Lipid nanoparticle formulations and uses thereof

Abstract

The present disclosure relates to polynucleotide formulations containing lipid nanoparticles and methods of administration, particularly in vaccine formulations.

Description

LIPID NANOPARTICLE FORMULATIONS AND USES THEREOFCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Application No.63 / 533,026, entitled “LNP FORMULATION AND USES THEREOF,” filed on August 16, 2023, the content of which is incorporated herein in its entirety.BACKGROUND

[0002] The approval of lipid nanoparticles (LNPs)-mRNA vaccines for use against the SARS-CoV2 virus has increased interest in the use of LNPs to deliver mRNA vaccines. LNPs such as ALC-0315 and SM-102 LNPs have been applied to deliver mRNAs encoding viral antigens against the SARS-CoV-2 virus. These LNPs are typically comprised of four distinct lipid components: ionizable lipids, phospholipids, cholesterol, and polyethylene glycol lipids (PEG lipids). The main function of PEG lipids in these vaccines is to stabilize and lengthen LNP circulation time after administration by reducing protein binding interactions. However, PEG lipids can stimulate the production of anti-PEG antibodies in some patients receiving them, particularly after repeated exposure. The production of anti- PEG antibodies can reduce the LNP delivery efficiency through an accelerated blood clearance (ABC) phenomenon, which occurs when the LNPs interact with the anti-PEG antibodies. In addition, anti-PEG antibodies have been attributed to a hypersensitivity reaction, eliciting severe allergic reactions which can potentially lead to critical anaphylaxis.

[0003] The widespread use of mRNA vaccines (particularly against SARS-CoV2) has increased patient exposure to PEG and raised the risk of adverse reactions to this ingredient. There is a need therefore to replace PEG in LNPs that are used in mRNA vaccines.SUMMARY

[0004] The present disclosure provides specific embodiments in the figures and examples described below. A person of skill would understand that the elements and principles of the present disclosure may be implemented using techniques and methods that are not explicitly disclosed herein. The present disclosure is not limited to the exemplary techniques and methods illustrated in the drawings and described below.

[0005] The present disclosure addresses the prior deficiencies by replacing PEG with a polypeptoid of the amino acid sarcosine (N-methylated glycine). A panel of different Polysarciosine (pSar) lipid formulations were engineered and compared them with PEG lipids in ALC-0315 and SM-102 based LNP formulations, which are approved by the U.S. Food and Drug Administration (FDA). The inventors assessed each formulation for mRNA delivery in vitro and in vivo. The results revealed that SM-102 based LNPs formulated with pSar lipids delivered mRNA as efficiently as PEG formulations, and, surprisingly, ALC- 0315 based LNPs formulated with pSar lipids were more efficient than PEG lipid-based formulations in mRNA delivery. Meanwhile, the immunogenicity profiles of pSar versus PEG LNP formulations were comparable.

[0006] Some formulations provide a lipid nanoparticle comprising a polysarcosine lipid combined with a polynucleotide. In a formulation, the polynucleotide is a messenger RNA. In a formulation, the polysarcosine lipid is selected from the group consisting of DMG- pSar25; N-hexadecyl-pSar25; N-octadexyl-pSar25; and N-TETAMINE-pSar25. In a formulation, the composition is a vaccine. In a formulation, the composition is essentially free of PEGylated lipids.

[0010] Some embodiments provide methods for administering to a subject a composition comprising a lipid nanoparticle comprising a polysarcosine lipid combined with a polynucleotide. In an embodiment, the polynucleotide is a messenger RNA. In an embodiment, the polysarcosine lipid is selected from the group consisting of DMG-pSar25; N-hexadecyl-pSar25; N-octadexyl-pSar25; and N-TETAMINE-pSar25. In an embodiment, the composition is a vaccine. In an embodiment, the composition is essentially free of PEGylated lipids.

