Avanti. The ionizable lipid SM-102 was bought from Broadpharm. RPMI 1640, Fetal Bovine Serum (FBS), LysoTracker Deep Red, trihydrochloride (Hoecht-33342, lOmg mL’1) were purchased from Life Technologies. CleanCap Firefly luciferase (FLuc) mRNA (5-moU) was obtained from TriLink Biotechnologies. EZ Cap Cy-5 Labeled FLuc mRNA was purchased from APEXBio. Quant-iT RiboGreen RNA assay kit, Alamar Blue Cell Viability Reagent was purchased from Thermo Fisher Scientific, USA. Bright-Glo Luciferase assay system was obtained from Promega. Pitstop2 was purchased from Abeam. Ultrapure water (Milli-Q) with a resistivity greater than 18.2 MQ cm was used in all experiments and obtained from a three stage Millipore Milli-Q Plus 185 purification system. All chemicals were used without further purification.

[0213] Synthesis of N-Sterol: 5 g of Cholesterol (12.93 mmol, 1 eq, Sigma Aldrich C8667) was added to a reaction flask containing 125 mL of Toluene. 2.42 mL of Propiolic Acid (32.33 mmol, 2.5 equivalents Sigma Aldrich P51400) was added to the reaction, immediately followed by 222 mg of / wc / -Toluenesulfonic Acid (1.29 mmol, 0.1 equivalents Sigma Aldrich 8.14725). The reaction was heated under reflux at 165 °C using a Dean-Stark apparatus for 24 h. The reaction was subsequently cooled, and the solvent was removed via rotary evaporation. The crude product then passed through a silica column with a gradient elution profile comprising hexane and diethylether (100:0 — > 96:4 — > 94:6) as the mobile phase to afford an intermediate with a yield of 64%. After purification, 9.18 mg of (2-Methoxyethyl)methylamine (0.103 mmol, 1.0 equivalents Sigma Aldrich 708917) was added into 0.5 mL of Chloroform and left to stir. A 0.5 mL solution of Chloroform containing the intermediate (45 mg, (0.103 mmol, 1.0 equivalents) was then added into the reaction flask and left to stir overnight at 45 °C. The solvent was then removed via rotary evaporation to yield N-Sterol.

[0214] Formulation of Spleen Targeting Lipid Nanoparticles: An ethanol phase was prepared by mixing the ionizable lipid SM-102, dioleoyl-sn-glycero-3-phosphoethanolamine, N-Sterol, l,2-dimyristoyl- / 7-glycero-3 and l,2-dimyristoyl- H-glycero-3-phosphoethanolamine-N- [methoxy-(polyethyleneglycol)] of molecular weight 1000 respectively. An aqueous phase was made by dissolving the 0.1 mg mL'1of FLuc / Cy-5 labeled FLuc mRNA in 10 mM citrate buffer (pH 3). Then, the aqueous and ethanol phases were mixed at a ratio of 3 : 1 into a microfluid chip device. Finally, the LNPs were dialyzed against lx PBS in a 20K MWCO dialysis cassette at 4 °C in a cold room for 2 h and stored at 4 °C prior for further use.

[0215] Characterization of LNPs: All LNPs from Library A, B and C were measured using dynamic light scattering (DLS) (NanoBrook 90 Plus Zeta, Brookhaven, USA). To prepare DLS and Zeta samples, LNPs were diluted in IX PBS (pH 7.4) and 0.1X PBS (pH 7.4) respectively. The FLuc / Cy-5 labeled FLuc mRNA encapsulation efficiency was quantified by Quant-iT RiboGreen RNA assay kit and read using a SpectraMax microplate reader (Molecular Device, USA) with 480 nm excitation wavelength and 520 nm emission wavelength. To characterize the overall mRNA concentration, the LNPs were fully dissolved with IX Triton X-100.

[0216] In Vitro Dose Response of FLuc Expression: J774A.1 cells were seeded at a density of 1 x 104cells per well in 96 well plates at 37 °C. The next day, the cell media was aspirated and 100 pL of 500 ng ml1, 1000 ng ml / 1or 2000 ng ml / 1of FLuc mRNA encapsulated LNPs A6, B7, Cl naked FLuc mRNA, or PBS as negative control were added into the seeded J774A.1 cells and were incubated for respective timepoints (2, 4, 24 and 48 h). Afterwards, 100 pL of Bright-Glo Luciferase assay buffer was added into each treated well, and the luminescence value was measured using a SpectraMax microplate reader using the luminescence function. Luminescence values were then divided by the percentage of live cells in each respective treatment group to achieve luminescence per live cell.

