Synthetic lipids for lipid nanoparticle compositions

Abstract

The present disclosure provides synthetic lipids comprising a 5-hydroxytryptamine type 3 (5-HT3) receptor ligand covalently linked to at least one lipid through an amino-containing linker. Such synthetic lipids are designed to enhance delivery of associated payloads, including nucleic acids, by supporting efficient encapsulation, membrane interaction, and intracellular trafficking. The disclosed lipids may include biodegradable or branched lipid tails and may be incorporated into lipid nanoparticles with additional excipients such as phospholipids, cholesterol, and polyethylene glycol–derived lipids, enabling applications in targeted delivery and therapeutic modulation.

Description

Attorney Docket No. 774757: MTST-875PCSYNTHETIC LIPIDS FOR LIPID NANOPARTICLE COMPOSITIONSCROSS REFERENCE TO RELATED APPLICATION

[0001] This patent application claims the benefit of U.S. Provisional Patent Application No. 63 / 763,171, filed on February 25, 2025, which is incorporated by reference in its entirety herein.INCORPORATION BY REFERENCE OF AN ELECTRONIC SEQUENCE LISTING

[0002] This application contains a sequence listing that has been submitted in a computer readable format and is hereby incorporated by reference in its entirety. The computer readable file, created on February 12, 2026, is named 774757_MTST-875PC.xml and is 26,238 bytes in size.BACKGROUND

[0003] Brain mRNA delivery holds immense potential to revolutionize treatments for central nervous system (CNS) diseases, such as neurodegenerative diseases and brain tumors. Lipid nanoparticles (LNPs) are FDA-approved non-viral vectors for mRNA delivery, as they enable safe and efficient transport of mRNA across various biological barriers. Recent advancements in LNP technology have enabled targeted mRNA delivery to specific organs, such as lung, spleen, bone marrow, etc. following intravenous (i.v.) administration. However, delivering mRNA to the brain remains a significant challenge due to the presence of the bloodbrain barrier (BBB), which effectively prevents most macromolecules such as mRNA from entering into the brain. To overcome this barrier, several strategies have been developed. For instance, nanoparticles are modified with antibodies or peptides targeting specific receptors on the BBB, including transferrin, low-density lipoprotein, glucose transporter and insulin receptors. Additionally, external stimuli such as magnetic fields, ultrasound and glucose fasting have been explored to enhance nanoparticle penetration into deep brain tissues. Previous studies have demonstrated that certain small molecules can cross the BBB via distinct mechanisms. For example, SR-57227 is a potent 5-hydroxytryptamine type 3 (5-HT3) receptor ligand with ability to cross the across the BBB. Inspired by these findings, it was hypothesized that synthetic lipids derived from SR-57227 may improve the ability of LNPs to cross the BBB, offering potential as mRNA carriers for brain delivery via systematic administration.182021256v1Attorney Docket No. 774757: MTST-875PCBRIEF SUMMARY

[0004] One aspect of the disclosure is a synthetic lipid comprising a head group and at least one lipid tail, wherein the head group is derived from a 5-hydroxytryptamine type 3 (5 HT3) receptor ligand and is covalently linked through at least one amino containing linker to the at least one lipid tail.

[0005] In an aspect, the 5-HT3 receptor ligand is SR-57227.

[0006] In an aspect, the SR-57227 has the structure of:

[0007]

[0008] In an aspect, the at least one lipid tail comprises an ester linkage.

[0009] In an aspect, the at least one lipid tail comprises at least one biodegradable ester linkage.

[0010] In an aspect, the lipid tail comprises two branched ester lipids.

[0011] In an aspect, the lipid tail is of Formula I:HN

[0012]

[0013] (i);

[0014] or a pharmaceutically acceptable salt thereof;

[0015] wherein:

[0016] X1is a bond, -OCH2O-, -OCH(CH3)O-, -OC(O)O- or -C(O)OCH(CH2CH3)-;

[0017] X2is a bond, -OCH2O-, -OCH(CH3)O-, -OC(O)O- -C(O)OCH(CH2CH3)-, or -C(O)OCH(C(CH3)2)-;

[0018] W1is CH2or NRW1

[0019]

[0020] X3is a bond, -OCH2O-, -OCH(CH3)O-, -OC(O)O- or -C(O)OCH(CH2CH3)-;

[0021] wl is 1 to 5;Attorney Docket No. 774757: MTST-875PC

[0022] w2 is 1 to 3;

[0023] a is 1 to 5;

[0024] b is 1 to 3:

[0025] c is 3 to 5;

[0026] d is 3 to 5; and

[0027] e is 1 to 3.

[0028] In an aspect, the at least one lipid tail is selected from the group consisting of:

[0029]

[0030]

[0031]

[0032]

[0033]

[0034]

[0035]

[0036]

[0037]

[0038]

[0039] Attorney Docket No. 774757: MTST-875PC

[0041]

[0042]

[0043]

[0044]

[0045]

[0046] and combinations thereof.Attorney Docket No. 774757: MTST-875PC

[0047] In an aspect, the lipid tail is:

[0048] In an aspect, the synthetic lipid is ionizable.

[0049] One aspect of the disclosure is a lipid nanoparticle comprising the synthetic lipid disclosed.

[0050] In an aspect, the lipid nanoparticle further comprises phospholipids, cholesterol, additional ionizable lipids, and / or polyethylene glycol-derived lipids (PEG-lipid).

[0051] In an aspect, the lipid nanoparticle comprises 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG2k), and / or 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyethylene glycol)-2000 (DSPE-PEG2k-Mal).

[0052] In an aspect, the phospholipid is DOPE, the PEG-lipid is DMG-PEG2k or DSPE-PEG2k-maleimide, and the cholesterol is present.

[0053] In an aspect, the lipid tail issynthetic lipid: DOPE:cholesterol: PEG-lipid is about 30-50 mol% synthetic lipid, about 30-50 mol% DOPE, about 50-70 mol% cholesterol, and about 0.5-1.5 mol% PEG-lipid, alternatively about 20-60 mol% synthetic lipid, about 30-50 mol% DOPE, about 50-70 mol% cholesterol, and about 0.5-2.0 mol% PEG-lipid.

[0054] In an aspect, the molar ratio of synthetic lipid: DOPE:cholesterol: PEG-lipid is about 40:40:60:0.75.

[0055] In an aspect, the lipid nanoparticle has a particle size of between about 70nm and about 180nm, alternatively at least about 80nm, alternatively at least about 90nm, alternatively at least about 100nm, alternatively at least about 110nm, alternatively at least about 120nm, alternatively at least about 130nm, alternatively at least about 140nm,Attorney Docket No. 774757: MTST-875PCalternatively at least about 150nm, alternatively at least about 160nm, or alternatively at least about 170nm.

[0056] In an aspect, the lipid nanoparticle has a polydispersity index (PDI) of between about 0.05 and about 0.3, alternatively at least about 0.1, alternatively at least about 0.15, alternatively at least about 0.2, or alternatively at least about 0.25.

[0057] In an aspect, the lipid nanoparticle has an encapsulation efficiency of at least about 50%, alternatively at least about 55%, alternatively at least about 60%, alternatively at least about 65%, alternatively at least about 70%, alternatively at least about 75%, or alternatively at least about 85%.

[0058] One aspect of the disclosure is a modified lipid nanoparticle comprising the lipid nanoparticle disclosed and at least one cell-penetrating peptide (CPP).

[0059] In an aspect, the CPP is covalently linked to the lipid nanoparticle via a maleimide-functionalized PEG-lipid.

[0060] In an aspect, the CPP is Tat or a Tat-derived peptide.

[0061] In an aspect, the at least one CPP comprises an amino acid sequence at least 80% identical to the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, or SEQ ID NO: 10.

[0062] In an aspect, the at least one CPP is selected from a peptide having an amino acid sequence identical to the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, or SEQ ID NO: 10.

[0063] One aspect of the disclosure is a lipid nanoparticle composition comprising the lipid nanoparticle disclosed.

[0064] One aspect of the disclosure is a lipid nanoparticle composition comprising the modified lipid nanoparticle disclosed.

[0065] In an aspect, the lipid nanoparticle composition further includes a polynucleotide.

[0066] In an aspect, the polynucleotide is selected from the group consisting of DNA, ssDNA, cDNA, RNA, mRNA, tRNA, rRNA, siRNA, sgRNA, and combinations thereof.

[0067] In an aspect, the polynucleotide encodes a protein, peptide, or antibody.

[0068] In an aspect, the polynucleotide encodes a therapeutic protein or a therapeutic peptide.Attorney Docket No. 774757: MTST-875PC

[0069] In an aspect, the polynucleotide encodes an engineered interleukin- 12 (eIL-12) comprising IL-12p40, IL-12p35, an MMP-cleavable linker, an IL-12Rpl (Q20-A261) domain, or a FLAG tag.

[0070] In an aspect, the lipid nanoparticle composition is a pharmaceutical composition or a therapeutic composition.

[0071] In an aspect, the lipid nanoparticle composition is configured to cross the blood brain barrier (BBB).

[0072] One aspect of the disclosure is a method of delivering a polynucleotide to cells in a subject in need thereof, the method comprising administering the lipid nanoparticle composition disclosed to the subject.

[0073] In an aspect, the cells are brain cells.

[0074] In an aspect, the cells selected from the group consisting of neurons, astrocytes, microglia, or brain endothelial cells, or a combination thereof.

[0075] In an aspect, the administration of the lipid nanoparticle composition results in at least a 9-fold increase of delivery of polynucleotide to cells as compared to a control.

[0076] One aspect of the disclosure is a method of treating or preventing cancer in a subject in need thereof, the method comprising administering the lipid nanoparticle composition disclosed to the subject.

[0077] One aspect of the disclosure is a method of treating or preventing a central nervous system (CNS) disease or disorder the method comprising administering the lipid nanoparticle composition disclosed to the subject.

[0078] In an aspect, the CNS disease or disorder is selected from the group consisting of glioblastoma, polyneuropathy, anaplastic astrocytoma, medulloblastoma, oligodendroglioma, primary CNS lymphoma, and metastatic brain tumors.

[0079] In an aspect, administration of the lipid nanoparticle composition suppresses tumor growth, increases survival of the subject, increases polynucleotide delivery to cells, and / or increases cellular translation.

[0080] In an aspect, the polynucleotide encodes an engineered cytokine.

[0081] In an aspect, the polynucleotide comprises eIL-12 mRNA.

[0082] In an aspect, the engineered cytokine is engineered IL- 12.

[0083] In an aspect, administration of the lipid nanoparticle composition is intravenous.

[0084] One aspect of the disclosure is a method of making a modified lipid nanoparticle, comprising:Attorney Docket No. 774757: MTST-875PC

[0085] (a) formulating a lipid nanoparticle containing the synthetic lipid disclosed, DOPE, cholesterol, and a PEG-lipid; and

[0086] (b) reacting the lipid nanoparticle with a cysteine-containing CPP to yield a maleimide -thiol conjugate on the lipid nanoparticle surface.

[0087] One aspect of the disclosure is a kit comprising:

[0088] (a) a unit dose containing the lipid nanoparticle composition disclosed; and

[0089] (b) instructions for administration to deliver a polynucleotide across the BBB.

[0090] These and other advantages, aspects, and novel features of the present disclosure, as well as details of an illustrated embodiment thereof, will be more fully understood from the following description and drawings.BRIEF DESCRIPTION OF THE DRAWINGS

[0091] Various aspects of the present disclosure will now be described, by way of example only, with reference to the attached Figures, wherein:

[0092] FIG. 1 is a schematic illustrating four components of LNPs (ionizable lipid, DOPE, cholesterol, and DMG-PEG); structures of SR-57227-derived lipids (SLs); and a workflow that incorporates mRNA into the LNPs followed by administration to cells.

[0093] FIG. 2 is a bar graph showing particle size and poly dispersity indexes (PDI) of SLNPs loaded with Fluc-mRNA. n = 3. Data are presented as mean ± SD.

[0094] FIG. 3 are bar graphs showing zeta potential and encapsulation efficiency of SLNPs loaded with Fluc-mRNA. n = 3. Data are presented as mean ± SD.

[0095] FIG. 4A are bar graphs showing mRNA delivery efficiency in b. End3, astrocytes, and N2a cells.

[0096] FIG. 4B is a bar graph showing normalized intensity in the brain of mice treated with MC3, SO, S4, and S6 LNPs (n = 3).

[0097] FIG. 4C is a series of representative images of brain tissues treated with MC3, SO, S4, and S6 LNPs. Statistical significance was analyzed by one-way ANOVA with the Tukey’s multiple comparisons test. ***P < 0.001 and ****P < 0.0001.

[0098] FIG. 5 is a bar graph showing luminescence intensity in major organs of mice treated with SO, S4, or S6 LNPs. For SO and S6, n = 2; for S4, n = 3. Data are presented as mean ± SD. S4 showed the highest intensity in liver, spleen, and lung followed by SO.Attorney Docket No. 774757: MTST-875PC

[0099] FIG. 6A is a 16 (4)4orthogonal table depicting the 1stRound and predicted formulations 2ndRound.

[0100] FIG. 6B is a series of graphs showing the results of each lipid component at different molar ratios on mRNA delivery efficiency of S4 LNP (n = 4 biological replicates).

[0101] FIG. 6C are bar graphs showing relative luminescence intensity in b. End.3 and N2a cell lines. The intensity was normalized to formulation 0 group. 0-16 are the first round of optimization and 17-25 are the second round of optimization.

[0102] FIG. 6D is a bar graph showing normalized luminescence intensity in the brain of mice treated with MC3, SM-102, ALC-0315, S4, or OS4 LNPs (n = 3). The intensity was normalized to MC3 LNP group. Data are presented as mean± SD.

[0103] FIG. 6E is a series of representative images of brain tissues treated with MC3, SM-102, ALC-0315, S4, or OS4 LNPs. Statistical significance was analyzed by one-way ANOVA with Tukey’s multiple comparisons test. **P < 0.01, ****P < 0.0001.

[0104] FIG. 7 is a table listing different cell-penetrating peptides (CPPs, SEQ ID NOs: 1-10).

[0105] FIG. 8 is a bar graph showing zeta potential of CPP-conjugated OS4 LNPs loaded with Fluc-mRNA. n = 3. Data are presented as mean ± SD.

[0106] FIG. 9A is a bar graph showing the size and PDI of CPP-conjugated OS4 LNPs.

[0107] FIG. 9B is a bar graph showing the normalized luminescence intensity in the brain from the mice treated with CPP-conjugated OS4 LNPs (n = 3 biological replicates). The intensity was normalized to unmodified OS4 LNP group.

[0108] FIG. 9C is a bar graph showing the quantification of luminescence intensity in the brain of mice treated with MC3, MC3T, or OS4T LNPs (n = 3 biological replicates). The intensity was normalized to MC3 LNP group.

[0109] FIG. 9D is a series of representative images of brain tissues treated with MC3, MC3T, or OS4T LNPs (n = 3).

[0110] FIG. 9E is a representative cryo-TEM image of OS4T LNP (Scale bar, 50 nm).