[0011] Some formulations provide a lipid nanoparticle (LNP) composition comprising: an ionizable lipid, a helper lipid, cholesterol, and a polysarcosine lipid. In a formulation, the polysarcosine lipid is selected from the group consisting of: DMG-pSar25; N-hexadecyl- pSar25; N-octadexyl-pSar25; and N-TETAMINE-pSar25. In a formulation, the LNP composition further comprises a polynucleotide. In a formulation the polynucleotide is an mRNA. In a formulation, the LNP composition further comprises a vaccine. In a formulation, the composition is essentially free of PEGylated lipids.

[0012] Some embodiments provide a method for administering to a subject a lipid nanoparticle (LNP) composition comprising: an ionizable lipid, a helper lipid, cholesterol, and a polysarcosine lipid. In an embodiment, the polysarcosine lipid is selected from the group consisting of: DMG-pSar25; N-hexadecyl-pSar25; N-octadexyl-pSar25; and N- TETAMINE-pSar25. In an embodiment, the LNP composition further comprises a polynucleotide. In an embodiment, the polynucleotide is an mRNA. In an embodiment, the LNP composition further comprises a vaccine. In an embodiment, the composition is essentially free of PEGylated lipids.BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Fig. 1 provides illustrations structures of ionizable lipids, DSPC, cholesterol, PEG lipids, and pSar lipids.

[0014] Fig. 2 provides a cytotoxicity assay of pSar or PEG LNPs in Hep3B and C2C12 cell lines: a) ALC-0315 based LNPs formulated with pSar or PEG lipids on Hep3B and C2C 12 cell lines; and b) SM-102 based LNPs formulated with pSar or PEG lipids on Hep3B and C2C12 cell lines. The cell viability was normalized to PBS treated cells. The data are presented as mean ± s.d.

[0015] Fig. 3 provides Particle properties of LNPs formulated with pSar lipids and mRNA delivery in cells: a) Size, PDI, encapsulation efficiency, and zeta potential of ALC- 0315 based LNPs; b) Size, PDI, encapsulation efficiency, and zeta potential of SM-102 based LNPs; c) ALC-0315 based LNPs relative luminescence intensity in C2C12 and Hep3B cells; and d) SM-102 based LNPs relative luminescence intensity in C2C12 and Hep3B cells. Data in a, b, c, and d are presented as mean ± s.d. and statistical significance was analyzed by two-tailed Student's t-test. *P < 0.05, **P < 0.01, ***P < 0.001, ****p < 0.0001.

[0016] Fig. 4 shows the results of a comparison in luminosity intensity of different lipid formulations: a) ALC-0159 PEG lipid was replaced with DMG-PEG2000 at the same molar ratios and formulated with FLuc mRNA; b) Size and PDI of ALC-0315 and SM-102 based LNPs formulated with varying length of pSar chains, 25, 35, and 45 with the same lipid tail; c) Relative luminescence intensity of ALC-0315 or SM-102 based LNPs formulated withthree different pSar chain length. Data in a, b, and c are presented as mean ± s.d. and statistical significance was analyzed by two-tailed Student’s t-test. *P < 0.05, **P < 0.01, ns, no significance.

[0017] Fig. 5 provides the results of in vivo administration of pSar and PEG LNPs: a) IVIS measurement of ALC-0315 based LNPs; b) IVIS measurement of SM-102 based LNPs; c) human EPO quantitation of ALC-0315 based LNPs in the blood and muscle; and d) human EPO quantitation of SM-102 based LNPs in the blood and muscle. All data are presented as mean ± s.d. and statistical significance was analyzed by two-tailed Student's t- test. *P < 0.05, **P < 0.01, ns, no significance.

[0018] Fig. 6 provides immunogenicity testing of different LNP formulations: a) Total flux measured on IVIS after intramuscular injection of ALC-0315 based LNPs formulated with either DMG-pSar25 or ALC-0159 PEG lipid; and b) IVIS images of mice injected with ALC-0315 or SM-102 based LNPs formulated with pSar or PEG lipids at 6 hours post intramuscular administration. Data in a are presented as mean ± s.d.