[0217] Cell Viability by Alamar Blue Cell Viability Reagent: J774A.1 cells were grown in RPMI containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin at 37 °C in a 5% CO2 incubator. The cells were seeded in 96-well well plates at a cell density of 1 * 104cells per well in 100 pL media and incubated overnight. The following day, the cell media was removed and 100 pL of fresh cell media (for cell control), 100 pL of ethanol in RPMI (as a negative “kill” control) and 100 pL of LNPs A6, B7, Cl and naked FLuc mRNA were dosed at 500 ng mL’11000 ng mL4or 2000 ng mL1of mRNA for respective timepoints (2, 4, 24 and 48 h). The next day, a 10% Alamar Blue Cell viability reagent was prepared. The spent media was aspirated and replaced with 100 pL of Alamar Blue Cell viability reagent for 3 h. Finally, the absorbance of the solution was measured using a SpectraMax microplate reader with 570 nm wavelength with a reference wavelength of 600 nm. Untreated cells containing only RPMI 1640 media were used as background controls. Viability results were then calculated by normalizing the absorbance of treatment groups to the untreated cells.

[0218] C 'ell Association: J774A.1 cells were seeded in a transparent treated 24-well plate at a density 5 * 104cells per well and incubated in RPMI media at 37 °C for 24 h. Subsequently, 400 pL of 500 ng mL Cy-5 Labeled FLuc mRNA encapsulated ST LNPs A6, B7, Cl and Moderna LNP were added to the cells followed by incubation for the respective timepoints (2 and 24 h) at 37 °C. After the desired timepoints, the cells were washed with DPBS three times and dissociated using a cell scraper. Samples were then diluted in RPMI, spun down at 300 ref and decanted to remove the spent media, and 300 pL of PBS was added to each sample. Finally, the samples were analyzed on Attune Flow Cytometer (Thermo Fisher Scientific, USA). The cell associationwas assessed by gating for the percentage of cells with stronger Cy-5 fluorescence intensity than the control untreated cells.

[0219] Mechanism of Internalization ofLNPs: J774A.1 cells were seeded in a 24-well transparent well plate at a density 8 x 104cells per well and incubated in complete RPMI media at 37 °C for 24 h. Subsequently, endocytosis inhibitors (pitstop 2, EIP A, fllipin from S. filipinensis, NaN3, and cytochalasin D) were added to the cells to achieve final concentrations of 10, 30, 5 pg mL ’, 33.4 mM, and 40 pM, respectively. After 15 min incubation with the endocytosis inhibitors, 300 pL of 500 ng mL'1LNPsA6, B7, Cl , and Moderna encapsulating Cy-5 labeled FLuc mRNA were added to the cells followed by incubation for 2 h at 37 °C. The cell media was then removed, and the cells were washed with DPBS three times and dissociated using a cell scraper. Samples were then diluted in RPMI, spun down, and decanted to remove excess media, and 300 pL of lx PBS was added to each sample. Finally, the cells were analyzed using an Attune Flow Cytometer (Thermo Fisher Scientific, USA). The extent of cellular inhibition (%) was measured by normalizing the Cy-5 fluorescence intensity of inhibited treatment groups to uninhibited treatment groups.

[0220] Endosomal Escape of Lipid Nanoparticles: J774A.1 cells were seeded at a density of 8 x 104cells per well in p-slide 8-well coverslip slides at 37 °C overnight. Next day, the cell media was aspirated and 300 pL ofLNPs A6, B7, Cl and Moderna encapsulating Cy-5 labeled FLuc mRNA were added into wells at a mRNA concentration of 500 ng mL1, and was incubated at 37 °C for 2 h. The cells were then washed with DPBS three times, and 300 pL 100 nM of LysoTracker Green, dissolved in culture media (prewarmed at 37 °C), was added and incubated for 2 h. Subsequently, cells were washed with DPBS three times, and 1 pg mL1of Hoechst 33342 was added to cells for 15 min to stain the nucleus. Finally, the live cell imaging was performed by confocal laser scanning microscope (CLSM) (Zeizz SPX980) with 40x oil immersion objective. The images were further processed by WCIF Image J software.Experiments were performed in triplicates; five representative cell images (> 50 cells) were used to calculate the Pearson Correlation Coefficient (PCC) value.

[0221] Animal Studies of Luc mRNA encapsulated LN Ps: All animal studies were approved by the UNC Institutional Animal Care and Use Committee, were consistent with local, state, and federal regulations as applicable, and were supported within the UNC Lineberger ASC at the University of North Carolina at Chapel Hill.