[0111] FIG. 10A is a bar graph showing the relative luminescence intensity compared to liver for OS4, OS4T, MC3, and MC3T LNPs in major organs after i.v. injection, n = 3 biological replicates. Data are presented as mean ± SD. For each organ the data is presented in the order MC3, MC3T, OS4, and OS4T.

[0112] FIG. 10B are bar graphs showing the distribution of hydrodynamic diameters (Dh) of LNPs conjugated with A5K (OS4A), LAH4 (OS4L) and THR (OS4Th) peptides, respectively.Attorney Docket No. 774757: MTST-875PC

[0113] FIGS. 11A-11C are cellular uptake and endosome escape assays. FIG.11A is a bar graph showing the percentage of Alexa-Fluor 647 positive cells, n = 3 biological replicates.FIG. 11B is a bar graph showing the analysis of colocalization coefficient between mRNA-alexa647 and calcein. FIG. 11C is a series of representative confocal images of N2a cells after 2 h of incubation with calcein and LNPs encapsulating Alexa-Fluor 647 RNA. Scale bar: 50 pm. Data in FIG. 11B are analyzed from n = 4 random views. Data are presented as mean ± SD. Statistical significance was analyzed by one-way ANOVA with Tukey’s multiple comparisons test, ns, not significant, ****P < 0.0001.

[0114] FIGS. 12A-12D are a schematic and bar graphs showing the transwell assay for studying BBB-crossing pathways.

[0115] FIG. 12A is a schematic illustrating the in vitro BBB transwell model. The bEnd.3 cells were pre-treated with meta-chlorophenylpiperazine (a 5-HT receptor ligand), EIPA (a micropinocytosis-mediated endocytosis inhibitor), CPZ (a clathrin-mediated endocytosis inhibitor) or MpCD (a caveolae-mediated endocytosis inhibitor).

[0116] FIG. 12B are bar graphs showing the normalized luminescence intensity in N2a cells relative to the OS4 or OS4T control group. Statistical significance was calculated by oneway ANOVA with the Tukey’s multiple comparisons test.

[0117] FIG. 12C is a bar graph showing t N2a cell viability after treatment with different inhibitors. The order on the x-axis (left to right) is meta-chlorophenylpiperazine, EIPA, CPZ, and MpCD.

[0118] FIG.12D is a bar graph showing the luminescence intensity of N2a cells treated with OS4 or OS4T LNPs without inhibitors. Statistical significance was calculated by T test. **P < 0.01, ***P < 0.001, ****p < 0.0001, ns, not significant.

[0119] FIG. 13A is a bar graph showing cell viability of N2a cells after treatment with different LNPs for 24 h (mRNA dose: 0.5 pg / mL). n = 4 biological replicates. Data are presented as mean ± SD.

[0120] FIG. 13B are bar graphs showing the levels of blood urea nitrogen (BUN), alanine transaminase (ALT), and aspartate aminotransferase (AST) in mice following i.v. administration of PBS, MC3, OS4, or OS4T LNPs (in order on the x-axis from left to right for both 24 hr and 48 hr time points) (n = 3 biological replicates). Data are presented as mean ± SD. Statistical significance was analyzed by one-way ANOVA with Tukey’s multiple comparisons test, n.s., not significant, **P < 0.01, ***P < 0.001, ****P < 0.0001.Attorney Docket No. 774757: MTST-875PC

[0121] FIG. 14 is a series of representative histopathological analysis (H&E stained tissue section) images of major organs from mice after treatment by i.v. administration of PBS, MC3, OS4, or OS4T LNPs (Scale bar, 250 pm).

[0122] FIG. 15 is a series of images of the gating strategy for flow cytometry analysis of brain cells. Forward and side scatter properties were utilized to define the ‘Cell’ gate, which was further validated by positive nuclear DAPI staining. Subsequent gating was performed using specific cell markers: NeuN+for neurons, GFAP+for astrocytes, CD11b+for microglia, and CD31+for brain capillary endothelial cells (BCECs).

[0123] FIGS. 16A-16G are bar graphs, schematic, and images showing that OST4 LNP enables effective mRNA delivery into different brain cells.

[0124] FIGS. 16A-16B are bar graphs showing flow cytometry analysis of GFP expression (FIG. 16A) or tdTomato expression (FIG. 16B) across different brain cells (NeuN+for neuron, GFAP+for astrocyte, CD11b+for microglia, and CD31+for brain capillary endothelial cell (BCEC) (for each cell type on the x-axis, the data are presented left to right as PBS, MC3, OS4, and OS4T for FIG. 16A and PBS, MC3 (IX dose), MC3 (3X dose), OS4T (IX dose), and OS4T (3X dose) for FIG. 16B. Data are presented as mean± SD. Statistical significance was analyzed by one-way ANOVA with Tukey’s multiple comparisons test.

[0125] FIG. 16C is a schematic illustrating the delivery of Cre mRNA to activate tdTomato expression, along with the administration regimen.

[0126] FIG. 16D is a series of images representing immunofluorescence sections of brain tissues of Ail4 mice after three administrations with PBS, MC3, or OS4T LNPs (Cre mRNA, 1 mg / kg). Scale bar: 2.5 mm.

[0127] FIG. 16E is a series of images showing the tdTomato expression in neurons after three administrations with PBS, MC3, or OS4T LNPs (Cre mRNA, 1 mg / kg). Scale bar: 50 pm. **P < 0.01, ***P < 0.001, ****p < 0.0001.

[0128] FIG. 16F is a series of images showing the tdTomato expression in microglia after three administrations with PBS, MC3, or OS4T LNPs (Cre mRNA, 1 mg / kg). Scale bar: 50 pm. **P < 0.01, ***P < 0.001, ****p < 0.0001.

[0129] FIG. 16G is a series of images showing the tdTomato expression in astrocytes after three administrations with PBS, MC3, or OS4T LNPs (Cre mRNA, 1 mg / kg). Scale bar: 50 pm. **P < 0.01, ***P < 0.001, ****p < 0.0001.

[0130] FIGS. 17A-17G are schematics, images, and plots showing the therapeutic efficacy of OS4T LNPs in an orthotopic mouse model of glioblastoma (GBM).Attorney Docket No. 774757: MTST-875PC

[0131] FIG. 17A is a schematic illustrating the engineered IL- 12 (eIL-12) expressed by OS4T LNPs can be activated by MMP9 within the tumor microenvironment.

[0132] FIG. 17B is an image of a western blot gel showing the cleavage of elL- 12 by MMP9.

[0133] FIG. 17C is a graph showing the body weight changes in C3H / HeJ mice (n = 5) following administration of OS4T loaded with IL- 12 or eIL-12 mRNAs (mRNA, 1 mg / kg). The arrows indicate the times of administration. PBS showed a slight weight gain; OS4T-eiL-12 showed about a 9% decrease in weight after 4 days; and OS4T-IL-12 showed about an 18% decrease in weight after 4 days. Data are presented as mean± SD.

[0134] FIG. 17D is a schematic illustrating the treatment regimen in the CT-2A-Luc GBM model.

[0135] FIG. 17E is a graph showing the luminescence intensity of orthotopic GBM tumor tissues in the mice i.v. treated with PBS, OS4T-Fluc mRNA, MC3T-eIL-12, OS4-eIL-12, or OS4T-eIL-12 LNPs, respectively (mRNA, 1 mg / kg). Data are presented as mean± SD. Statistical significance was analyzed by two-way ANOVA with Tukey’s multiple comparisons test. OS4T-Fluc and OS4-eiL-12 showed the highest intensity on day 12 followed by PBS and MC3T-eiL-12 and OS4T-eil-12 had the lowest intensity.

[0136] FIG. 17F is a graph showing the survival proportions over time (n = 7 biological replicates). Statistical significance was analyzed by Gehan-Breslow-Wilcoxon test. PBS had the lowest percent survival rate followed by OS4T-Fluc, MC3T-eiL-12, OS4-eiL-12, and OS4T-eil-12, respectively.

[0137] FIG. 17G is a series of representative IVIS images of tumor-bearing mice. The missing image indicates that the mouse had already succumbed at this time. **P < 0.01, ***P < 0.001, ****p < 0.0001.

[0138] FIG. 18A are bar graphs showing cytokine measurements. C57BL / 6J mice were i.v. treated with PBS, Flue mRNA LNPs (OS4T-Fluc), IL-12 mRNA LNPs (OS4T-IL-12) or eIL-12 mRNA LNPs (OS4T-eIL-12), respectively (mRNA, 1 mg / kg). After 24 h, the IFN-y levels in serum (a), liver (b) and spleen (c) were measured (n = 3 biological replicates). Data are presented as mean± SD. Statistical significance was analyzed by two-way ANOVA with Tukey’s multiple comparisons test, (d) ELISA analysis results of cytokine in GBM-bearing brain tissues, including IFN-y, IL-6 and IL-12p70 (n = 4 biological replicates). Data are presented as mean ± SD. Statistical significance was calculated by unpaired T test. *P < 0.05, **P < 0.01, ns, not significant.Attorney Docket No. 774757: MTST-875PC

[0139] FIG. 18B is a series of graphs showing body weight changes in each mouse with an orthotopic GBM model treated with various formulations.

[0140] FIG. 18C is a schematic showing the gating strategy for flow cytometry analysis of immune cells in brain tissues.

[0141] FIGS. 19A-19D are bar graphs showing immune cell activation in the brain tissues of GBM-bearing mice following treatment.

[0142] FIG. 19A is a series of bar graphs showing the flow cytometric analysis of activation markers of macrophages quantified by mean fluorescence intensity (MFI, n = 5 biological replicates).

[0143] FIG. 19B is a series of bar graphs showing the flow cytometric analysis of activation markers of macroglia quantified by mean fluorescence intensity (MFI, n = 5 biological replicates).

[0144] FIG. 19C is a bar graph showing the percentage of regulatory T cells (Tregs, n = 5 biological replicates).

[0145] FIG. 19D is a series of bar graphs showing percentage of TNF-a+and CD69+CD8+T cells (n = 5 biological replicates). Data are presented as mean ± SD. Statistical significance was calculated by unpaired T test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

[0146] FIG. 20 is chemical structures, bar graphs, and images showing comparison of mRNA delivery efficiency between OS4 LNP and amino lipid (AL) LNP. (a) Chemical structures of ionizable lipids used in OS4 and AL LNP, respectively, (b) Particle size and PDI of OS4 and AL LNPs before and after Tat conjugation, (c) Representative images of brain tissues and quantification of normalized luciferase intensity in the brains of mice treated with OS4 or AL LNPs (loaded with Flue mRNA, 0.6 mg / kg). n = 3 biological replicates. Statistical significance was calculated by unpaired T test. *P < 0.05.

[0147] FIG. 21 are images and heat maps showing the therapeutic efficacy of LNP -Gria3 mRNA treatment in a SCZ mouse model, (a) Representative immunofluorescence images of mouse brains with or without Gria3-LNP treatment. GRIA3 expression (light grey) is shown in whole -brain sections and specific brain regions, including the cortex, hippocampus, thalamus, and hypothalamus. Scale bars: 2.5 mm (whole brain) and 100 pm (zoomed-in images), (b) Heat maps showing movement traces and latency time of SCZ mice in the open field test. Gria3-LNP treatment significantly reduced locomotor activity in SCZ mice. Flue, a reporter mRNA, was used as a control mRNA. Data in (a) are from n = 3 mice. Data in (b) are from n = 20 biologically independent mice. **p < 0.01, ****p < 0.0001;Attorney Docket No. 774757: MTST-875PCstatistical analysis was performed using one-way ANOVA with Tukey’s multiple comparisons test.

[0148] FIG. 22 are images showing the safety evaluation of LNP treatment by H&E staining. Hematoxylin and eosin (H& E) staining of major organs (heart, liver, spleen, lung, and kidney) from mice treated with LNPs. No obvious pathological changes were observed following treatment. Scale bar 100 pm. Data are from n = 3 mice.DETAILED DESCRIPTIONI. Introduction

[0149] The application of LNP-mRNA in treatment and prophylaxis has gained unprecedented advancements, particularly following the rapid development of mRNA vaccines during the COVID- 19 pandemic. Although LNP-mRNA systems have emerged as a transformative platform for addressing conditions such as protein deficiencies, genetic mutations, or functional impairments, efficient mRNA delivery to the brain remains a formidable challenge due to the need to overcome several critical physiological barriers. The first is the restrictive nature of the BBB, a highly restrictive layer of endothelial cells connected by tight junctions. While some small molecules can traverse the BBB via passive diffusion, macromolecules such as LNPs rely on transcytosis, a process wherein they are encapsulated in vesicles on one side of the endothelial cell, transported across, and released on the opposite side. Beyond crossing the BBB, LNPs must navigate the interstitial space to reach target cell populations. Further challenges include achieving efficient cellular uptake and ensuring endosomal escape, particularly for mRNA, as successful protein translation occurs only when the mRNA is released into the cytoplasm. Overcoming these interconnected barriers is critical to achieving effective brain mRNA delivery.

[0150] The present disclosure relates to synthetic lipids bearing 5-hydroxytryptamine type 3 (5-HT3) receptor ligands that are covalently coupled to lipid tails through at least one amino-containing linker, and to lipid nanoparticles (LNPs) that incorporate these synthetic lipids for the delivery of polynucleotides, including messenger RNA (mRNA), across biological barriers such as the blood-brain barrier (BBB). In certain embodiments, the 5-HT3 ligand is derived from SR-57227, and the synthetic lipid is ionizable and includes biodegradable (e.g., ester) tail(s). In further embodiments, the LNP can be post-modified with a cell-penetrating peptide (CPP) (e.g., Tat) via a maleimide-functionalized PEG-lipid to enhance brain delivery.Attorney Docket No. 774757: MTST-875PCII. Definitions

[0151] Before continuing to describe the present disclosure in further detail, it will be understood that the materials, methods, and examples are illustrative only and not intended to be limiting. Methods and materials are described herein for use in the present invention and other, suitable methods and materials known in the art can also be used. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control.

[0152] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the methods described herein belong. Any reference to standard methods refers to the most recent available version of the method at the time of filing of this disclosure unless otherwise indicated.

[0153] For any method disclosed herein that includes discrete steps, the steps may be conducted in any feasible order. And, as appropriate, any combination of two or more steps may be conducted simultaneously.

[0154] All headings are for the convenience of the reader and should not be used to limit the meaning of the text that follows the heading, unless so specified.

[0155] The words "preferred" and "preferably" refer to embodiments of the invention that may afford certain benefits, under certain circumstances. However, other embodiments may also be preferred, under the same or other circumstances. Furthermore, the recitation of one or more preferred embodiments does not imply that other embodiments are not useful and is not intended to exclude other embodiments from the scope of the invention.

[0156] The term "comprises" and variations thereof do not have a limiting meaning where these terms appear in the description and claims. Such terms will be understood to imply the inclusion of a stated step or element or group of steps or elements but not the exclusion of any other step or element or group of steps or elements.

[0157] By "consisting of' is meant including, and limited to, whatever follows the phrase "consisting of." Thus, the phrase "consisting of' indicates that the listed elements are required or mandatory, and that no other elements may be present. By "consisting essentially of' is meant including any elements listed after the phrase, and limited to other elements that do not interfere with or contribute to the activity or action specified in the disclosure for theAttorney Docket No. 774757: MTST-875PClisted elements. Thus, the phrase "consisting essentially of indicates that the listed elements are required or mandatory, but that other elements are optional and may or may not be present depending upon whether or not they materially affect the activity or action of the listed elements.