[0019] Fig. 7 provides systemic immunogenicity and safety' evaluation of different LNP formulations: a) Heatmap showing log fold change compared to PBS-treated mice for 32 different cytokines and chemokines; b) SM-102 based LNPs, comparison between DMG- pSar25 and DMG-PEG2000 LNPs; c) ALC-0315 based LNPs, comparison between DMG- pSar25 and ALC-0159 LNPs; d) Comparison of relative cytokine and chemokine expression of DMG-pSar25 formulated with SM-102 or ALC-0315; and e) ALT and AST quantitation. Data in b to e are presented as mean ± s.d. and statistical significance was analyzed by two- tailed Student's t-test. *P < 0.05, **P < 0.01, ***P < 0.001, ns, no significance.

[0020] Fig. 8 provides systemic immunogenicity and safety' evaluation of different LNP formulations after multiple injections: a) Heatmap of log fold change compared to PBS treated mice for 32 different cytokines and chemokines after a repeat administration of pSar or PEG formulated LNPs; b) Circulating total IgG levels after a repeat administration of pSar or PEG formulated LNPs or untreated mice; and c) Anti-PEG IgG detected through ELISA in plasma of mice after a repeat administration of pSar or PEG formulated LNPs. Data in b and c are presented as mean ± s.d. and statistical significance was analyzed by two-tailed Student’s t-test. *P < 0.05. ns. no significance.DETAILED DESCRIPTION

[0021] Unless otherwise defined, all other scientific and technical terms used in the description, figures and claims have their ordinary meaning as commonly understood by one of ordinary skill in the art. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirely. In case of conflict, the present specification, including definitions, will prevail. If two or more documents incorporated by reference include conflicting and / or inconsistent disclosure with respect to each other, then the document having the later effective date shall control. The materials, methods, and examples are illustrative only and not intended to be limiting.EXAMPLES

[0022] The present disclosure will be more fully understood by reference to the following examples. They should not, however, be construed as limiting the scope of the present disclosure. It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are included scope of this application.Example 1: Identification of pSar lipids for use with LNPs

[0023] The inventors chose a panel of pSar lipids, which are shown in Figure 1. Two distinct LNP groups were formed based on two ionizable lipids, SM-102 and ALC-0315. SM-102 based LNPs consist of SM-102, DSPC, cholesterol, and DMG-PEG2000. ALC- 0315 based LNPs are comprised of ALC-0315, DSPC, cholesterol, and ALC-0159 PEG lipid. The molar ratio and LNP composition were kept consistent with FDA-approved formulations, except for the replacement of the PEG lipid with a pSar lipid at an equivalent molar ratio. pSar lipids with a repeat of 25 sarcosine moieties were selected. These lengths were chosen because the resulting molecular weight of the pSar lipids is approximately 2000 g / mol, which is analogous to the molecular weight of DMG-PEG2000 and ALC-0159. The pSar lipids vary in the structure of the lipid chain that is linked to the pSar repeat chain (Fig. 1). C14-pSar25 (N-Tetradecyl pSar2s), CI6-pSar25 (N-Hexadecyl pSar2s), and C18- pSar25 (N-Octadecyl pSar25) are comprised of a single saturated lipid tail. Other pSar25 lipids have a double lipid tail chain. Of the double lipid tails, DOPE-pSar25 lipid has monounsaturated lipid tails.

[0024] Materials and Methods: SM-102, ALC-0315, and ALC-0159 PEG lipids were purchased from MedKoo Biosciences. Distearoylphosphatidylcholine (DSPC). and the various polysarcosine lipids were purchased from Avanti Lipids. Cholesterol was purchased from Sigma®. l,2-dimyristoyl-rac-glycero-3-methoxypolyethylene gly col-2000 (DMG- PEG2000) was purchased fromNof America. Hep3B and C2C12 cell lines were purchased from ATCC.