[0222] FLuc mRNA encapsulated LNP A6, B7, Cl , and Moderna were prepared. Only FLuc mRNA injection was used as a positive control and PBS injection was used as a negative control. Each group was then injected via the tail vein of female C57 black 6 mice (Jackson Laboratory, 18-21 g) at a dose of 0.6 mg kg'1. After 24 h, mice were injected intraperitoneally with 130 pL of D-luciferin (30 mg mL'1in PBS). After 15 min, mice were euthanized and organs (pancreas, spleen, liver, kidneys, ovaries, lung and heart) were removed and imaged with an In Vivo imaging system (IVIS) (Perkin Elmer, Waltham, MA). Luminescence was quantified using AuRA software (Spectral instruments imaging). The liver, spleen, and lung were then fixed in 10% neutral buffered formalin after imaging and were then routinely processed to paraffin. Histological sections were evaluated using hematoxylin and eosin (H&E) stain. Briefly, Paraffin embedded tissue blocks were sectioned at 5 pm onto positively charged slides. In order to proceed with histological staining, samples were first baked at 60 °C for 1 h at a minimum and were then deparaffinized in xylene and hydrated with graded ethanol before continuing with the H&E stain. H&E stains were performed using the autostainer XL from Leica Biosystems. The sections were stained with Hematoxylin (Richard-Allen Scientific, 7211) for 2 mins and Eosin-Y (Richard-Allen Scientific, 7111) for 1 min. Clarifier 2 (7402) and Bluing (7111) solutions from Richard-Allen Scientific were used to differentiate the reaction. After staining, slides were then dehydrated and coverslipped with Cytoseal 60 (8310-4, Thermo Fisher Scientific, USA). Histological analysis was then performed on each sample.Example 3. Spleen-Expressing mRNA LNP Platform With Optimized Ratios

[0223] Additional formulations were prepared using compounds A8 (Table 9), A9 (Table 10),A16 (Table 11), or A18 (Table 12).Table 9. Formulation ratios of SE-LNPs using compound A8.* Represented as molar ratios.

[0224] SE-LNPs were successfully formulated with reproducible size, zeta potential, mRNA and encapsulation efficiency. The sizes ranging from -180 nm to 310 nm; zeta potentials ranging from — 25 mV to -3 mV, and mRNA encapsulation efficiency ranging from -20% to -80% (FIG. 34). The SE-LNP formulations resulted in an increase in FLuc expression in DC 2.4 cells (FIG. 35A) and were well tolerated, as evidenced by the observed cell viability being similar to untreated cells (FIG. 35B).Table 10. Formulation ratios of SE-LNPs using compound A9.* Represented as molar ratios.

[0225] SE-LNPs were successfully formulated with reproducible size, zeta potential, mRNA and encapsulation efficiency. The sizes ranging from -200 nm to 300 nm; zeta potentials ranging from — 24 mV to -3 mV, and mRNA encapsulation efficiency ranging from -20% to -85% (FIG. 36). The SE-LNP formulations resulted in an increase in FLuc expression in DC 2.4 cells(FIG. 37A) and were well tolerated, as evidenced by the observed cell viability being similar to untreated cells (FIG. 37B).Table 11. Formulation ratios of SE-LNPs using compound A16.* Represented as molar ratios.

[0226] SE-LNPs were successfully formulated with reproducible size, zeta potential, mRNA and encapsulation efficiency. The sizes ranging from -200 nm to 310 nm; zeta potentials ranging from —23 mV to -3 mV, and mRNA encapsulation efficiency ranging from -20% to -84% (FIG. 38). The SE-LNP formulations resulted in an increase in FLuc expression in DC 2.4 cells (FIG. 39A) and were well tolerated, as evidenced by the observed cell viability being similar to untreated cells (FIG. 39B).Table 12. Formulation ratios of SE-ENPs using compound Al 8.* Represented as molar ratios.

[0227] SE-LNPs were successfully formulated with reproducible size, zeta potential, mRNA and encapsulation efficiency. The sizes ranging from -200 nm to 310 nm; zeta potentials ranging from — 40 mV to 0 mV, and mRNA encapsulation efficiency ranging from -10% to -70% (FIG. 40). The SE-LNP formulations resulted in an increase in FLuc expression in J774A.1 cells (FIG. 41A) and were well tolerated, as evidenced by the observed cell viability being similar to untreated cells (FIG. 41B).

[0228] Representative in vivo images of tissue specific delivery of exemplary LNPs comprising cholesterol derivatives A8, A9, A16, or A18 are shown in FIG. 42.

[0229] The foregoing examples are illustrative of the present invention, and are not to be construed as limiting thereof. Although the invention has been described in detail with reference to preferred embodiments, variations and modifications exist within the scope and spirit of the invention as described and defined in the following claims.