[0158] The singular form "a", "an" and "the" include plural referents unless the context clearly dictates otherwise. These articles refer to one or to more than one (i.e., to at least one). As used herein, the term "or" is generally employed in its usual sense including "and / or" unless the content clearly dictates otherwise. The term "and / or" means any one or more of the items in the list joined by "and / or". As an example, "x and / or y" means any element of the three-element set {(x), (y), (x, y)}. In other words, "x and / or y" means "one or both of x and y". As another example, "x, y, and / or z" means any element of the seven-element set {(x), (y), (z), (x, y), (x, z), (y, z), (x, y, z)}. In other words, "x, y and / or z" means "one or more of x, y and z".

[0159] Where ranges are given, endpoints include all numbers subsumed within that range (e.g., 1 to 5 includes 1, 1.5, 2, 2.75, 3, 3.80, 4, 5, etc.). Furthermore, unless otherwise indicated or otherwise evident from the context and understanding of one of ordinary skill in the art, values that are expressed as ranges can assume any specific value or subrange within the stated ranges in different embodiments of the disclosure, to the tenth of the unit of the lower limit of the range, unless the context clearly dictates otherwise. Herein, "up to" a number (for example, up to 50) includes the number (for example, 50). The term "in the range" or "within a range" (and similar statements) includes the endpoints of the stated range.

[0160] Reference throughout this specification to "one aspect,” "an aspect,” "certain aspects," or "some aspects," etc., means that a particular feature, configuration, composition, or characteristic described in connection with the aspect is included in at least one aspect of the disclosure. Thus, the appearances of such phrases in various places throughout this specification are not necessarily referring to the same embodiment of the disclosure. Furthermore, the particular features, configurations, compositions, or characteristics may be combined in any suitable manner in one or more aspects.

[0161] Unless otherwise indicated, all numbers expressing quantities of components, molecular weights, and so forth used in the specification and claims are to be understood as being modified in all instances by the term "about." As used herein in connection with a measured quantity, the term "about" refers to that variation in the measured quantity as wouldAttorney Docket No. 774757: MTST-875PCbe expected by the skilled artisan making the measurement and exercising a level of care commensurate with the objective of the measurement and the precision of the measuring equipment used. The term "about" as used in connection with a numerical value throughout the specification and the claims denotes an interval of accuracy, familiar and acceptable to a person skilled in the art. In general, such interval of accuracy is + / - 10%. Accordingly, unless otherwise indicated to the contrary, the numerical parameters set forth in the specification and claims are approximations that may vary depending upon the desired properties sought to be obtained by the present invention. At the very least, and not as an attempt to limit the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.

[0162] Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the invention are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. All numerical values, however, inherently contain a range necessarily resulting from the standard deviation found in their respective testing measurements.

[0163] The term "exemplary" means serving as a non-limiting example, instance, or illustration. As utilized herein, the terms "e.g.," and "for example" set off lists of one or more non-limiting aspects, examples, instances, or illustrations.

[0164] As used herein, the term "substantially" refers to the qualitative condition of exhibiting total or near-total extent or degree of a characteristic or property of interest. Biological and chemical phenomena rarely, if ever, go to completion and / or proceed to completeness or achieve or avoid an absolute result. The term "substantially" is therefore used herein to capture the potential lack of completeness inherent in many biological and chemical phenomena. For example, "substantially" may refer to being within at least about 20%, alternatively at least about 10%, alternatively at least about 5% of a characteristic or property of interest.

[0165] The term "nucleic acid" or “polynucleotide” refers to any polymeric chain of nucleotides. A nucleic acid may be DNA, RNA, or a combination thereof. In some embodiments, a nucleic acid comprises one or more natural nucleic acid residues. In some embodiments, a nucleic acid comprises of one or more nucleic acid analogs. In some embodiments, nucleic acids are prepared by one or more of isolation from a natural source,Attorney Docket No. 774757: MTST-875PCenzymatic synthesis by polymerization based on a complementary template (in vivo or in vitro), reproduction in a recombinant cell or system, and chemical synthesis. In some embodiments, a nucleic acid is at least 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 20, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000 or more residues long (e.g., 20 to 100, 20 to 500, 20 to 1000, 20 to 2000, or 20 to 5000 or more residues). In some embodiments, a nucleic acid is partly or wholly single stranded; in some embodiments, a nucleic acid is partly or wholly double stranded. In some embodiments a nucleic acid has a nucleotide sequence comprising at least one element that encodes, or is the complement of a sequence that encodes, a polypeptide.

[0166] A polynucleotide, vector, polypeptide, cell, or any composition disclosed herein which is “isolated” is a polynucleotide, vector, polypeptide, cell, or composition which is in a form not found in nature. Isolated polynucleotides, vectors, polypeptides, or compositions include those that have been purified to the degree that they are no longer in a form in which they are found in nature. In some aspects, a polynucleotide, vector, polypeptide, or composition that is isolated is substantially pure.

[0167] Nucleotides are referred to by their commonly accepted single-letter codes. Unless otherwise indicated, nucleic acids are written left to right in 5' to 3' orientation. Nucleotides are referred to herein by their commonly known one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Accordingly, A represents adenine, C represents cytosine, G represents guanine, T represents thymine, and U represents uracil.

[0168] Messenger RNA (mRNA): As used herein, the term “messenger RNA” (mRNA) refers to any polynucleotide that encodes a polypeptide of interest and is capable of being translated to produce the encoded polypeptide in vitro, in vivo, in situ, or ex vivo.

[0169] Lipid nanoparticle (LNP): As used herein, the term lipid nanoparticle (LNP), refers to a nanoscale delivery system composed of lipid-based molecules designed to encapsulate and transport therapeutic agents, such as mRNA, small molecules, or nucleic acids, into target cells. LNPs typically consist of four main components: ionizable lipids, which aid in cargo encapsulation and cellular uptake; phospholipids, which provide structural stability; cholesterol, which enhances membrane fluidity; and polyethylene glycol (PEG)-lipids, which improve circulation time and reduce immune detection. LNPs herein encapsulate polynucleotides and are suitable for parenteral administration (e.g., intravenous).Attorney Docket No. 774757: MTST-875PC

[0170] Identity: As used herein, the term “identity” refers to the overall monomer conservation between polymeric molecules, e.g., between polynucleotide molecules (e.g., DNA molecules and / or RNA molecules) and / or between polypeptide molecules. Calculation of the percent identity of two polynucleotide sequences, for example, can be performed by aligning the two sequences for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second nucleic acid sequence for optimal alignment and nonidentical sequences can be disregarded for comparison purposes). In certain aspects, the length of a sequence aligned for comparison purposes is at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, or at least about 100% of the length of the reference sequence. The nucleotides at corresponding nucleotide positions are then compared. When a position in the first sequence is occupied by the same nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which needs to be introduced for optimal alignment of the two sequences. The comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm. When comparing DNA and RNA, thymine (T) and uracil (U) can be considered equivalent.

[0171] Suitable software programs are available from various sources and for alignment of both protein and nucleotide sequences. One suitable program to determine percent sequence identity is bl2seq, part of the BLAST suite of programs available from the U. S. government's National Center for Biotechnology Information BLAST website (blast.ncbi.nlm.nih.gov). B12seq performs a comparison between two sequences using either the BLASTN or BLASTP algorithm. BLASTN is used to compare nucleic acid sequences, while BLASTP is used to compare amino acid sequences. Other suitable programs are, e.g., Needle, Stretcher, Water, or Matcher, part of the EMBOSS suite of bioinformatics programs and also available from the European Bioinformatics Institute (EBI).

[0172] Sequence alignments can be conducted using methods such as, but not limited to, MAFFT, Clustal (ClustalW, Clustal X or Clustal Omega), or MUSCLE.

[0173] Different regions within a single polynucleotide or polypeptide target sequence that aligns with a polynucleotide or polypeptide reference sequence can each have their own percent sequence identity.Attorney Docket No. 774757: MTST-875PC

[0174] As used herein, “synthetic lipid” refers to a lipidic molecule that is not found in nature in the described chemical form and is prepared by chemical synthesis. In one aspect, the synthetic lipids herein comprise: (i) a 5-HT3 receptor derived ligand head group; (ii) one or more amino-containing linkers (e.g., aminoalkyl); and (iii) one or more lipid tails, preferably biodegradable tails that comprise at least one ester linkage.

[0175] As used herein, “ionizable lipid” means a lipid having a pH-dependent apparent charge, typically neutral at physiological pH and protonated under acidic conditions (e.g., endosomal pH), which facilitates polynucleotide complexation / encapsulation and endosomal escape following cellular uptake.

[0176] As used herein, “Amino-containing linker” means a linker moiety bearing at least one amine (primary, secondary, or tertiary), including C2-C8 aminoalkyl or aminooxyalkyl chains. In certain embodiments, the linker is derived from aminohexanoic acid or similar bifunctional reagents enabling amide formation to the head group followed by reductive amination to install the tails.

[0177] As used herein, “5-HT3 receptor ligand” refers to a small-molecule entity that binds to a 5-HT3 receptor (agonist, partial agonist, antagonist). SR-57227 and derivatives thereof are exemplary head groups that exhibit BBB-relevant transport interactions and serve as a targeting head in the disclosed ionizable lipids.

[0178] As used herein, “Biodegradable tail” means a lipid tail featuring a cleavable linkage (e.g., ester) to enable intracellular degradation and facilitate cargo release, enhancing potency and reducing persistence. Branched ester-containing tails (e.g., two branched ester chains) are exemplary.

[0179] As used herein, “Cell-penetrating peptide (CPP)” refers to short peptides that promote cellular entry and / or barrier traversal (e.g., Tat, pVEC, RVG). DSPE-PEG-maleimide enables covalent CPP attachment (Michael-type addition to cysteine residues).

[0180] The term "administering" as used herein refers to the physical introduction of an agent to a subject, such as a lipid nanoparticle disclosed herein, using any of the various methods and delivery systems known to those skilled in the art. Exemplary routes of administration for the formulations disclosed herein include intravenous, intramuscular, subcutaneous, intraperitoneal, spinal, or other parenteral routes of administration, for example by injection or infusion. The phrase "parenteral administration" means modes of administration other than enteral and topical administration, usually by injection, and includes,Attorney Docket No. 774757: MTST-875PCwithout limitation, intravenous, intramuscular, intraarterial, intrathecal, intralymphatic, intralesional, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrastemal injection and infusion, as well as in vivo electroporation. In some embodiments, the formulation is administered via a non-parenteral route, e.g., orally. Other non-parenteral routes include a topical, epidermal or mucosal route of administration, for example, intranasally, vaginally, rectally, sublingually or topically. Administering can also be performed, for example, once, a plurality of times, and / or over one or more extended periods.

[0181] As used herein, a ‘lipid nanoparticle composition’ refers to a composition comprising one or more lipids assembled into particulate structures capable of encapsulating, complexing, or otherwise associating with a therapeutic payload, including but not limited to nucleic acids (e.g., mRNA, siRNA, miRNA, DNA), proteins, peptides, small molecules, or combinations thereof. In certain embodiments, the lipid nanoparticle composition comprises ionizable lipids, cationic lipids, phospholipids, sterols (e.g., cholesterol), PEGylated lipids, helper lipids, or any combination thereof. The lipid nanoparticle composition may further include buffers, excipients, stabilizers, cryoprotectants, or other pharmaceutically acceptable components.”

[0182] As used herein, a ‘pharmaceutical composition’ refers to a lipid nanoparticle composition that is combined with one or more pharmaceutically acceptable carriers, excipients, or diluents, and that is formulated for administration to a subject. Pharmaceutical compositions may be formulated for any suitable route of administration, including parenteral (e.g., intravenous, intramuscular, subcutaneous), oral, intranasal, pulmonary, transdermal, or mucosal delivery.

[0183] As used herein, a ‘therapeutic composition’ refers to a lipid nanoparticle composition that is formulated or intended for use in the treatment, prevention, amelioration, or modulation of a disease, disorder, or physiological condition in a subject. In certain embodiments, the therapeutic composition comprises a lipid nanoparticle encapsulating or associated with a biologically active payload effective to produce a therapeutic effect upon administration.

[0184] Thus, in some embodiments, the lipid nanoparticle composition described herein is a pharmaceutical composition. In some embodiments, the lipid nanoparticle composition is aAttorney Docket No. 774757: MTST-875PCtherapeutic composition. In further embodiments, the composition is both a pharmaceutical composition and a therapeutic composition.

[0185] The term "pharmaceutically acceptable" refers to a molecule or composition that, when administered to a recipient, is not deleterious to the recipient thereof, or that any deleterious effect is outweighed by a benefit to the recipient thereof. With respect to a carrier, diluent, or excipient used to formulate a composition as disclosed herein, a pharmaceutically acceptable carrier, diluent, or excipient must be compatible with the other ingredients of the composition and not deleterious to the recipient thereof, or any deleterious effect must be outweighed by a benefit to the recipient. The term "pharmaceutically acceptable carrier" means a pharmaceutically- acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, or solvent encapsulating material, involved in carrying or transporting an agent from one portion of the body to another (e.g., from one organ to another). Each carrier present in a pharmaceutical composition must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not deleterious to the patient, or any deleterious effect must be outweighed by a benefit to the recipient. Some examples of materials which may serve as pharmaceutically acceptable carriers comprise: sugars, such as lactose, glucose and sucrose; starches, such as corn starch and potato starch; cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients, such as cocoa butter and suppository waxes; oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, com oil and soybean oil; glycols, such as propylene glycol; polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; esters, such as ethyl oleate and ethyl laurate; agar; buffering agents, such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogenfree water; isotonic saline; Ringer's solution; ethyl alcohol; pH buffered solutions; polyesters, polycarbonates and / or polyanhydrides; and other non-toxic compatible substances employed in pharmaceutical formulations.

[0186] " Treatment" or "treating" of a subject refers to any type of intervention or process performed on, or the administration of an active agent to, the subject with the objective of reversing, alleviating, ameliorating, inhibiting, slowing down or preventing the onset, progression, development, severity or recurrence of a symptom, complication or condition, or biochemical indicia associated with a disease. In one embodiment, "treatment" or "treating" includes a partial remission. In another embodiment, "treatment" or "treating" includes a complete remission. In some embodiments, treatment may be of a subject who does not exhibitAttorney Docket No. 774757: MTST-875PCsigns of the relevant disease, disorder and / or condition and / or of a subject who exhibits only early signs of the disease, disorder, and / or condition. In some embodiments, such treatment may be of a subject who exhibits one or more established signs of the relevant disease, disorder and / or condition. In some embodiments, treatment may be of a subject who has been diagnosed as suffering from the relevant disease, disorder, and / or condition. In some embodiments, treatment may be of a subject known to have one or more susceptibility factors that are statistically correlated with increased risk of development of the relevant disease, disorder, and / or condition.