[0025] LNP-mRNA were prepared by rapid mixing of an ethanol solution containing lipids and a citrate solution containing mRNA via NanoAssemblr (Precision NanoSystems). The total flow rate was 12 mL / min at a ratio of 1:3 ethanol phase to aqueous phase. The lipid components, ionizable lipids, DSPC, cholesterol, and pSar lipids or PEG lipids were dissolved in ethanol. The lipid ratios and the mRNA to lipid mass ratio were kept consistent with published FDA-approved formulations for the respective ionizable lipids. For SM-102 based LNPs, the ionizable lipid to mRNA mass ratio was 11.03, with a molar ratio of 50: 10:38.5: 1.5. For ALC-0315 based LNPs. the ionizable lipid to mRNA mass ratio was 14.3, with a molar ratio of 46.3:9.4:42.7: 1.6. LNPs were dialyzed in 1 x PBS with Slide-A- Lyzer dialysis cassette and 0.22 pm fdtered with a PES membrane filter before administration in vivo.

[0026] Materials and Methods: The size, polydispersity index (PDI), and zeta potential were measured by a NanoZS Zetasizer® instrument (Malvern). The encapsulation efficiency was calculated by a Quant-iT™ RiboGreen assay. Briefly, LNPs were incubated with 1 x TE or 1 x TE + 1% Triton-X solution in a black 96-well plate at 37 °C for 15 min. The fluorescence intensity was measured on a BioTek® Cytation 5 plate reader at excitation wavelength 485 nm and emission wavelength 528 nm.Example 2: Characterization of LNP Properties

[0027] All LNPs were formed with a microfluidic instrument and dialyzed in 1 x PBS before characterization analyses. Upon preliminary evaluation, the hydrodynamic diameter of C14-pSar25 lipid formulated LNPs measured greater than 500 nm. The C14-pSar25 lipid was excluded from further investigations since its size is much larger than those of the LNP- PEG formulations. Figure 3a shows that ALC-0315 based LNPs with single-tail lipids form bigger particles with hydrodynamic diameters greater than 200 nm. Compared to the LNPs formulated with PEG lipid, which has a diameter of approximately 85 nm. the size doubleswhen the LNPs were formulated with either C16-pSar25 or C18-pSar25 (Figure 3a). The three other particles formulated with double lipid chained pSar lipids form smaller-sized particles. The sizes of LNPs formulated with TETAMINE-pSa^s, DMG-pSar25, and DOPE- pSar25 were about 150 nm, 100 nm, and 80 nm, respectively (Figure 3a). The measured encapsulation efficiency was from 80% to 90% across all the different pSar LNPs, except ALC-0315 based LNPs with DOPE-pSar25 (70%, Figure 3a). The zeta potential of ALC- 0315 based LNPs was mostly slightly negatively charged, except for TETAMINE- pSar25 (Figure 3a). For SM-102 based LNPs, the trends of sizes, PDI, and encapsulation efficiency were similar to those in ALC-0315 based LNPs (Figure 3b). The zeta potential of SM-102 based LNPs were all slightly positively charged (Figure 3b). Overall, various physicochemical properties of the LNPs formulated with pSar or PEG lipids were comparable, except for the C16-pSar25 and C18-pSar25 LNPs, which formed larger particles for both ionizable lipids.

[0028] ALC-0315 and SM-102 based LNPs were formulated to be assessed in vitro across two representative cell lines: C2C12 and Hep3B cells. C2C12 is a mouse myoblast cell line, which was chosen to represent intramuscular administration. Hep3B is a human liver epithelial carcinoma, which was chosen to represent the hepatic tropism of systemically administered LNPs. All LNPs were formulated with FLuc mRNA. Luminescence intensity was measured as a surrogate marker of mRNA delivery efficiency, which was normalized relative to the LNPs formulated with PEG lipid (Fig. 3c and d). Across both ionizable lipid-based particles, pSar formulated LNPs showed comparable cytotoxicity compared to PEG formulated LNPs (Figure 2). Furthermore, the inventors discovered that LNPs formulated with DMG-pSans, C16-pSar25. C18-pSar25. and TETAMINE-pSar25 exhibited enhanced mRNA delivery efficiency compared to the control PEG LNPs (Fig. 3 c and d).