Claims

WHAT IS CLAIMED IS:

1. A cholesterol derivative according to Formula I:Formula I wherein Ri and R2are independently H, C1-C8 alkyl, C2-C8 alkenyl, C2-C8 alkynyl, -(CH2)n-NRaRb, -(CH2)nO(CH2)n, -(CH2)nOH, or,Ri and R2together with the N atom to which they are bonded form a 4-15 membered ring, each member independently selected from the group consisting of C, N, O, S, CH, CH2, and NH, the ring being saturated, partially unsaturated or aromatic, and optionally substituted by one to three substituents selected from the group consisting of: C1-C8 alkyl, C2-C8 alkenyl, C2- C8 alkynyl, C1-C8 alkoxyl, (CH2)n(NRaRb), (CH2)nO(CH2)n, (CH2)nOH, and a 4-8 membered ring, each member independently selected from C, N, O, S, CH, CH2, and NH, the ring being saturated, partially unsaturated, or aromatic, wherein each of the substituents can be further substituted with a 3-8 membered ring each member independently selected from the group consisting of C, N, O, S, CH, CH2, and NH, the ring being saturated, partially unsaturated, or aromatic; wherein Ra and Rb are each independently selected from H, C1-C8 alkyl, C2-C8 alkenyl, C2-C8 alkynyl, -(CH2)n-NRaRb, -(CH2)nO(CH2)n, -(CH2)nOH; and each n is independently an integer between 1 and 8.

2. The cholesterol derivative of claim 1, wherein Ri and R2together with the N atom to which they are bonded form a 4-15 membered ring, the ring selected from:wherein the ring is optionally substituted by one to three substituents selected from the group consisting of: C1-C8 alkyl, C2-C8 alkenyl, C2-C8 alkynyl, C1-C8 alkoxyl, (CH2)n(NRaRb), (CH2)nO(CH2)n„ -(CH2)nOH, and a 4-8 membered ring, each member independently selected from C, N, O, S, CH, CH2, and NH, the ring being saturated, partially unsaturated, or aromatic, wherein each of the substituents can be further substituted with a 4-8 membered ring each member independently selected from the group consisting of C, N, O, S, CH, CH2, and NH, the ring being saturated, partially unsaturated or aromatic.

3. The cholesterol derivative of claim 1, wherein the derivative is selected from4. A composition comprising an ionizable lipid, a phospholipid, a poly(ethylene glycol) (PEG)-lipid and the cholesterol derivative according to any one of claims 1-3.

5. The composition of claim 4, comprising about 5-95% ionizable lipid, about 5-30% phospholipid, about 20-60% of the cholesterol derivative, and about 1-5 mol% PEG-lipid6. The composition of claim 4, comprising about 45-65 mol% ionizable lipid, about 20-50 mol% cholesterol derivative, about 5-25 mol% phospholipid, and about 0.4-2.0 mol% PEG-lipid.

7. The composition of claim 4, comprising about 46-48 mol% ionizable lipid, about 44-46 mol% cholesterol derivative, about 5-25 mol% phospholipid, and about 0.5-0.6 mol% PEG-lipid.

8. The composition of any one of claims 4-7, further comprising a polyphenol.

9. The composition of any one of claims 4-8, wherein the ionizable lipid is selected from 9- heptadecanyl 8-{(2-hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino}octanoate (SM-102) and / or [(4-hydroxybutyl)azanediyl]di(hexane-6,l-diyl) bis(2-hexyldecanoate)(ALC-0315).

10. The composition of any one of claims 4-9, wherein the phospholipid is dioleoylphosphatidylethanolamine (DOPE), dioleoylphosphatidylcholine (DOPC), distearoylphosphatidylcholine (DSPC), or any combination thereof.

11. The composition of any one of claims 4-10, wherein the PEG-lipid is DMG-PEG(2000) or 14:0 PEG IK.

12. A nanoparticle comprising the cholesterol derivative of any one of claims 1-3, or the composition of any one of claims 4-11, and a cargo.

13. The nanoparticle of claim 12, wherein the cargo is a biologically active agent, imaging agent, or therapeutic agent.

14. The nanoparticle of claim 12 or 13, wherein the cargo is a nucleic acid, a protein, a complex of a nucleic acid and a protein, a carbohydrate, a lipid, or a small molecule.

15. The nanoparticle of claim 14, wherein the nucleic acid is an RNA.

16. The nanoparticle of claim 14 or 15, wherein the nucleic acid is an mRNA, antisense oligonucleotide, guide RNA, or siRNA.