[0187] A "disease", as used herein, is a state of health of a subject wherein the subject cannot maintain homeostasis, and wherein if the disease is not ameliorated, the subject's health continues to deteriorate. In contrast, a "disorder" is a state of health in which the subject is able to maintain homeostasis, but in which the subject's state of health is less favorable than it would be in the absence of the disorder. Left untreated, a disorder does not necessarily cause a further decrease in the subject's state of health. A disease or disorder is "alleviated" if the severity of a sign or symptom of the disease or disorder, the frequency with which such a sign or symptom is experienced by a subject, or both, is reduced.

[0188] As used herein, the terms “subject”, “individual”, and “patient” are interchangeable, and relate to vertebrates, preferably mammals. For example, mammals in the context of the disclosure are humans, non-human primates, domesticated animals such as dogs, cats, sheep, cattle, goats, pigs, horses, etc., laboratory animals such as mice, rats, rabbits, guinea pigs, etc., as well as animals in captivity such as animals in zoos. The term "animal" as used herein includes humans. The term "subject" may also include a patient, i.e., an animal, having a disease. In exemplary aspects, a subject, individual, or patient refers to a human (e.g., a man, a woman, or a child).

[0189] As used herein, the term “preventing a disease” in a subject means, for example, to stop the development of one or more clinical symptoms of a disease or disorder in a subject before they occur or are detectable. Preferably, the disease or disorder does not develop at all, i.e., no symptoms of the disease or disorder are detectable. In some aspects, it can also mean delaying or slowing of the development of one or more symptoms of the disease or disorder. Alternatively, or in addition, it can mean decreasing the severity of one or more subsequently developed symptoms.Attorney Docket No. 774757: MTST-875PC

[0190] The invention is defined in the claims. However, below is a non-exhaustive listing of non-limiting exemplary aspects. Any one or more of the features of these aspects may be combined with any one or more features of another example, embodiment, or aspect described herein.III. Synthetic Lipids

[0191] Disclosed herein are series of synthetic lipids comprising a 5-hydroxytryptamine type 3 (5-HT3) receptor ligand and at least one lipid, wherein the 5-HT3 receptor ligand is linked to the at least one lipid via at least one amino containing linker.

[0192] In one aspect, a synthetic lipid is provided comprising a 5-HT3 receptor ligand head group linked via at least one amino-containing linker to at least one lipid tail. In certain embodiments, the head group is derived from SR-57227 (e.g., through amide formation to an amino-functionalized linker) and the tail region comprises biodegradable ester linkages. In some embodiments, the lipid includes two branched ester-containing tails, which enhance degradability and facilitate endosomal escape when formulated into LNPs.

[0193] In some embodiments, the lipid tail is of Formula I:(i);

[0194] or a pharmaceutically acceptable salt thereof;

[0195] wherein:

[0196] X1is a bond, -OCH2O-, -OCH(CH3)O-, -OC(O)O- or -C(O)OCH(CH2CH3)-;

[0197] X2is a bond, -OCH2O-, -OCH(CH3)O-, -OC(O)O- -C(O)OCH(CH2CH3)-, or -C(O)OCH(C(CH3)2)-;

[0198] W1is CH2or NRW1

[0199] RW1isX w1 w2

[0200] X3is a bond, -OCH2O-, -OCH(CH3)O-, -OC(O)O- or -C(O)OCH(CH2CH3)-;

[0201] wl is 1 to 5;Attorney Docket No. 774757: MTST-875PC

[0202] w2 is 1 to 3;

[0203] a is 1 to 5;

[0204] b is I to 3:

[0205] c is 3 to 5;

[0206] d is' ^ 3 too 5; and

[0207] e is I to 3. Z o=

[0208] The 4 synthetic lipid may be ionizable, balancing encapsulation and circulation oproperties with intracellular release.o o

[0209] SR-57227-derived lipids (SLs) incorporating three building blocks: SR-57227Z y - head, amino linkers, and biodegradable lipid tails i \s ° szhown in FIG. 1. These SLs with DOPE, cholesterol, and DMG-PEG may be effectively encapsulated mRNA to form SR-57227- derived LNPs (SLNPs, FIG. 1)z—' O /

[0210] Illustrative structures of SR-57227 and exemplary lipid tails (SO, SI, S2, S3, S4, o oS5, S6, S7, S8 and S9) are shown in Table 1.

[0211] Table 1ID Com zpo=oundSR-57227 y q ° \SOSI0 4 o0S2S3Attorney Docket No. 774757: MTST-875PC

[0212] In some aspects, the SR-57227-derived lipids are prepared by amide coupling of the SR-57227 derivative to a Boc-protected amino acid linker, Boc deprotection, and reductive amination with a tail-bearing aldehyde to deliver the final ionizable lipids.Attorney Docket No. 774757: MTST-875PC

[0213] Leveraging the capacity of certain small molecules to penetrate the BBB via high-affinity interactions with specific receptors or transporters, a novel strategy was proposed for brain delivery by conjugating BBB-permeable molecules to ionizable lipids. This approach aims to enhance the ability of LNPs to effectively traverse the BBB. The results demonstrated that SR-57227-derived LNP, OS4, achieved markedly improved delivery efficiency to the brain following systemic administration (FIG. 6D, FIG. 6E). The chemical structure of S4 in OS4 LNPs comprising an SR-57227-derived head, an amino linker, and two branched ester tails (FIG. 1), has shown unexpected and superior brain-delivery performance among the tested series. Previous studies have suggested that the head group of ionizable lipids can incorporate small-molecule ligands, thereby incorporating specific biological functions. As for the tail of ionizable lipid, the inclusion of biodegradable ester bonds has been reported to enhance delivery efficiency, potentially by accelerating intracellular release kinetics. Thus, the superior delivery efficiency of S4 is likely a result of the combined effects of both its head and tail structures.

[0214] In another aspect, LNPs are provided that include any of the synthetic lipids described herein together with phospholipids (e.g., DOPE), cholesterol, and PEG-lipids (e.g., DMG-PEG2k and / or DSPE-PEG2k-maleimide). Such LNPs may optionally include additional ionizable lipids. The LNPs are formulated using established ethanol / aqueous mixing procedures at a desired lipid-to-polynucleotide mass ratio (e.g., ~10: 1) and dialyzed or buffer- exchanged into physiological media.

[0215] The LNPs typically exhibit hydrodynamic diameters of about 70-180 nm (e.g., 100-150 nm) with PDI of about 0.05-0.30, and encapsulation efficiency of >50%, e.g., >70% or >85%, depending on formulation and polynucleotide. Size, PDI, and zeta potential may be measured by DLS, and morphology by cryo-TEM.

[0216] In certain embodiments, when the synthetic lipid is S4, the molar ratio of S4: DOPE: cholesterol: PEG-lipid is selected from:

[0217] (i) about 40:40:60:0.75;

[0218] (ii) about 30-50: 30-50: 50-70: 0.5-1.5; or

[0219] (iii) about 20-60: 30-50: 50-70: 0.5-2.0.

[0220] In particular embodiments, S4 and DOPE are present at substantially equal molar percentages, and cholesterol is present at a higher percentage than either S4 or DOPE.

[0221] To further improve brain delivery efficiency, the impact of conjugating various CPPs onto the surface via post-modification on the ability of OS4 LNP to cross the BBB was investigated. In some embodiments, the LNP is post-modified with a CPP via maleimide-thiolAttorney Docket No. 774757: MTST-875PCconjugation. For example, LNPs formulated using DSPE-PEG2k-maleimide can be reacted with a cysteine-containing CPP (e.g., Tat) at a peptide:maleimide molar ratio of ~2:1 for >1 hour at room temperature to afford stable CPP-decorated LNPs.

[0222] CPPs are a group of short peptides capable of traversing biological membranes, promoting the cellular uptake of otherwise membrane-impermeable cargoes. While studies have shown that certain CPPs can facilitate the transport of small drugs, biomacromolecules, and nanoparticles across the BBB, their potential to enhance the brain delivery of LNP-mRNA formulations via systemic administration remains relatively unexplored. The ten different CPPs modification exhibited markedly different effects on LNP-mRNA formulations for brain delivery. Some CPPs, including A5K, LAH4, and THR peptides, caused LNP aggregation, while some others failed to noticeably improve the brain delivery efficiency of OS4 LNP. Among the tested CPPs, the Tat peptide emerged as the optimal choice, significantly enhancing mRNA delivery to the brain without compromising the stability of OS4 LNP. Following i.v. administration, Tat-modified OS4 (OS4T) LNP achieved a 12.7-fold increase in brain mRNA delivery efficiency compared to unmodified OS4 LNP (FIG. 6D). All the CPPs converted the OS4 LNP surface charge from negative to positive; however, Tat modification did not result in the highest surface charge (FIG. 8). Notably, although OS4B and OS4T exhibited similar zeta potentials (FIG. 8), their brain delivery efficiencies differed significantly, with OS4T outperforming OS4B (FIG. 9B). This observation indicates that the improvement in delivery efficiency is not solely dependent on the surface charge of OS4 LNP. Indeed, OS4 LNPs inherently exhibit high endosomal escape efficiency, and Tat modification did not further enhance the cellular uptake or endosomal escape of OS4 LNPs (FIG. 11A, FIG. 11B, FIG. 11C). However, the transwell assay revealed that OS4T LNPs exhibited a stronger BBB-crossing ability compared to unmodified OS4 LNP (FIG. 12A, FIG. 12B, FIG.12C).

[0223] To investigate the effects of the small molecular ligand in S4 structure of ionizable lipids on brain delivery, a control ionizable lipid was synthesized, referred to as “Amino Lipid (AL)”, which lacks the small molecule head group but contains the same tail group (FIG. 20).Using the same formulation as OS4 LNPs, AL LNPs were prepared, which had a larger average particle size compared to OS4 LNPs (FIG. 20). That was then conjugated to the AL LNPs; however, upon Tat conjugation, significant aggregation and precipitation was obserbed, with particle sizes exceeding 1 pm (FIG. 20). Additionally, OS4 LNPs and AL LNPs were compared without Tat conjugation in an in vivo delivery study. As shown in FIG.20, OS4 LNPs induced significantly higher luciferase intensity in the brain compared to ALAttorney Docket No. 774757: MTST-875PCLNPs, indicating that the small molecular head group plays a critical role in promoting brain delivery.

[0224] These findings suggest that the high brain delivery efficiency of OS4T is attributable to the combined contributions of both Tat and OS4 LNP. As a result, OS4T LNP can more effectively deliver mRNA into brain cells, including neurons, astrocytes, microglia, and BCECs compared to MC3 LNP following i.v. injection (FIGS. 16A 16G). The therapeutic potential of OS4T LNP was further demonstrated in an orthotopic glioblastoma (GBM) mouse model by delivering eIL-12 mRNA. Three doses of OS4T LNP -mRNA encoding eIL-12 significantly inhibited GBM growth and extended the median survival by over 2-fold to 37 days compared to the PBS group. The use of OS4T LNPs for efficient mRNA delivery to the brain provides new insights into the design of biomaterials capable of crossing the BBB, potentially paving the way for more effective CNS-targeted therapies. Consistent with its superior brain delivery efficiency, OS4T LNP encapsulating eIL-12 mRNA more effectively suppressed tumor growth and prolonged survival compared to treatments with MC3T or OS4 LNPs carrying eIL-12 mRNA (FIG. 17E, FIG. 17F).

[0225] LNPs can be characterized by dynamic light scattering (DLS) for size and PDI, zeta potential for surface charge, cryo-TEM for morphology, and RiboGreen or analogous assays for encapsulation efficiency. In vivo biocompatibility can be assessed by serum AST, ALT, BUN at 24-48 h and H& E histopathology of major organs (brain, heart, liver, lung, spleen, kidney), which have shown no significant pathological changes under tested conditions.

[0226] Lipid nanoparticle compositions comprising the LNPs or CPP -modified LNPs described herein can further include a polynucleotide, selected from DNA, ssDNA, cDNA, RNA, mRNA, tRNA, rRNA, siRNA, sgRNA, and combinations thereof. The compositions can be formulated with pharmaceutically acceptable excipients (buffers, tonicity agents) and supplied as ready -to-use or as concentrates for dilution prior to administration.

[0227] In particular embodiments, the polynucleotide encodes a protein, peptide, or antibody, including a therapeutic protein or peptide. In some embodiments, the polynucleotide encodes an engineered interleukin- 12 (eIL-12) comprising IL-12p40, IL-12p35, an MMP-cleavable linker, an IL-12Rpl (Q20-A261) domain, and optionally a FLAG tag to enable tumor-restricted activation and improved tolerability upon systemic administration.

[0228] The compositions may be configured to cross the BBB upon intravenous administration, facilitating delivery to brain cells. In some embodiments, delivery is associated with 5-HT receptor-mediated transcytosis, with contributions from macropinocytosis and / or caveolae-mediated pathways, as evidenced by pharmacological inhibition in transwell models.Attorney Docket No. 774757: MTST-875PC

[0229] In one aspect, a method is provided for delivering a polynucleotide to cells in a subject by administering any of the lipid nanoparticle compositions described herein, preferably by intravenous injection. Target cells can include brain cells such as neurons, astrocytes, microglia, and brain endothelial cells. In certain embodiments, administration results in at least a 9-fold increase in polynucleotide delivery relative to a control LNP (e.g., an MC3-based comparator).

[0230] In some embodiments, multiple administrations (e.g., three doses) may further enhance expression / editing readouts across the indicated brain cell types relative to a single administration, while maintaining an acceptable tolerability profile in standard rodent models.

[0231] Therapeutic methods include treating or preventing cancer (e.g., glioblastoma) or treating CNS diseases or disorders by administering the lipid nanoparticle compositions described herein. In some embodiments, OS4T LNPs encapsulating eIL-12 mRNA suppress tumor growth, extend survival compared with control groups, and activate antitumor immunity in brain tissues, while exhibiting reduced systemic cytokine induction compared with wild-type IL- 12 mRNA. Indications include glioblastoma, anaplastic astrocytoma, medulloblastoma, oligodendroglioma, primary CNS lymphoma, metastatic brain tumors, and polyneuropathy.

[0232] In certain embodiments, treatment suppresses tumor growth, increases survival, increases polynucleotide delivery, and / or increases cellular translation in the target tissue. Exemplary dosing regimens include multiple intravenous administrations spaced by several days (e.g., day 3, 8, and 13 post-tumor implantation in murine models).

[0233] In a further aspect, the disclosure provides kits comprising: (a) a unit dose of a lipid nanoparticle composition as described herein (e.g., OS4 or OS4T LNP encapsulating a polynucleotide) and (b) instructions for administration to deliver a polynucleotide across the BBB. Kits may optionally include a cysteine-containing CPP supplied separately for on-site conjugation to maleimide -bearing LNPs prior to administration.IV. Synthesis, characterizations, and optimization of SR-57227-derived LNPs (SLNPs)

[0234] 5-HT3 receptors are extensively distributed throughout the central nervous system. Here, SR-57227, a potent 5-HT3 receptor ligand, was used to develop ionizable lipids for mRNA delivery to the brain. The series of SR-57227-derived lipids (SLs) was designed with three structural modules: the SR-57227 core, two amino linkers, and five lipophilic tails (FIG.1). The lipids, SO, SI, S2, S3, S4, S5, S6, S7, S8 and S9, were synthesized and validated by 'll NMR and mass spectrometry (Supplementary information). Next, each lipid wasAttorney Docket No. 774757: MTST-875PCformulated with cholesterol, l,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG2k), and firefly luciferase (FLuc) mRNA to generate SLNPs. The particle sizes of these SLNPs ranged from approximately 120 nm to 180 nm, with polydispersity indices (PDI) below 0.3 (FIG. 2).