[0029] For ALC-0315 based LNPs, replacing ALC-0159 PEG with DMG- PEG2000 resulted in a decreased trend of luminescence intensity across both cell lines (Figure 4a). Surprisingly, the replacement of ALC-0159 PEG with DMG-pSar25 enhanced the mRNA delivery' efficiency of ALC-0315 LNPs (Fig. 3c). This led to the conclusion that even when the same lipid structure is used, the pSar25-lipid promotes enhanced mRNA delivery efficiency compared to the PEG lipid. To further investigate the effect of the length of the pSar chain on LNP delivery efficiency, two different pSar-lipids, TET AMINE-pSar35 and TETAMINE-pSa s were included and compared with TETAMINE-pSar25. All three lipids have the same lipid tail structure, but the length of the pSar repeat is changed from 25, 35, and 45 pSar units. As shown in Figure 4b, SM-102 and ALC-0315 formulated with TETAMINE-pSar with different length exhibit similar particle size and PDI. The results revealed that increasing the chain length of the pSar moiety reduced the delivery7efficiency in both SM-102 and ALC-0315 based LNPs (Figure 4c).

[0030] The inventors also discovered that the lipid tail structure of pSar lipids plays a role in mRNA delivery7efficiency. For example, single lipid chained pSar lipids exhibited high mRNA delivery efficiency, especially in the ALC-0315 based LNPs. As exhibited in Figure 3c, ALC-0315 based LNPs formulated with C18-pSar25 lipid reached greater than a 4-fold and an 8-fold higher delivery efficiency in C2C12 and Hep3B cells, respectively. DOPE-pSar25 consistently showed low mRNA delivery7efficiency across both cell lines for both ionizable lipids. LNPs formulated with DOPE-pSar25 and ALC-0315 lipid had the lowest mRNA encapsulation efficiency (Figure 3a), which reveals the low delivery efficiency observed in vitro for ALC-0315 based LNPs formulated with DOPE-pSar25, but not for the SM-102 based LNPs. This discovery reveals that the ability of the LNPs to release the mRNA into the cytosol for translation plays a greater role than the encapsulation efficiency. The inventors determined, therefore, that the low delivery efficiency is attributed to the difference in the saturation state of the hydrocarbon lipid tails. Overall, in vitro results revealed that direct substitution of the PEG lipids with one pSar lipid is a viable option for LNP formulation and can improve delivery7efficiency.

[0031] Materials and Methods: The linear dsDNA of firefly luciferase (FLuc) and human EPO (hEPO) were obtained from Integrated DNA Technologies, which were cloned into our NASAR vector plasmid through HiFi assembly. All mRNAs were synthesized by in vitro transcription, consistent with previously published protocol. All mRNAs were synthesized with full Al-Methyl-pseudouridine substitution. The mRNAs were enzy matically capped with Vaccinia Capping Enzy me (New7England Biolabs) and mRNA Cap 2'-(9-Methyltransferase (New England Biolabs).

[0032] C2C12 cells were grown in DMEM with 10% FBS. Hep3B cells were grown inEMEM supplemented with 10% FBS. The cells were incubated at 37 °C in a 5% CO2 environment. Cells were seeded at a density of 2 x 104cells per w ell in a white 96-well plate, cultured overnight, and then treated with 50 ng FLuc mRNA-loaded LNPs per well.Luciferase substrate (Bright-Glo reagent, Promega) was added to each well after 18 h of incubation. After 5 min, the luminescence intensity was measured by a BioTek Cytation5 plate reader.Example 3:

[0033] In view of the positive discoveries from the in vitro analysis, the inventors selected four pSar lipids (DMG-pSar25, C16-pSar25, C18-pSar25, and TETAMINE-pSar2s) to compare with the control PEG LNPs in mice for delivery efficiency and immunogenicity'. ALC-0315 and SM-102 based LNPs were formulated with FLuc mRNA and were then injected intramuscularly into the flank of mice at an mRNA dose of 0.06 pg per injection. A time course evaluation of luminescence intensity in vivo showed that peak radiance is observed 6 h post intramuscular administration (Figure 6). Therefore, the delivery' efficiency of the different pSar or PEG formulated LNPs at 6 h post LNPs administration was evaluated. Confirming the in vitro results, the inventors discovered that ALC-0315 based pSar LNPs resulted in statistically higher luminescence intensity than the PEG formulated LNPs (Figure 5a, Figure 6b). LNPs formulated with C16-pSar25 and DMG-pSar25 generated greater than 5-fold higher total flux compared to PEG LNPs. The use of SM-102 based pSar LNPs. on the other hand, led to results that were comparable to the result for the PEG LNPs (Figure 5b, Figure 6b).

[0034] A closer comparison between pSar lipids and PEG lipids as vehicles for LNPs was pursued next. The inventors, therefore, focused on comparing DMG-pSar25 with PEG2000, based in part on their results from cellular data, LNPs characterization, and in vivo IVIS data. Since DMG-pSar25 has the same lipid backbone as DMG-PEG2000, and it shares similar 14-carbon lipid tails with ALC-0159, the inventors determined that the differential mRNA delivery efficiency of compositions using these lipids reflect the effects of the PEG or pSar chains, rather than the lipid backbone.

[0035] For this study, synthesized mRNA encoding for human erythropoietin (hEPO), a therapeutically relevant cargo, was synthesized. Six hours post intramuscular injection, the mice were euthanized, and the flank muscle and blood were collected for hEPO quantitation through ELISA. In concordance with the in vitro data, these studies revealed that ALC-0315 based LNPs formulated with DMG-pSar25 resulted in significantly higher levels of hEPO in the muscle compared to ALC-0159 PEG LNPs (Figure 5c), whichconfirms the enhanced mRNA delivery efficiency as measured through luminescence intensity. SM-102 based LNPs showed similar results in that pSar LNPs and PEG LNPs led to the comparable translation of the hEPO protein (Figure 5 d).

[0036] The results vary slightly between the different ionizable lipids. For ALC-0315 based LNPs, replacement of the PEG lipid with DMG-pSar25 lipid increases the expression of the encoded mRNA. thereby indicating enhanced delivery efficiency. The statistically significant higher expression across both mRNAs supports this finding. By comparison, SM-102 based LNPs did not show a statistically significant increase in mRNA delivery efficiency between DMG-pSa s and DMG-PEG2000 LNPs. These results indicate that DMG-pSar25 is superior to PEG lipids in certain LNP formulations.

[0037] Materials and Methods: IVIS imaging — C57BL6 mice were used for IVIS imaging experiments. LNPs encapsulating FLuc mRNA were administered via intramuscular injection at a dose of 0.06 pg mRNA / kg / flank. 6 h after LNP administration, the mice were intraperitoneally injected with D-Luciferin substrate and were imaged on Biophotonic IVIS-Spectrum for total flux emission.

[0038] hEPO quantitation — Mouse flank muscles were isolated and frozen upon isolation. The tissue was homogenized with T-PER™ Tissue Protein Extraction Reagent (Thermo Scientific, 78510) with protease inhibitors (Thermo Scientific, 87785) at a ratio of 1 g of tissue to 10 mL of T-PER™ reagent. The sample was centrifuged at 10,000xg for 5 min. and the supernatant was collected for ELISA assay for hEPO quantitation (BioLegend, 442907). Mouse whole blood was collected 6 h post-injection in citrate-treated tubes. Blood was centrifuged at 1500xg for 10 min at 4 °C to obtain plasma for hEPO quantitation.Example 4:

[0039] The main functional role of LNPs is to mediate mRNA delivery for expression. However, LNPs can elicit immunogenic responses. This can affect the overall innate immune responses to the LNPs administration. Therefore, cytokine and chemokine induction profiles of pSar and PEG LNPs were evaluated. Blood from mice either injected with DMG-pSar25 or PEG LNPs were collected 6 h post-administration and analyzed for a broad panel cytokine and chemokine analysis with a Luminex Assay. The results revealed that expression profiles for the 32 different cytokines and chemokines were similarregardless of LNPs formulated with pSar or PEG lipids (Figure 7a). For instance, CXCL10, an interferon-y induced chemokine, increased approximately 5-fold for both pSar and PEG LNPs treated mice, indicating the activation of the innate immune system. Similarly, the upregulation of CXCL10 was also observed in patients administered with the BNT162b2 mRNA vaccine, which is a marker of active humoral immunity following vaccination. Additionally, the level of IL-6 increased dramatically, close to 10-fold, regardless of pSar or PEG LNPs. IL-6 is known to induce a robust T follicular helper dependent germinal center B cell response, important for effective antibody responses.

[0040] Some cytokines or chemokines showed differential expressions between PEG and pSar LNPs (Figure 7b and c). For example, SM-102 based LNPs formulated with DMG- pSar25 lipid show higher expression of cytokines or chemokines such as G-CSF, IL-6, and CXCL1 compared to the PEG counterpart (Figure 7b). Recent clinical findings indicate that the heightened levels of G-CSF and CXCL1 may stem from the adjuvant effect of a vaccine. Moreover, the upregulation of G-CSF, IL-6, and CXCL1 was observed in another study where mice were treated with an LNP-mRNA vaccine.

[0041] As previously noted, lipid components can trigger different immunogenic responses. Comparing SM-102 and ALC-0315 based LNPs both formulated with DMG- pSar25, some cytokines or chemokines were higher for SM-102 based LNPs whereas some levels w ere lower (Figure 7d). The difference in the upregulation of specific cytokines across SM-102 and ALC-0315 based LNPs revealed that the overall LNPs composition is important for eliciting different immunogenic responses.

[0042] To further compare the immunogenicity of pSar or PEG formulated LNPs, the inventors assessed whether a repeat administration of LNPs would elicit a heightened immune response by analyzing the systemic cytokine release profiles. Repeated injections elicited a pro-inflammatory pattern similar to the first-time administration (Figure 7a, Figure 8). The levels of total circulating IgG were slightly elevated in mice that received either pSar or PEG formulated LNPs compared to untreated mice but were statistically insignificant (Figure 8b).

[0043] Additionally, an ELISA was performed to detect anti-PEG mouse IgG after the second time administration. This revealed that pSar LNP treated mice did not induce the production of antibodies against PEG, whereas PEG LNP injected mice showed aheightened O.D. signal, indicating the presence of anti-PEG antibodies (Figure 8c). This further revealed that administration of pSar LNPs will not react with existing anti-PEG antibodies. This ELISA study is similar to methods reported previously that assessed the production of antibodies against PEG in a liposome format, which found that pSar formulated liposomes induced lower levels of antibodies than PEG formulated liposomes. Taken together, these results show that the replacement of PEG lipids with a pSar lipid in current FDA-approved LNP formulations based on ALC-0315 and SM-102 can achieve functional mRNA delivery while evading concerns regarding existing anti-PEG antibodies.

[0044] Lastly, hepatic toxicity was assessed by quantifying the levels of aspartate transaminase (AST) and alanine transaminase (ALT) in the plasma. The levels of AST and ALT were quantified from mouse plasma obtained 6 h after intramuscular injection of either LNPs containing pSar lipid or PEG lipid. Compared to the PBS-treated mouse, administration of both ALC-0315 or SM-102 based LNPs formulated with pSar or PEG lipids showed no significant increase in both enzymes, revealing that the liver function was not compromised after the single administration (Figure 7e).