17. The nanoparticle of claim 12, wherein the cargo is a gene modulating agent.

18. The nanoparticle of claim 17, wherein the gene modulating agent is a gene editing system comprising a CRISPR-Cas system, a zinc finger nuclease, a TALEN, a base editor, or a meganuclease.

19. A method of making the cholesterol derivative according to any one of claims 1-3, comprising: reacting a molecule according towith a secondary amine compound at room temperature to thereby yield the compound according to Formula I.

20. The method of claim 19, wherein the secondary amine is selected from the group consisting of:

21. A method of delivering a cargo to a target cell, comprising contacting the target cell with the nanoparticle of any one of claims 12-18.

22. A method of modulating expression of a gene in a target cell, comprising contacting the target cell with the nanoparticle of any one of claims 12-18.

23. The method of claim 21 or 22, wherein the contacting occurs in vivo.

24. The method of claim 21 or 22, wherein the contacting occurs ex vivo.

25. The method of claim 21 or 22, wherein the contacting occurs in vitro.

26. The method of any one of claims 21-25, wherein the target cell is a spleen cell.

27. A method of delivering a cargo to a subject in a tissue specific manner, comprising contacting the tissue with the nanoparticle of any one of claims 12-18.

28. The method of claim 27, wherein the tissue is spleen.

29. The method of claim 27 or 28, wherein the contacting comprises administering the nanoparticle intravenously.

30. The method of claim 27 or 28, wherein the contacting comprises systemic or local administration.

31. The method of claim 30, wherein the administration comprises administering intravenously, intramuscularly, subcutaneously, or topically.

32. A method of treating a disease or disorder in a subject in need thereof, comprising administering to the subject the nanoparticle of any one of claims 12-18, wherein the cargo provides a therapeutic effect for the disease or disorder.

33. The method of claim 32, wherein the disease or disorder is cancer, pathogen infection, or an autoimmune disease, optionally type I diabetes, systemic lupus erythematosus, autoimmune encephalitis, or multiple sclerosis.

34. A method of vaccinating a subject in need thereof, comprising administering to the subject the nanoparticle of any one of claims 12-18, wherein the cargo provides an immunostimulatory effect.

35. A method of making a cargo-loaded nanoparticle, comprising: adding an organic phase comprising: the cholesterol derivative of any one of claims 1-3, a PEG lipid, an ionizable lipid and a phospholipid to an aqueous phase comprising a cargo to make a mixture, wherein the nanoparticle self-assembles via electrostatic interactions between the cargo and the cholesterol derivative, the PEG lipid, the ionizable lipid and the phospholipid.

36. The method of claim 35, wherein the cargo is a biologically active agent.

37. The method of claim 35 or 36, wherein the cargo is an imaging agent.

38. The method of claim 35 or 36, wherein the cargo is a therapeutic agent.

39. The method of claim 35 or 36, wherein the cargo is a nucleic acid, a protein, a complex of a nucleic acid and a protein, a carbohydrate, a lipid, or a small molecule.

40. The method of claim 39, wherein the nucleic acid is an RNA.

41. The method of claim 39 or 40, wherein the nucleic acid is an mRNA, antisense oligonucleotide, or siRNA.

42. The method of claim 39, wherein the cargo is a gene modulating agent.

43. The method of claim 42, wherein the gene modulating agent is a gene editing system comprising a CRISPR-Cas system, a zinc finger nuclease, a TALEN, a base editor or a meganuclease.

44. A method of making a nanoparticle that targets the spleen, comprising:using the cholesterol derivative of any one of claims 1-3 in a nanoparticle formulation.

45. The method of claim 44, wherein the nanoparticle formulation comprises the cholesterol derivative, a PEG-lipid, an ionizable lipid and a phospholipid.

46. The method of claim 44 or 45, wherein the nanoparticle further comprises a cargo.

47. The method of claim 46, wherein the cargo is a biologically active agent.

48. The method of claim 46 or 47, wherein the cargo is an imaging agent.

49. The method of claim 46 or 47, wherein the cargo is a therapeutic agent.

50. The method of claim 46 or 47, wherein the cargo is a nucleic acid, a protein, a complex of a nucleic acid and a protein, a carbohydrate, a lipid, or a small molecule.

51. The method of claim 50, wherein the nucleic acid is an RNA.

52. The method of claim 50 or 51, wherein the nucleic acid is an mRNA, antisense oligonucleotide, or siRNA.

53. The method of claim 50, wherein the cargo is a gene modulating agent.

54. The method of claim 53, wherein the gene modulating agent is a gene editing system comprising a CRISPR-Cas system, a zinc finger nuclease, a TALEN, a base editor or a meganuclease.

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