[0235] Most of these SLNPs exhibited negative charges, and their mRNA encapsulation efficiency ranged from 68.3 ± 9.1% to 83.2 ± 2.5% (FIG. 3). To assess mRNA delivery efficiency, FLuc SLNPs were incubated with three representative types of mouse brain-associated cells: Neuro-2a (N2a), a neuroblastoma cell line; primary astrocytes; and bEnd.3, a brain endothelial cell line. As shown in FIG. 4A, SO, S4, and S6 LNPs exhibited the highest luminescence intensity across all three cell types, outperforming other SLNPs. This led to their selection for further in vivo validation of mRNA delivery efficiency in the brain. Six hours after i.v. injection of FLuc SLNPs (mRNA, 0.6 mg / kg), S4 LNP induced the highest luminescence intensity in brain tissues, which was 4.5-fold and 16.3-fold higher than SO LNP and S6 LNP, respectively (FIG. 4B, FIG. 4C).

[0236] These SLNPs showed similar distribution across other major organs, including the liver, lung, spleen, kidney, and heart (FIG. 5). Consequently, S4 LNP was selected for further optimization to enhance mRNA delivery efficiency in the brain.

[0237] An orthogonal assay was employed to refine the ratio of each lipid component (S4, DOPE, cholesterol, and DMG-PEG2k) in the S4 LNP formulation. In the first round, 16 formulations were prepared based on an L16 (4)4orthogonal table (FIG. 6A), and their mRNA delivery efficiency was evaluated in N2a and bEnd.3 cell lines. Formulation 0 served as the baseline control, derived from initial evaluation prior to the optimization study. Each data point in FIG. 6B represents the average luminescence intensity of all LNP formulations sharing the same molar ratio for a specific lipid component (e.g., all formulations containing 20% S4). As shown in FIG. 6B, the trends of luminescence intensity for each lipid component were similar in both cell lines at different molar ratios. Based on these results, the following ratios were selected for further refinement: S4 mol% = 20, 40, 60, DOPE mol% = 40, cholesterol mol% = 60, and PEG mol% = 0.75, 1.0, 1.5. To identify the optimal formulation, additional predicted formulations (formulations 17-25 in FIG. 6A) were prepared in the second round. As exhibited in FIG. 6C, formulation 24 (S4 / DOPE / Chol / DMG-PEG2k = 40:40:60:0.75) demonstrated superior mRNA delivery efficiency compared to other predicted formulations in both cell lines. Specifically, formulation 24 enhanced 3.4-fold (P < 0.001) and 5.2-fold (P < 0.001 ) mRNA delivery efficiency compared to the initial formulation 0 in bEnd.3 and N2a cell lines, respectively (FIG. 6C). Here, formulation 24, the optimized S4 LNP, wasAttorney Docket No. 774757: MTST-875PCnamed as OS4 LNP. To validate the mRNA delivery efficiency in vivo, mice were i.v. treated with FLuc OS4 LNP (mRNA, 0.6 mg / kg) and other control LNPs including S4 LNP and FDA-approved formulations, MC3, SM-102, and ALC-0315. Six hours after administration, FLuc OS4 LNP resulted in about 13.3-fold, 12.5-fold, 9.6-fold, and 2-fold higher luminescence intensity in the brain than MC3, SM-102, ALC-0315, and S4 LNPs, respectively (FIG. 6D, FIG. 6E).V. Enhancing brain delivery efficiency through CPP conjugation

[0238] Although CPPs have been reported to facilitate the transport of various cargoes across the BBB, few studies have specifically examined their impact on the BBB permeability of LNP-mRNA formulations following systemic administration. To further improve the brain delivery efficiency of OS4 LNP, the LNP was modified with a series of CPPs (FIG.7, Table 2), originating from synthetic peptides and naturally occurring viral peptides.

[0239] Table 2SEQ ID NO: 1 CGLFEKIEGFIENGWEGMIDGWYGYGRKKRRQRRSEQ ID NO: 2 CVPALRSEQ ID NO: 3 CTVSALKSEQ ID NO: 4 LRRERQSRLRRERQSRCSEQ ID NO: 5 CRQVTIWFQNRRVKEKKSEQ ID NO: 6 KKALLALALHHLAHLALALKKACSEQ ID NO: 7 CLLIILRRRIRKQAHAHSKSEQ ID NO: 8 YTIWMPENPRPGTPCDIFTNSRGKRASNGSEQ ID NO: 9 GRKKRRQRRRPPQCSEQ ID NO: 10 THRPPMWSPVWPC

[0240] Each peptide was conjugated onto OS4 LNP through a Michael addition reaction. All CPP-conjugated OS4 LNPs exhibited positive charges (FIG. 8). Based on the changes in size and PDI (FIG.9A), conjugation of the A5K, THR, or LAH4 peptides caused aggregation of OS4 LNP, as the sizes of OS4A, OS4L, and OS4Th LNPs exceeded 450 nm. Therefore, the remaining seven CPP-conjugated OS4 LNPs were selected for in vivo evaluation of brain delivery efficiency. Although all these CPPs have been reported to possess BBB-penetrating capabilities, they exhibited varying potency in enhancing the brain delivery efficiency of OS4 LNP. As exhibited in FIG. 9B, pVEC, RVG, and Tat conjugation significantly increased the luminescence intensity of OS4 LNP in the brain. Among them, Tat- conjugated OS4 LNPAttorney Docket No. 774757: MTST-875PC(OS4T) demonstrated the highest mRNA delivery efficiency to the brain, showing a 12.7-fold, 6.6-fold and 2.0-fold increase compared to OS4, OS4R and 0S4P LNPs, respectively (FIG.9B). Notably, OS4T LNP exhibited 59.1-fold and 17.2-fold greater brain delivery efficiency than MC3 LNP and Tat-conjugated MC3 (MC3T) LNP, respectively (FIG. 9C, FIG. 9D).OS4T showed a spherical morphology under cryo-TEM imaging (FIG. 9E). Additionally, it was observed that Tat- conjugated LNPs, OS4T, and MC3T, induced higher luminescence signals in the lung compared to their unmodified counterparts, OS4 and MC3, respectively (FIG. 10). This effect may be attributed to the increased positive charge, which has been reported to enhance LNP accumulation in the lung following systemic injection.

[0241] To further understand OS4- and OS4T-mediated mRNA delivery, cellular uptake and endosomal escape assays were performed using LNPs encapsulating Alexa Fluor 647-labeled RNA. As shown in FIG. 11A, MC3, OS4, and OS4T LNPs achieved comparable levels of cellular uptake. The in vitro biocompatibility of OS4 and OS4T LNPs was evaluated in N2a cells at the test dose (FIG. 13A), showing no significant cytotoxicity. To study the endosomal escape of these LNPs, N2a cells were incubated with calcein, a fluorescent dye that localizes within endosomes, and LNPs encapsulating Alexa Fluor 647-labeled RNA. Confocal imaging analysis revealed that green fluorescence appeared as punctate spots in the control group, which is consistent with calcein being trapped in endosomes. In the MC3 group, the prominent overlap of green and red fluorescence signals indicated limited endosomal escape, with LNPs largely retained within the endosomal compartments. In contrast, cells treated with OS4 and OS4T LNPs exhibited reduced co-localization with endosomes and a more diffused calcein signal (FIG. 11B, FIG. 11C), suggesting stronger endosomal escape. This observation aligns with the mRNA delivery efficiency results (FIG. 4A). To investigate the potential mechanisms of transport by OS4 and OS4T LNPs, a transwell migration assay was utilized featuring bEnd.3 cells to model the BBB, following previously reported methods (FIG. 12A). In this assay, bEnd.3 cells were pretreated with or without metachlorophenylpiperazine, a 5-HT receptor ligand, followed by incubation with FLuc OS4 or OS4T LNPs. The luminescence intensity of N2a cells in the lower chamber served as an indicator of the extent of LNPs transport across the BBB. The transcytosis efficiency of OS4 and OS4T LNPs decreased in the presence of meta-chlorophenylpiperazine compared to the condition without the inhibitor (FIG. 12B). Importantly, none of these inhibitors used in this study had a significant impact on cell viability at the test dose (FIG. 12C). This highlights the critical role of the BBB-crossing moiety, SR-57227, in the S4 lipid, indicating that both OS4 LNP and OS4T LNP can cross endothelial cells by 5-HT receptor-mediated transcytosis. To investigateAttorney Docket No. 774757: MTST-875PCthe pathways, bEnd.3 cells were pretreated with EIPA (a macropinocytosis inhibitor), CPZ (a clathrin-mediated endocytosis inhibitor), or MpCD (a caveolae -mediated endocytosis inhibitor) before incubation with OS4 or OS4T LNPs in the transwell migration assay. For OS4 LNP, transcytosis was reduced by over 90% with MpCD and more than 70% with EIPA, whereas CPZ did not significantly affect transcytosis compared to the condition without the inhibitor (FIG. 12B). These findings suggest that macropinocytosis- and caveolae-mediated pathways are critical for OS4 LNP to penetrate the BBB. For OS4T LNP, transcytosis was inhibited by approximately 75% with MpCD, 95% with EIPA, and 25% with CPZ, indicating that OS4T LNPs utilize multiple pathways to cross the BBB (FIG. 12B). Notably, without any inhibitors, the luminescence intensity of N2a cells in the OS4T group was 1.4-fold higher than in the OS4 group (FIG. 12C). This aligns with the observation that OS4T LNP demonstrated significantly greater mRNA delivery efficiency in the brain compared to OS4 LNP in vivo (FIG. 9B), highlighting the critical role of Tat in crossing the BBB. Based on these results, OS4T LNP was selected for the following studies.

[0242] To evaluate the in vivo biosafety profile, mice were i.v. administered PBS, MC3 LNP, OS4 LNP, or OS4T LNP (1.0 mg / kg). The blood levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT), and urea nitrogen (BUN) were measured at 24 and 48 hours. As shown in FIG. 13, the levels of AST, ALT, and BUN in LNP -treated groups were comparable to those in the PBS-treated group. Furthermore, histopathological analysis of tissues, including the brain, heart, liver, lung, spleen, and kidney, at 48 hours post-injection revealed no significant pathological changes (FIG. 14). These findings indicate the biocompatibility and safety of both OS4 and OS4T LNPs.VI. Efficient delivery of mRNA in different brain cells

[0243] To assess the delivery efficiency and biodistribution of OS4T LNPs across different brain cell populations, OS4T LNP containing GFP mRNA were i.v. injected into mice (mRNA, 1.0 mg / kg). At 12 hours post-administration, brain tissues were dissociated into single-cell suspensions, and the percentage of GFP+ cells was analyzed by flow cytometry following the gating strategy (FIG. 15). Compared to MC3 LNP, OS4 LNP increased about 8-fold, 4-fold, 6-fold, and 3-fold mRNA delivery efficiency in neurons, astrocytes, microglia, and BCECs, respectively (FIG. 16A). After Tat conjugation, OS4T LNP displayed improved mRNA delivery efficiency in neurons, microglia and BCECs compared with OS4 LNP.

[0244] Next, Ail 4 mice were utilized to validate mRNA delivery efficiency within the brain (FIG. 16B). Seven days after a single i.v. injection of Cre mRNA-loaded LNPs (1.0Attorney Docket No. 774757: MTST-875PCmg / kg), tdTomato expression in different brain cell types was analyzed by flow cytometry. Compared to the MC3 group, which showed less than 2.0% tdTomato+ cells, the injection of OS4T LNP resulted in 13.1 ± 1.2% tdTomato+ neurons, 9.9 ± 1.2% tdTomato+ astrocytes, 10.2 ± 0.4% tdTomato+ microglia, and 11.6 ± 1.6% in tdTomato+ BCECs (FIG. 16C).Whether multiple injections could enhance gene editing efficiency in Ail 4 mice was also studied. Flow cytometry analysis revealed that triple injections of OS4T LNP resulted in a 1.6-fold, 2.0-fold, 1.5-fold, and 1.7-fold increase in tdTomato expression in neurons, astrocytes, BCECs, and microglia, respectively, compared to the single injection group (FIG.16C). However, triple injections of MC3 LNP induced a limited increase of tdTomato signals across these cells (FIG. 16C). Immunofluorescence imaging further confirmed the enhanced mRNA delivery efficiency of OS4T LNP in the brain. In PBS and MC3 LNP groups, tdTomato signals in the brain were negligible on day 21 (FIG. 16D). In contrast, OS4T LNP containing Cre mRNA resulted in widespread tdTomato expression across most brain regions (FIG. 16D). Next, immunofluorescence staining on neurons (FIG. 16E), microglia (FIG.16F), and astrocytes (FIG. 16G) were performed, revealing that tdTomato expression levels induced by OS4T LNP were significantly higher compared to MC3 LNP.VII. Therapeutic efficacy of OS4T in an orthotopic mouse model of GBM

[0245] IL- 12 is a promising anti-tumor cytokine due to its ability to activate multiple immune cell populations, including antigen-presenting cells (APCs) and T cells. Despite encouraging preclinical results, systemic administration of IL- 12 has been associated with severe immune-related adverse events in clinical trials, leading to early termination of many studies. One approach involves the intratumoral administration of IL- 12, delivered either as a fusion with a tumor extracellular matrix-binding domain or through exosome or LNP encapsulating mRNA encoding IL-12. While intratumoral injections have demonstrated improved tolerability, their repeated application in the treatment of brain cancers presents substantial clinical challenges. Recently, Hubbell and colleagues developed an engineered IL-12 (eIL-12) by fusing it to the domain of its receptor (FIG. 17A), IL-12Rpl(Q20-A261), through a matrix metalloproteinase (MMP)-sensitive linker. Given the high expression of MMP in tumor tissues, the linker is cleaved upon reaching the tumor site, locally releasing active IL- 12. This design confines IL-12’s pro-inflammatory effects to the tumor site, reducing systemic side effects following i.v. administration. Thus, an mRNA transcript was developed encoding this eIL-12, containing sequences encoding IL- 12, MMP-cleavable linkers, IL-12Rpl(Q20-A261), and a Flag tag (FIG. 17A). Next, HEK293T cells were treated with OS4T LNP encapsulating eIL-12 mRNA, collected the supernatant, and performed western blotAttorney Docket No. 774757: MTST-875PCanalysis to detect the expressed protein. As shown in FIG. 17B, the molecular weight of intact eIL-12 matched its theoretical value. Furthermore, after incubation with MMP9, a molecular weight corresponding to the IL-12Rpl (Q20-A261) segment was observed, confirming that cleavage had occurred. Whether the eIL-12 could mitigate systemic immune-related adverse events was then evaluated. For this study, healthy C3H / HeJ mice were used, which are a more representative model for human sensitivity to IL-12 compared to C57BL / 6 mice. The mice were treated with two doses of OS4T LNP encapsulating either IL- 12 mRNA or eIL-12 mRNA, administered every other day, while monitoring body weight changes (FIG. 17C).Treatment with IL- 12 mRNA resulted in approximately 20% body weight loss compared to PBS-treated controls, whereas eIL-12 mRNA induced less weight loss, demonstrating improved in vivo tolerability (FIG. 17C). Considering that LNPs may mediate delivery to off-target organs such as the liver and spleen following systemic administration, IFN-y levels were evaluated, a key downstream cytokine indicative of IL- 12 bioactivity, in the serum, liver, and spleen of mice treated with OS4T LNPs encapsulating either wild-type IL- 12 or eIL-12 mRNA. As shown in Supplementary Fig. 12, mice treated with eIL-12 mRNA LNPs exhibited lower IFN-y levels compared to those receiving wild-type IL- 12 mRNA, suggesting that although off-target delivery may occur, the engineered IL- 12 construct helps to mitigate systemic immune-related adverse effects relative to the wild-type IL- 12.