[0045] As previously noted, lipid components can trigger different immunogenic responses. When comparing SM-102 and ALC-0315 based LNPs. both formulated with DMG-pSar25. some cytokines or chemokines were higher for SM-102 based LNPs whereas some levels were lower (Figure 7d). The difference in the upregulation of specific cytokines across SM-102 and ALC-0315 based LNPs revealed that the overall LNPs composition is critical for eliciting different immunogenic responses. This reflects the significance of ionizable lipids in the overall immunogenicity of LNPs.

[0046] Materials and Methods: Luminex analysis for cytokines and chemokines — Mouse whole blood was collected 6 h post-injection in citrate-treated tubes. Blood was centrifuged at 1500xg for 10 min at 4 °C to obtain the plasma. The plasma was diluted 1: 1 in 1 x PBS and stored at -80 °C. Mouse cytokines and chemokines were detected by mouse cytokine / chemokine discovery assay (Eve Technologies).

[0047] ELISA for anti-PEG IgG quantitation — A 96-well plate (Thermo Scientific, 456529) was coated with PEG lipid at a coating densify of 2.5 pg / well overnight at 4 °C. The plate was washed 3 times with 1 x PBS, then blocked with 5% BSA in 1 x PBS for 4 h at room temperature, followed by 3 times washing with 1 x PBS. Then, 50 x diluted plasmawas added to each well, and incubated at 37 °C for 2 h. The plate was washed with 1 x PBS +0.05% Tween-20 three times, then incubated with HRP conjugated goat anti-mouse IgG antibody (Abeam, ab7068) for 1 h at 37 °C. The plate was washed with 1 x PBS +0.05% Tween-20 three times, then freshly prepared OPT substrate was added (Sigma-Aldrich, P4664). The reaction was stopped with 3 M Sulfuric Acid and read at 492 nm on a plate reader.

[0048] All mouse studies were approved by the Institutional Animal Care and Use Committee at The Icahn School of Medicine at Mount Sinai (IPROT0202200000134) and complied with local, state, and federal regulations. C57BL / 6J mice were ordered from Jackson Laboratories (Strain #000664).

Claims

CLAIMSWhat is claimed is:

1. A lipid nanoparticle composition, wherein a lipid component of the composition comprises a polysarcosine lipid.

2. The composition of claim 1, wherein the polysarcosine lipid is selected from the group consisting of: DMG-pSar25; N-hexadecyl-pSar25; N-octadexyl-pSar25; and N- TETAMINE-pSar25.

3. A vaccine comprising the lipid nanoparticle composition of claim 1.

4. The composition of claim 3, wherein the composition is essentially free of PEGylated lipids.

5. A lipid nanoparticle (LNP) composition comprising: an ionizable lipid, a helper lipid, cholesterol, and a polysarcosine lipid.

6. The LNP composition of claim 5. wherein the polysarcosine lipid is selected from the group consisting of: DMG-pSar25; N-hexadecyl-pSar25; N-octadexyl-pSar25; and N- TETAMINE-pSar25.

7. A vaccine composition comprising the LNP composition of claim 5.

8. The LNP composition of claim 5. wherein the composition is essentially free of PEGylated lipids.

9. A method of administering a polynucleotide using the LNP composition of claim 5.

10. The method of claim 9, wherein the poly sarcosine lipid is selected from the group consisting of: DMG-pSar25; N-hexadecyl-pSar25; N-octadexyl-pSar25; and N- TETAMINE-pSar25.

11. The method of claim 10, wherein the polynucleotide is an mRNA.

12. The method of claim 10, wherein the composition comprises a vaccine.

13. The method of claim 10, wherein the composition is essentially free of PEGylated lipids.

14. A method of administering a polynucleotide to a subject using a lipid nanoparticle composition comprising a polysarcosine lipid.

15. The method of claim 14, wherein the polynucleotide is a messenger RNA.

16. The method of claim 15, wherein the polysarcosine lipid is selected from the group consisting of DMG-pSar25; N-hexadecyl-pSar25; N-octadexyl-pSar25; and N- TETAMINE-pSar25.

17. The method of claim 15 comprising a vaccine.

18. The method of claim 17, wherein the vaccine is essentially free of PEGylated lipids.

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