[0246] To evaluate the therapeutic potential of OS4T LNP encapsulating eIL-12 mRNA (OS4T-eIL-12), a GBM mouse model was established using CT-2A-Luc cells, a highgrade glioma characterized by an immunosuppressive tumor microenvironment. Mice were i.v. injected with OS4T-eIL-12 (1 mg / kg) for three doses, administered every five days (FIG.17D). Control groups included PBS, OS4T-FLuc mRNA, MC3T-eIL-12, and OS4-eIL-12 LNPs. Compared to control treatments, OS4T-eIL-12 LNP significantly inhibited tumor growth and extended survival (FIG. 17E, FIG. 17F). Remarkably, the OS4T group exhibited an improvement in median survival, reaching 37 days — double that of the PBS group, which showed a median survival of 17 days (FIG. 17F). Moreover, no sustained body weight reduction was observed after the third treatment (FIG. 18). These findings highlight the potential of systemic delivery of OS4T-eIL-12 LNP as a promising and tolerated strategy for effective GBM treatment.

[0247] To further investigate the immune response, immune cell populations in tumor tissues were analyzed (FIG. 18C). A single treatment with OS4T-eIL-12 led to the upregulation of Ml phenotype markers, including CD80, CD86, and iNOS, in macrophages and microglia within brain tissues (FIG. 19A, FIG. 19B). The treatment also decreased theAttorney Docket No. 774757: MTST-875PCpercentage of regulatory CD4+ T cells (FIG. 19C) and activated CD8+ T cells in brain tissues, as indicated by an increased frequency of TNFa+ and CD69+ CD8+ T cells (FIG. 19D). A comparison of cytokine levels further demonstrates that OS4T-eIL-12 markedly enhances immune activation in the brain tissue of GBM-bearing mice (FIG. 18A).

[0248] The presently described technology and its advantages will be better understood by reference to the following examples. These examples are provided to describe specific implementations of the present technology. By providing these specific examples, it is not intended limit the scope and spirit of the present technology. It will be understood by those skilled in the art that the full scope of the presently described technology encompasses the subject matter defined by the claims appending this specification, and any alterations, modifications, or equivalents of those claims.EXAMPLES

[0249] Methods

[0250] Animal studies

[0251] The animal experiments conducted in this study adhered to the guidelines approved by the Icahn School of Medicine at Mount Sinai (IPROT0202200000134). All relevant ethical regulations were followed as applicable. Both male and female C57BL / 6J (Strain #:000664), C3 / Hej (Strain #:000659) and Ail4 (Strain #:007914) mice (6-10 weeks old) obtained from the Jackson Laboratory, were used for the experiments. Animals were randomly assigned to different groups.

[0252] Materials

[0253] All chemicals and solvents were purchased from Fisher Scientific unless otherwise listed. DLin-MC3-DMA (MC3), SM-102, ALC-0315 and ALC-0159 were purchased from MedKoo Biosciences (NC, USA). Cholesterol (Choi), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dimyristoyl-rac-glycero-3 -methoxy (poly (ethylene glycol))-2000 (DMG-PEG2k), and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyethylene glycol)-2000 (DSPE-PEG2k-Mal) were purchased from Avanti Polar Lipids (AL, USA). Aldehydes were synthesized according to previously reported procedures. Amino lipid (AL) was synthesized according to previously reported procedures.

[0254] Synthesis of ionizable lipidsAttorney Docket No. 774757: MTST-875PC

[0255] All chemicals and solvents were purchased from Fisher Scientific unless otherwise listed. Aldehydes were synthesized according to previously reported procedures (7). Ionizable lipids were purified by column chromatography using a CombiFlash Rf system with a RediSep Gold Resolution silica column (Teledyne Isco) with gradient elution. All!H NMR spectra were run on a Bruker Avance 400 MHz instrument. Mass spectrometric measurements were performed by microflex LRF MALDLTOF mass spectrometer (Bruker) at Icahn School of Medicine at Mount Sinai.Aldehydes NaBH(OAc)3

[0256] Scheme SI. Synthesis of S4.

[0257] To a solution of Boc-6-aminohexanoic acid (116 mg, 0.5 mmol), DMF (5 mL) and A, A-diisopropylethylamine (145 pL, 0.8 mmol), hexafluorophosphate azabenzotriazole tetramethyl uronium (300 mg, 0.8 mmol) was added. The resulting mixture was stirred for 20 min at room temperature. Then SR57227 (100 mg, 0.47 mmol) was added, and the solution was stirred at room temperature overnight. The resulting mixture was washed with Nal ICCL (50 mL) 3 times and brine (50 mL), and further purified by Combiflash column chromatography with a RediSep Gold Resolution silica column with gradient elution from 100% CH2CI2 to CEbCh / MeOH (90 / 10, v / v) to give Boc protected intermediate (156 mg, 79%). Then, the Boc protected intermediate was dissolved in 5 mL of CH2CI2 and 0.5 mL of CF3COOH and the mixture stirred for 3 h. After the reaction was completed, the resulting mixture was purified by Combiflash column chromatography with a RediSep Gold Resolution silica column with gradient elution from 100% CH2CI2 to CI LCF / McOI I (70 / 30, v / v) to give compound 1 (102 mg, 81%). Subsequently, to a solution of compound 1 (32 mg, 0.1 mmol) in anhydrous THF (10 mL) was added aldehyde (142 mg, 0.5 mmol), and sodium triacetoxyborohydride (106 mg, 0.5 mmol). The reacting mixture was stirred at room temperature overnight and evaporated under reduced pressure. The resulting mixture was washed with NaHCCh (50 mL) and brine (50 mL), purified by Combiflash columnAttorney Docket No. 774757: MTST-875PCchromatography with a RediSep Gold Resolution silica column using gradient elution from 100% CH2CI2 to and CH2Cl2 / MeOH / NH4OH (90 / 10 / 0.3, v / v / v) to afford S4 (40 mg, 46%) having 'HNMR (400 MHz, CDCh) 87.39 (m, 1H), 6.58 (d, J= 7.4 Hz, 1H), 6.52 (d,8.4 Hz, 1H), 4.83 (m, 2H), 4.24 (m, 2H), 4.00 - 3.74 (m, 1H), 3.05 - 2.97 (m, 1H), 2.50 - 2.19 (m, 10H), 2.16 (m, 2H), 2.09 - 1.00 (m, 52H), 0.90 (t, 6.7 Hz, 12H) and MALDI-MS for C50H89CIN4O5 ([M+H]+) calculated: 861.7, found: 861.9.

[0258] S0-S9 were synthesized using similar methods.

[0259] SO (36 mg, 55%) NMR (400 MHz, CDCh) 8 7.37 (t, J 7.9 Hz, 1H), 6.57 (d, J 7.4 Hz, 1H), 6.50 (d, 8.4 Hz, 1H), 4.22 (d, 13.4 Hz, 2H), 4.09 - 3.92 (m, 1H), 2.99 (t, J= 12.5 Hz, 2H), 2.50 - 2.27 (m, 6H), 2.16 (t, J= 7.5 Hz, 2H), 2.06 - 1.85 (m, 4H), 1.71 - 1.54 (m, 2H), 1.51 - 1.10 (m, 54H), 0.88 (t, J = 6.4 Hz, 6H) and MALDI-MS for C40H73CIN4O ([M+H]+) calculated: 661.6, found: 661.5.

[0260] SI (38 mg, 51%) NMR (400 MHz, CDCh) 8 7.37 (t, J 7.9 Hz, 1H), 6.57 (d, 7.5 Hz, 1H), 6.50 (d, 8.4 Hz, 1H), 4.22 (d, J= 13.4 Hz, 2H), 4.12 (t,6.7 Hz, 9H), 4.04 - 3.88 (m, 1H), 2.99 (t, J 12.5 Hz, 2H), 2.37 (m, 6H), 2.16 (t, J= 7.5 Hz, 2H), 2.01 (m, 2H), 1.78 - 1.05 (m, 46H), 0.89 (t, 6.6 Hz, 6H) and MALDI-MS for C42H73CIN4O7 ([M+H]+) calculated: 781.5, found: 781.6.

[0261] S2 (33 mg, 44%) NMR (400 MHz, CDCh) 8 7.37 (t, J 7.8 Hz, 1H), 6.57 (d, J= 7.4 Hz, 1H), 6.50 (d, J= 8.3 Hz, 1H), 4.66 (s, 4H), 4.22 (m, 2H), 4.12 - 3.91 (m, 1H), 3.50 (m, 10H), 2.99 (t, J= 12.5 Hz, 2H), 2.45 (m, 8H), 2.25 - 1.01 (m, 49H), 0.88 (t, J 6.5 Hz, 6H) and MALDI-MS for C42H77CIN4O5 ([M+H]+) calculated: 753.6, found: 753.6.

[0262] S3 (31 mg, 39%) 'll NMR (400 MHz, CDCh) 8 7.37 (t, J 7.9 Hz, 1H), 6.57 (d, 7.4 Hz, 1H), 6.50 (d, J 8.4 Hz, 1H), 4.66 (q, 2H), 4.22 (d, 13.4 Hz, 2H), 4.09 - 3.92 (m, 1H), 3.56 (m, 4H), 3.41 (m, 4H), 2.99 (t, 12.5 Hz, 2H), 2.57 - 2.27 (m, 8H), 2.16 (t, J= 7.5 Hz, 2H), 2.06 - 1.85 (m, 4H), 1.71 - 1.54 (m, 2H), 1.51 - 1.10 (m, 54H), 0.88 (t, J = 6.4 Hz, 6H) and MALDI-MS for C44H81CIN4O5 ([M+H]+) calculated: 781.6, found: 781.8.

[0263] S5 (37 mg, 42%)NMR (400 MHz, CDCh) 8 7.37 (t, J 7.9 Hz, 1H), 6.57 (d, J 7.4 Hz, 1H), 6.50 (d, 8.4 Hz, 1H), 4.22 (d, 13.4 Hz, 2H), 4.10 - 3.92 (m, 1H), 2.99 (t, J= 12.3 Hz, 2H), 2.40 (m, 12H), 2.16 (t, 7.5 Hz, 2H), 2.01 (m, 2H), 1.75 - 1.03 (m, 74H), 0.88 (t, J= 6.5 Hz, 9H) and MALDI-MS for C55H104CIN5O ([M+H]+) calculated: 886.8, found: 887.0.

[0264] S6 (48 mg, 45%) 'll NMR (400 MHz, CDCh) 8 7.37 (t, J 7.9 Hz, 1H), 6.57 (d, 7.4 Hz, 1H), 6.50 (d, 8.4 Hz, 1H), 4.22 (d, J= 13.4 Hz, 2H), 4.11 (t,6.7 Hz, 12H), 4.02 (m, 1H), 2.99 (t, 12.3 Hz, 2H), 2.37 (m, 12H), 2.15 (t, J= 7.5 Hz, 2H), 2.07 -Attorney Docket No. 774757: MTST-875PC1.95 (m, 2H), 1.83 - 1.05 (m, 61H), 0.89 (t, J = 6.4 Hz, 9H) and MALDI-MS for C58H104CIN5O10 ([M+H]+) calculated: 1066.8, found: 1067.1.

[0265] S7 (35 mg, 34%) NMR (400 MHz, CDCh) 8 7.37 (t, J 7.9 Hz, 1H), 6.57 (d, 7.4 Hz, 1H), 6.50 (d, J= 8.4 Hz, 1H), 4.65 (s, 6H), 4.22 (d, J= 13.3 Hz, 2H), 4.10 - 3.94 (m, 1H), 3.50 (m, 12H), 2.99 (t, J 12.3 Hz, 2H), 2.46 (m, 12H), 2.16 (t, J 7.4 Hz, 2H), 2.01 (d,.7= 11.8 Hz, 2H), 1.95 - 1.06 (m, 61H), 0.88 (t, J- 6.0 Hz, 9H) and MALDI-MS for C58H110CIN5O7 ([M+H]+) calculated: 1024.8, found: 1025.1.

[0266] S8 (32 mg, 30%) 'll NMR (400 MHz, CDCh) 8 7.38 (t, J - 7.9 Hz, 1H), 6.57 (d, J- 7.4 Hz, 1H), 6.50 (d, J - 8.4 Hz, 1H), 4.75 - 4.56 (q, 3H), 4.22 (d, J - 13.4 Hz, 2H), 4.08 - 3.90 (m, 1H), 3.56 (m, 6H), 3.45 - 3.28 (m, 6H), 2.99 (t, J - 12.4 Hz, 2H), 2.67 - 2.49 (m, 6H), 2.47 - 2.33 (m, 8H), 2.26 -1.05 (m, 88H), 0.89 (t, J - 6.2 Hz, 9H) and MALDI-MS for C61H116CIN5O7 ([M+H]+) calculated: 1066.9, found: 1066.9.

[0267] S9 (41 mg, 35%) NMR (400 MHz, CDCh) 8 7.38 (t, J - 7.9 Hz, 1H), 6.57 (d, J - 7.4 Hz, 1H), 6.51 (d, J - 8.4 Hz, 1H), 4.83 (t, J - 6.0 Hz, 3H), 4.24 (d, J - 13.5 Hz, 2H), 4.00 - 3.74 (m, 1H), 3.84 - 3.62 (m, 6H), 3.51 (m, 6H), 3.11 - 2.97 (m, 6H), 2.50 - 2.19 (m, 12H), 2.09 - 1.00 (m, 89H), 0.89 (t, J - 6.7 Hz, 18H) and MALDI-MS for C70H128CIN5O7 ([M+H]+) calculated: 1187.0, found: 1187.2.

[0268] Characterization of LNPs and peptide conjugated LNPs

[0269] To prepare mRNA-loaded lipid nanoparticles (LNPs), a previously established method was employed. Initially, the organic solution was prepared by dissolving different lipids in ethanol at a defined molar ratio. Subsequently, the mRNA aqueous solution was mixed with the ethanol solution, maintaining a lipid- to-mRNA mass ratio of 10:1. For MC3 LNPs, a mixture of MC3 lipid, DSPC, Choi, and DMG-PEG2k is used in a molar ratio of 50 / 10 / 38.5 / 1.5. Similarly, SM-102 LNPs utilized SM-102, DSPC, Choi, and DMG-PEG2k in a molar ratio of 50 / 10 / 38.5 / 1.5. For ALC-0315 LNPs, the ethanol phase contained ALC-0315, DSPC, Choi, and ALC-0159 at a molar ratio of 45.8 / 10.4 / 42.2 / 1.6.

[0270] To conjugate peptides onto LNPs, the DMG-PEG2k was replaced by DSPE-PEG2k-Mal, enabling covalent attachment to the cysteine residue in peptides through a Michael addition reaction. After formulation, the LNPs were dialyzed in PBS prior to conjugation. Each candidate peptide contains a cysteine residue within its sequence. Peptides were then mixed with the LNPs in a 2:1 molar ratio (cysteine / Mal) to facilitate the Michael addition reaction, which was allowed to proceed for at least 1 hour at room temperature.Attorney Docket No. 774757: MTST-875PC

[0271] The hydrodynamic diameter and polydispersity index (PDI) of the different LNP formulations are measured using a NanoZS Zetasizer (Malvern). The morphology of LNPs is then analyzed via Cryo-TEM (Thermo Scientific Glacios), allowing for the detailed observation of particle structure and uniformity.

[0272] In vitro assays of LNPs loaded with firefly luciferase mRNA (Fluc-mRNA)

[0273] Neuro-2a (N2a) cell line (CCL-131) and bEnd.3 cell line (CRL-2299) were obtained from the American Type Culture Collection (Manassas, VA). Primary astrocytes harvested from C57BL / 6J (male / female) mice. Cells were seeded in 96-well plates at a concentration of 2 × 103cells / well in Dulbecco’s modified Eagle’s medium (DMEM) with 10% fetal bovine serum (FBS) and incubated at 37°C in a humidified atmosphere containing 5% CO2. After an overnight incubation, different LNPs were added to the wells. Following 18 h of incubation, 50 pL of Bright-Glo luciferase substrate was added to each well. The plate was briefly mixed using a microplate shaker, and luminescence intensity was measured using a microplate reader.

[0274] Cellular uptake and endosome escape assay

[0275] N2a cells were seeded into 24-well plates at a concentration of 1 × 104cells / well overnight. Subsequently, LNPs containing Alexa647-tagged mRNA were added to each well. After 3 h of incubation to allow uptake, the cells were washed with PBS, harvested, and analyzed using flow cytometry. For the endosome escape assay, N2a cells were seeded in each well of glass-bottom dishes containing DMEM (37 °C with 5% CO2). After overnight incubation, Calcein (Invitrogen) and LNPs loaded with Alexa647-tagged mRNA were added to each well. After 2 h of incubation to allow uptake, cells were washed with PBS and stained with Hoechst for 10 minutes to label the nucleus. Imaging was performed using a confocal laser scanning microscope (CLSM, Leica DiM8).

[0276] In vivo assays of LNPs loaded with Fluc-mRNA

[0277] Fluc-mRNA loaded LNPs were intravenously injected into C57BL / 6J mice (Fluc-mRNA, 0.6 mg / kg). After 6 h, the mice administered an intraperitoneal injection of D-luciferin substrate. After 7 min, the mice were sacrificed, and major organs were collected. Bioluminescence signals were quantified using regions of interest (ROIs) by using the in vivo imaging system (IVIS, PerkinElmer).

[0278] In vivo biocompatibility evaluation

[0279] Mouse plasma and major organs were harvested 24 and 48 h after intravenous administration of PBS, MC3 LNPs, OS4 LNPs or OS4T LNPs (FLuc mRNA, 1 mg / kg). Whole blood was collected in sodium citrate- coated tubes and centrifuged to isolate plasma.Attorney Docket No. 774757: MTST-875PCLevels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), and blood urea nitrogen (BUN) were determined using an enzyme-linked immunosorbent assay (ELISA) kit. To assess potential systemic or local toxicity, tissue sections were stained with hematoxylin and eosin (H& E) and scanned under NanoZoomer S60 digital slide scanner (Hamamatsu Photonics K. K.).

[0280] Flow cytometry of brain cells

[0281] GFP-mRNA or Cre-mRNA-loaded LNPs were prepared following established protocols and administered intravenously to C57BL / 6J or Ail4 mice, respectively. For GFP-mRNA studies, brain tissues were harvested 12 h post-injection, while for Cre-mRNA studies, brain tissues were collected 5 days after the final dose. Following isolation, single-cell suspensions from the brain tissue were prepared. Red blood cells were lysed using RBC lysis buffer, and myelin was removed using Myelin Removal Beads (MACS 130-096-433), following the manufacturer’s instructions. The purified single-cell suspensions were subsequently stained with specific markers for cell type identification: NeuN (1:50 dilution, MACS 130-119-494) for neurons, GFAP (1:50, MACS 130-123-846) for astrocytes, CD11b (1:50, MACS 130-113-802) for microglia, CD31 (1:50, MACS 130-111-541) for brain capillary endothelial cells (BCECs). For the GFP-mRNA studies, Rb pAb to GFP (1:100, ab6556) and Donkey Anti- Rabbit IgG H& L (1:100, ab150073) were used for GFP staining. Prior to immunofluorescence staining for NeuN and GFAP, the samples were incubated with a fixation-permeabilization buffer to enable intracellular staining. After the staining and washing, the cells were stained with DAPI staining solution (Miltenyi Biotec) and analyzed using a flow cytometer.

[0282] Immunofluorescence staining of brain tissues

[0283] After the designated treatments, mice were anesthetized and perfused sequentially with PBS and 4% paraformaldehyde (PFA). The brains were harvested and fixed in 4% PFA, followed by preparation of paraffin section slides. Antigen retrieval was performed on the slides prior to staining. The sections were first incubated in blocking buffer for 1 h and subsequently incubated overnight at 4 °C with primary antibodies. After washing, the sections were incubated at room temperature for 2 h with secondary antibodies. The following antibodies were used: NeuN antibody (1:50 dilution, Miltenyi Biotec, Clone: REA1131) for neurons; GFAP antibody (1:300 dilution, Cell Signaling Technology, Cat. #: 3655S) for astrocytes; Anti-Ibal antibody (1:5000 dilution, abeam, ab283346) and Goat antiRat IgG H& L (FITC) (1:1000 dilution, abeam, ab6840) for microglia; TdTomato (E3G5L) Rabbit mAb (1:300 dilution, Cell Signaling Technology, Cat. #: 20163) and Goat anti-rabbitAttorney Docket No. 774757: MTST-875PCIgG H& L (Cy3®) (1:100 dilution, abeam, ab6939) for tdTomato. The sections were mounted using a mounting medium containing DAPI to stain cell nuclei. Imaging was performed using a NanoZoomer S60 digital slide scanner (Hamamatsu Photonics K. K.).

[0284] Engineered IL- 12 (eIL-12) mRNA

[0285] Although IL-12 exhibits potent anticancer activity, dose-limiting immune-related adverse events significantly hinder its clinical application, particularly for systemic administration. To minimize the systemic toxicity, IL- 12 mRNA sequence was constructed based on the previously reported fused IL- 12 construct. The eIL-12 mRNA sequence was produced by incorporating IL-12p40, flexible linkers (such as Glycine-Glycine-Glycine-Serine, GGGS (SEQ ID NO: 25)), IL-12p35, a cleavable peptide sequence ((HPVGLLARVPLSLYSG)2(LSGRSDNH)(SEQ ID NO: 18), specifically recognized by MMP-9), IL-12R01 (Q20-A261) and Flag tag (DYKDDDDK) (SEQ ID NO: 23). The complete sequence details are provided as Table 3.Attorney Docket No. 774757: MTST-875PCTable 3. eIL-12 sequencesSequence Identifier Atgtgtcctcagaagctaaccatctcctggtttgccatcgttttgctggtgtctccac Signaltcatggcc (SEQ ID NO: 11) Atgtgggagctggagaaagacgtttatgttgtagaggtggactggactcccgatg IL12p40 cccctggagaaacagtgaacctcacctgtgacacgcctgaagaagatgacatca cctggacctcagaccagagacatggagtcataggctctggaaagaccctgacca tcactgtcaaagagtttctagatgctggccagtacacctgccacaaaggaggcga gactctgagccactcacatctgctgctccacaagaaggaaaatggaatttggtcca ctgaaattttaaaaaatttcaaaaacaagactttcctgaagtgtgaagcaccaaatta ctccggacggttcacgtgctcatggctggtgcaaagaaacatggacttgaagttc aacatcaagagcagtagcagttcccctgactctcgggcagtgacatgtggaatgg cgtctctgtctgcagagaaggtcacactggaccaaagggactatgagaagtattc agtgtcctgccaggaggatgtcacctgcccaactgccgaggagaccctgcccat tgaactggcgttggaagcacggcagcagaataaatatgagaactacagcaccag cttcttcatcagggacatcatcaaaccagacccgcccaagaacttgcagatgaag cctttgaagaactcacaggtggaggtcagctgggagtaccctgactcctggagca ctccccattcctacttctccctcaagttctttgttcgaatccagcgcaagaaagaaaa gatgaaggagacagaggaggggtgtaaccagaaaggtgcgttcctcgtagaga agacatctaccgaagtccaatgcaaaggcgggaatgtctgcgtgcaagctcagg atcgctattacaattcctcatgcagcaagtgggcatgtgttccctgcagggtccgatcc (SEQ ID NO: 12)GGS(GGGS)3(SEQ ID NO: 13)Tagggggggagcggtggcggcagtgggggtggatcaggtggagggtct(SEQ ID NO: 14)Agggtcattccagtctctggacctgccaggtgtcttagccagtcccgaaacctgc IL12p35 tgaagaccacagatgacatggtgaagacggccagagaaaaactgaaacattatt cctgcactgctgaagacatcgatcatgaagacatcacacgggaccaaaccagca cattgaagacctgtttaccactggaactacacaagaacgagagttgcctggctact agagagacttcttccacaacaagagggagctgcctgcccccacagaagacgtct ttgatgatgaccctgtgccttggtagcatctatgaggacttgaagatgtaccagacagagttccaggccatcaacgcagcacttcagaatcacaaccatcagcagatcattctAttorney Docket No. 774757: MTST-875PCagacaagggcatgctggtggccatcgatgagctgatgcagtctctgaatcataat ggcgagactctgcgccagaaacctcctgtgggagaagcagacccttacagagt gaaaatgaagctctgcatcctgcttcacgccttcagcacccgcgtcgtgaccatca acagggtgatgggctatctgagctccgcc (SEQ ID NO: 15)GS(GGGS)2(SEQ ID NO: 16)Tgagggtcaggtggcggcagtggcggagggtcc (SEQ ID NO: 17)(HPVGLLARVPLSLYSG)2(LSGRSDNH) (SEQ ID NO:18)Catccagttggtttgctcgcacgcgttcctttgtcattgtactccggtcacccagtcg gccttcttgcccgcgtgccactgagtctgtactctgggctctctgggcgatcagataatcat (SEQ ID NO: 19)(GGGS)2(SEQ ID NO: 20)Ggagggggttccgggggtgggagc (SEQ ID NO: 21)Cagctaggcgcctcaggcccaggtgatggctgctgcgttgagaagacatcgttc IL-12R01 (Q20-A261) ccagagggagcctcaggctcacccttaggacccaggaacttgagttgctacagg gtttccaagacagactatgagtgctcctggcagtatgatggccctgaggacaatgt ttctcacgtcctgtggtgctgctttgtccctccgaaccatacccacaccggccagg agcgctgccgctacttctcctcaggcccagaccgcactgtgcagttctgggaaca ggacggtatccctgtgctgtccaaggtcaacttctgggtggagtctcggcttggga accgaaccatgaagtcccagaagatatcccagtacctgtacaactggaccaaga cgacccctcccctgggacacatcaaggtgtcacaatcacaccggcagttgcgaa tggactggaatgtgtctgaagaggccggtgctgaggtacagttcaggcgccgtat gcccacaacgaattggaccttgggtgactgcggacctcaggttaactctggctca ggtgtgcttggtgacattcgtgggagcatgtctgagtcctgcctctgcccttctgag aacatggcccaagagatccagatacggaggaggaggcggctctcctcaggagc ccctggaggtccctggagtgattggagcatgcctgtgtgtgttccacctgaagtccttccccaggcc (SEQ ID NO: 22)DYKDDDDK Flag tagAttorney Docket No. 774757: MTST-875PC(SEQ ID NO: 23)Gattacaaggacgatgatgataaataa (SEQ ID NO: 24)

[0286] Cleavage assays of eIL-12 translated by mRNA

[0287] For in vitro detection of eIL-12, HEK293 cells seeded into a 6-well plate at a density of 1 × 105cells per well. After 24 h, treat the cells with LNPs loaded with elL- 12 (mRNA at 1 pg / mL). Incubate for an additional 24 h, then collect the proteins from supernatant and lyse the cells to extract the proteins. Protein purification was performed using anti-FLAG® M2 Magnetic Beads according to the manufacturer’s protocol.

[0288] Recombinant mouse MMP-9 protein was activated by p-aminophenylmercuric acetate (1 mM) before using. For cleavage assays, the purified proteins were incubated with activated MMP-9 (5 pg / mL) for 30 min at 37 °C. The samples were denatured and subjected to Western blot analysis. Protein separation was carried out on a Mini-PROTEAN TGX 4- 15% precast gel (Bio-Rad), followed by transfer to a nitrocellulose membrane (Bio-Rad). IL- 12 cleavage was detected via primary incubation with an anti-DDDDK (SEQ ID NO: 26) tag (Binds to FLAG® tag sequence) antibody (Abeam, ab1162, 1:1000 dilution) and secondary incubation with anti-rabbit IgG, Anti-rabbit IgG, HRP-linked Antibody (Cell Signaling, 1:1000 dilution). Visualization was performed using a ChemiDocTM MP Imaging System (Bio-Rad).

[0289] Body weight changes in C3H / HeJ mice

[0290] C3H / HeJ healthy mice (8-12 weeks old) were weighed on day 0. Mice were divided into three groups: PBS, OS4T-IL-12 and OS4T-eIL-12. Administrations were performed via tail vein on day 0 and day 2 (mRNA at 1.0 mg / kg). Body weight measurements were performed daily until day 4.

[0291] Treatment in an orthotopic glioblastoma model

[0292] The orthotopic glioblastoma (GBM) model was established by implanting CT-2A- luc cells into the brains of C57BL / 6J mice. All mice received a painkiller (buprenorphine, 0.1 mg / kg) prior to undergoing deep anesthesia (induced with 4% isoflurane and maintained with 1.5% isoflurane). Before surgery, the skin was disinfected three times with povidone-iodine. Using a stereotaxic instrument, the coordinate origin was located, followed by a movement of 2 mm to the right and 1 mm upward. A needle was then slowly inserted to a depth of 3 mm into the brain and left in place for at least 60 seconds before beginning the injection. CT-2A-Attorney Docket No. 774757: MTST-875PCluc cells (1×105cells) were injected at a constant rate of 300 nL / min (2 pL per site). Upon completing the injection, the needle was left in the brain for at least 3 minutes before being withdrawn at a controlled rate of 0.75 mm / min. After surgery, the mice were closely monitored in a heating chamber set to 37 °C until fully awake.

[0293] The tumor implantation was performed on day 0. The treatment began after 3 days of post-implantation. The mice were divided into five groups: PBS, OS4T-control mRNA, MC3T-eIL-12, OS4-eIL-12, OS4T-eIL-12. Treatments were intravenously administered via tail vein on day 3, day 8 and day 13 (eIL-12 mRNA, 1.0 mg / kg). From day 3 to day 12, the mice were imaged by IVIS every three days to monitor the tumor growth. For the survival evaluation, mice were monitored for signs indicating an endpoint, including cranial protuberance, neurological deficits such as seizures or abnormal gait, and a body weight reduction exceeding 20%. Mice were euthanized either at the study's conclusion or upon the first signs of reaching an endpoint.

[0294] Immune cell populations and cytokine levels within brain tissue were analyzed following treatment. GBM bearing C57BL / 6J mice (n = 5 per group) were intravenously injected with a single dose of PBS or OS4T-eIL-12 (1.0 mg / kg). Four days of post-treatment, cytokine profiling and flow cytometry analysis were conducted on brain tissues. For IFN-y, IL-6 and IL-12p70 cytokine measurements, brain tissue samples were analyzed by Eve Technologies. For flow cytometry, single-cell suspensions were prepared from brain tissues followed by myelin removal to enrich for immune cell populations prior to staining and analysis. Antibodies used in this study include: CD45, APC (30-F11, Invitrogen, 17-0451-82); CD11b, Pacific blue (MI / 70.15, Invitrogen, RM2828); F4 / 80, PE-eFluor™ 610 (BM8, Invitrogen, 61-4801-82); Ly-6G / Ly-6C, Alexa Fluor™ 700 (RB6-8C5, Invitrogen, 56-5931-80); P2RY12-PE (S16007D, BioLegend, 848003); iNOS, PE-Cy7 (CXNFT, Invitrogen, 25-5920-80); CD80, FITC (B7-1, Invitrogen, 11-0801-81); CD86, FITC (B7-2, Invitrogen, 11-0862-81); CD206, Alexa Fluor 488 (MR6F3, 53-2061-80); CD3e, PE (17A2, BioLegend, 100205); CD4, Pacific blue (RM4-5, Invitrogen, MCD0428); CD25, Alexa Fluor 700 (PC61.5, Invitrogen, 56-0251-82); FOXP3, PE-eFluor™ 610 (FJK-16s, Invitrogen, 61-5773-80); CD69, FITC (H1.2F3, Invitrogen, 11-0691-82); CD8a, APC-eFluor™ 780 (53-6.7, Invitrogen, 47-0081-82); TNF-a, eFluor™ 450 (MP6-XT22, Invitrogen, 48732180). The cells were analyzed by an LSRFortessa flow cytometer (BD Biosciences) and FlowJo (Version 10.8) using the following gating strategies.

[0295] Macrophages: CD45+CD1 lb+F4 / 80+Ly6C Ly6G P2ry12-

[0296] Microglia: CD45+CD1 lb+F4 / 80+Ly6C Ly6G P2ry12+Attorney Docket No. 774757: MTST-875PC

[0297] Tregs: CD45+CD3+CD4+CD25+Foxp3+

[0298] CD8+T cells: CD45+CD3+CD8+

[0299] In vivo cytokine profiling after IL- 12 mRNA LNPs administration in healthy mice

[0300] C57BL / 6J mice were intravenously injected with OS4T LNPs encapsulating either wild- type IL- 12 mRNA, engineered IL- 12 mRNA (eIL-12), or Flue- mRNA as a negative control (mRNA, 1 mg / kg). A PBS group was also included (n = 3 per group). After 24 h, serum, liver, and spleen tissues were collected, and levels of IFN-y were measured by mouse IFN-y ELISA Kit (Invitrogen, Cat # BMS606-2).

[0301] Statistical analysis

[0302] Data were statistically analyzed using GraphPad Prism. All relevant information on the statistical details of experiments is provided in the figure legends. For in vitro studies, experiments were performed twice with similar results. Data are presented as mean ± SD unless otherwise indicated. Sample sizes (n) for in vivo studies are also provided in the figure legends.

[0303] Immunofluorescence staining of brain slices

[0304] After 24 h of LNP injection, mice were transcardially perfused with 20 mL of PBS and 20 mL of 4% paraformaldehyde (PFA). The mouse brains were fixed in 4% PFA at 4 °C overnight and then dehydrated sequentially in 20% and 30% sucrose solution at 4 °C. Coronal brain sections were prepared (15 pm thick) using a cryotome.

[0305] For staining, brain slices were incubated in 0.3% Triton X-100 in PBS for 15 min and then blocked with blocking buffer (CST) for 30 mins. Due to the Gria3 with a V5 tag, slices were incubated with anti-V5 tag (1:200, Thermo Fisher Scientific) overnight at 4 °C, followed by incubation with secondary antibodies (Anti-mouse IgG (H+L), F(ab')2 Fragment (Alexa Fluor® 594 Conjugate) (1:200, CST) for 60 min at 37°C the following day. 4',6-Diamino-2-phenylindole dihydrochloride (DAPI, Thermo) was used to counterstain the nuclei. All sections were imaged using fluorescence microscopy (Leica DiM8).

[0306] Open field test

[0307] Mice received MK-801 (O.lmg / kg) daily for 4 days via intraperitoneal (i.p.) injection. To evaluate the efficacy of LNP treatments on general motor activity on day 5, mice were placed in a chamber (40 cm x 40 cm x 50 cm) equipped with recording devices. Locomotor activity was quantified by measuring the total distance traveled within the chamber over a 10-min session using an automated tracking system Fusion 5.6 (SuperFlex).Attorney Docket No. 774757: MTST-875PCReferences1. A. J. Barbier, A. Y. Jiang, P. Zhang, R. Wooster, D. G. Anderson, The clinical progress of mRNA vaccines and immunotherapies. Nat Biotechnol 40, 840-854 (2022).2. K. A. Hajj, K. A. Whitehead, Tools for translation: non-viral materials for therapeutic mRNA delivery. Nat Rev Mater 2, 17056 (2017).3. D. Loughrey, J. E. Dahlman, Non-liver mRNA Delivery. Acc. Chem. Res. 55, 13-23 (2022).4. X. Hou, T. Zaks, R. Langer, Y. Dong, Lipid nanoparticles for mRNA delivery. Nat Rev Mater 6, 1078-1094 (2021).5. B. Sun, W. 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[0308] It will be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims

Attorney Docket No. 774757: MTST-875PCCLAIMS1. A synthetic lipid comprising a head group and at least one lipid tail, wherein the head group is derived from a 5 -hydroxytryptamine type 3 (5 HT3) receptor ligand and is covalently linked through at least one amino containing linker to the at least one lipid tail.

2. The synthetic lipid of claim 1, wherein the 5-HT3 receptor ligand is SR-57227.

3. The synthetic lipid of claim 2, wherein the SR-57227 has the structure of:

4. The synthetic lipid of any one of claim 1 to 3, wherein the at least one lipid tail comprises an ester linkage.

5. The synthetic lipid of claim 4, wherein the at least one lipid tail comprises at least one biodegradable ester linkage.

6. The synthetic lipid of claim 4 or claim 5, wherein the lipid tail comprises two branched ester lipids.

7. The synthetic lipid of any one of claims 1 to 6, wherein the lipid tail is of Formula I:or a pharmaceutically acceptable salt thereof;wherein:X1is a bond, -OCH2O- -OCH(CH3)O- -OC(O)O-, or -C(O)OCH(CH2CH3)-;Attorney Docket No. 774757: MTST-875PCX2is a bond, -OCH2O- -OCH(CH3)O- -OC(O)O-, -C(O)OCH(CH2CH3)-, or -C(O)OCH(C(CH3)2)-;W1is CH2or NRW1RW1isX3is a bond, -OCH2O- -OCH(CH3)O- -OC(O)O- or -C(O)OCH(CH2CH3)-;wl is 1 to 5;w2 is 1 to 3;a is 1 to 5;b is 1 to 3:c is 3 to 5;d is 3 to 5; ande is 1 to 3.

8. The synthetic lipid of any one of claims 1 to 7, wherein the at least one lipid tail is selected from the group consisting of:NNAttorney Docket No. 774757: MTST-875PCAttorney Docket No. 774757: MTST-875PCthereof.10Attorney Docket No. 774757: MTST-875PC9. The synthetic lipid of claim 8, wherein the lipid tail is:

10. The synthetic lipid of any one of claims 1 to 9, wherein the synthetic lipid is ionizable.

11. A lipid nanoparticle comprising the synthetic lipid of any one of claims 1 to 10.

12. The lipid nanoparticle of claim 11, wherein the lipid nanoparticle further comprises phospholipids, cholesterol, additional ionizable lipids, and / or polyethylene glycol-derived lipids (PEG-lipid).

13. The lipid nanoparticle of claim 12, wherein the lipid nanoparticle comprises 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG2k), and / or 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyethylene glycol)-2000 (DSPE-PEG2k-Mal).

14. The lipid nanoparticle of claim 13, wherein the phospholipid is DOPE, the PEG-lipid is DMG-PEG2k or DSPE-PEG2k-maleimide, and the cholesterol is present.

15. The lipid nanoparticle of claim 14, wherein the lipid tail issynthetic lipid: DOPE:cholesterol: PEG-lipid is about 30-50 mol% synthetic lipid, about 30-50 mol% DOPE, about 50-70 mol% cholesterol, and about 0.5-1.5 mol% PEG-lipid, alternatively about 20-60 mol% synthetic lipid, about 30-50 mol% DOPE, about 50-70 mol% cholesterol, and about 0.5-2.0 mol% PEG-lipid.

16. The lipid nanoparticle of claim 15, wherein the molar ratio of synthetic lipid: DOPE:cholesterol: PEG-lipid is about 40:40:60:0.75.Attorney Docket No. 774757: MTST-875PC17. The lipid nanoparticle of any one of claims 11 to 16, wherein the lipid nanoparticle has a particle size of between about 70nm and about 180nm, alternatively at least about 80nm, alternatively at least about 90nm, alternatively at least about 100nm, alternatively at least about 110nm, alternatively at least about 120nm, alternatively at least about 130nm, alternatively at least about 140nm, alternatively at least about 150nm, alternatively at least about 160nm, or alternatively at least about 170nm.

18. The lipid nanoparticle of any one of claims 11 to 17, wherein the lipid nanoparticle has a polydispersity index (PDI) of between about 0.05 and about 0.3, alternatively at least about 0.1, alternatively at least about 0.15, alternatively at least about 0.2, or alternatively at least about 0.25.

19. The lipid nanoparticle of any one of claims 11 to 18, wherein the lipid nanoparticle has an encapsulation efficiency of at least about 50%, alternatively at least about 55%, alternatively at least about 60%, alternatively at least about 65%, alternatively at least about 70%, alternatively at least about 75%, or alternatively at least about 85%.

20. A modified lipid nanoparticle comprising, the lipid nanoparticle of any one of claims 11 to 19 and at least one cell-penetrating peptide (CPP).

21. The modified lipid nanoparticle of claim 20, wherein the CPP is covalently linked to the lipid nanoparticle via a maleimide-functionalized PEG-lipid.

22. The modified lipid nanoparticle of claim 20 or claim 21, wherein the CPP is Tat or a Tat-derived peptide.

23. The modified lipid nanoparticle of any one of claims 20 to 22, wherein the at least one CPP comprises an amino acid sequence at least 80% identical to the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, or SEQ ID NO: 10.

24. The modified lipid nanoparticle of any one of claims 20 to 22, wherein the at least one CPP is selected from a peptide having an amino acid sequence identical to the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, or SEQ ID NO: 10.

25. A lipid nanoparticle composition comprising the lipid nanoparticle of any one of claim 11 to 19.Attorney Docket No. 774757: MTST-875PC26. A lipid nanoparticle composition comprising the modified lipid nanoparticle of any one of claim 20 to 24.

27. The lipid nanoparticle composition of claim 25 or claim 26, further comprising a polynucleotide.

28. The lipid nanoparticle composition of claim 27, wherein the polynucleotide is selected from the group consisting of DNA, ssDNA, cDNA, RNA, mRNA, tRNA, rRNA, siRNA, sgRNA, and combinations thereof.

29. The lipid nanoparticle composition of claim 27 or claim 28, wherein the polynucleotide encodes a protein, peptide, or antibody.

30. The lipid nanoparticle composition of claim 27 or claim 28, wherein the polynucleotide encodes a therapeutic protein or a therapeutic peptide.

31. The lipid nanoparticle composition of claim 27 or claim 28, wherein the polynucleotide encodes an engineered interleukin- 12 (eIL-12) comprising IL-12p40, IL-12p35, an MMP-cleavable linker, an IL-12Rpl (Q20-A261) domain, or a FLAG tag.

32. The lipid nanoparticle composition of any one of claims 25 to 31, wherein the lipid nanoparticle composition is a pharmaceutical composition or a therapeutic composition.

33. The lipid nanoparticle composition of any one of claims 25 to 32, wherein the lipid nanoparticle composition is configured to cross the blood brain barrier (BBB).

34. A method of delivering a polynucleotide to cells in a subject in need thereof, the method comprising administering the lipid nanoparticle composition of any one of claims 27 to 33 to the subject.

35. The method of claim 34, wherein the cells are brain cells.

36. The method of claim 35, wherein the cells selected from the group consisting of neurons, astrocytes, microglia, or brain endothelial cells, or a combination thereof.

37. The method of any one of claims 34 to 36, wherein the administration of the lipid nanoparticle composition results in at least a 9-fold increase of delivery of polynucleotide to cells as compared to a control.Attorney Docket No. 774757: MTST-875PC38. A method of treating or preventing cancer in a subject in need thereof, the method comprising administering the lipid nanoparticle composition of any one of claims 25 to 33 to the subject.

39. A method of treating or preventing a central nervous system (CNS) disease or disorder the method comprising administering the lipid nanoparticle composition of any one of claims 25 to 33 to the subject.

40. The method of claim 39, wherein the CNS disease or disorder is selected from the group consisting of glioblastoma, polyneuropathy, anaplastic astrocytoma, medulloblastoma, oligodendroglioma, primary CNS lymphoma, and metastatic brain tumors.

41. The method of any one of claims 38 to 40, wherein administration of the lipid nanoparticle composition suppresses tumor growth, increases survival of the subject, increases polynucleotide delivery to cells, and / or increases cellular translation.

42. The method of any one of claims 34 to 41, wherein the polynucleotide encodes an engineered cytokine.

43. The method of claim 42, wherein the polynucleotide comprises eIL-12 mRNA.

44. The method of claim 43, wherein the engineered cytokine is engineered IL- 12.

45. The method of any one of claims 34 to 44, wherein the administration of the lipid nanoparticle composition is intravenous.

46. A method of making a modified lipid nanoparticle, comprising:(a) formulating a lipid nanoparticle containing the synthetic lipid according to any of claims 1 to 10, DOPE, cholesterol, and a PEG-lipid; and(b) reacting the lipid nanoparticle with a cysteine- containing CPP to yield a maleimide -thiol conjugate on the lipid nanoparticle surface.

47. A kit comprising:(a) a unit dose containing the lipid nanoparticle composition of claim 33; and(b) instructions for administration to deliver a polynucleotide across the BBB.

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