Anionic lipid nanoparticles for nucleic acid delivery

Abstract

The disclosure provides novel endosomal escape sequences that can be attached, e.g., covalently, to the outer surface of anionic lipid particles (aLNP) capable of encapsulating therapeutic payloads, e.g., nucleic acids. Nucleic acid payloads are embedded in the hydrophobic core of the lipid bilayers of the aLNPs, and additional payloads can be carried in the aqueous lumen of the aLNP, the internal (inner) surface of the bilayers, the external (outer) surface of the bilayers, the bilayer's hydrophobic core, or any combination thereof. The disclosed aLNPs are negatively charged and do not contain ionizable cationic lipids. The disclosure also provides methods to manufacture the aLNP, pharmaceutical compositions, kits, and methods of treatment comprising the administration of the aLNP to a subject in need thereof, for example, a subject having cancer.

Description

ANIONIC LIPID NANOPARTICLES FOR NUCLEIC ACID DELIVERYCROSS-REFERENCE TO RELATED APPLICATIONS AND INCORPORATION BY REFERENCE

[0001] This PCT application claims the priority benefit of U.S. Provisional Application No. 63 / 661,435, filed on June 18, 2024, which is herein incorporated by reference in its entirety.REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY

[0002] The content of the electronically ST.26 sequence listing in XML format (Name 5512_002PC01_SequenceListing_ST26.xml; Size: 1,178,358 bytes; and Date of Creation: June 18, 2025) filed with the application is hereby incorporated by reference in its entirety.FIELD

[0003] The present disclosure relates to stable nanosized anionic lipid nanoparticles with enhanced endosomal escape functionality for delivery of nucleic acids and other therapeutic and diagnostic payloads.BACKGROUND

[0004] Nucleic-acid-based therapies hold promise for the treatment of various diseases. However, their physicochemical properties, pharmacokinetics and toxicology profiles have limited their therapeutic applications. This is because nucleic acids are hydrophilic highly negatively charged molecules with poor cell entry capability. When administered intravenously, they have a short half-life and are rapidly degraded by blood nucleases. These unfavorable aspects often force the use of high doses that can induce non-specific and even toxic effects, especially under systemic administration (Damase et al., Frontiers in Bioengineering and Biotechnology 9:629137, 2021).

[0005] To overcome the challenges of the use of nucleic acids, non-viral and viral gene delivery vectors have been developed (Sung and Kim, Biomaterials Research 23:8, 2019). After administration, vectors are taken up by the target cells via the endocytic pathway, resulting in the entrapment of the vectors in endocytic vesicles. These endocytic vesicles then fuse with early endosomes in which the vectors can start to release their nucleic acid content. The early endosomesmature to late endosomes, which then transform to lysosomes as final stage. During this maturation process, the luminal pH decreases steadily reaching an acidic pH of about 4.5-5 in the lysosomes. In addition, endosomal ion concentrations (e.g. calcium ions) change strongly during the maturation from early endosomes to lysosomes. The lysosomal lumen is a very harsh and degradative environment containing many enzymes such as nucleases, lipases, and proteases.

[0006] Viral gene delivery vectors, such as adenovirus, herpesvirus, retrovirus, or lentivirus, have been extensively utilized in clinical trials and are approved as gene therapy products. These vectors utilize specific proteins that enhance the fusion of viral particles with the endosomal membrane to enable the endosomal escape of nucleic acids. Despite their utility, viral gene delivery vectors present several limitations. The manufacturing process is complex, involving cell culture systems that necessitate stringent biosafety requirements. Achieving high viral titers is challenging, and there is a risk of random genomic integration. Moreover, these vectors are inherently immunogenic, and repeated administration can provoke an immune response. Additionally, the use of viral gene delivery vectors is associated with immunogenicity against viral proteins complicates the re-dosing of viral vector systems, posing significant constraints (Lundstrom, Viruses 15: 698, 2023).

[0007] Non-viral systems for the delivery of nucleic acids are generally based on the use of cationic molecules interacting with the negatively charged phosphate groups of the nucleic acids, allowing their encapsulation and forming nano or microparticles. Cationic liposomes and cationic polymers have been widely used for in vitro transfection of cells, successfully protecting the nucleic acids from nuclease degradation and enhancing cellular uptake (Ren, Polymer 13:3307, 2021). Some of these delivery systems are highly toxic in vivo and not physically stable in plasma, limiting their application for systemic administration of nucleic acids.

[0008] Lipid nanoparticles (LNPs) have been extensively used for systemic administration of nucleic acids. LNPs for therapeutic nucleic acid delivery comprise ionizable cationic lipids, which are positively charged at acidic pH (<6.5) but not at physiological pH (7.4). Including ionizable cationic lipids in the LNPs results in improved blood serum stability, efficient nucleic acid encapsulation, efficient intracellular nucleic acid release, and endosomal escape of the nucleic acid. However, the presence of ionizable cationic lipids negatively impacts nucleic acid stability (Packer et al, Nat Commun 12:6777, 2021) and can induce dose-limiting inflammatory responses (Ndeupen et al, iScience. 24(12): 10347, 2021; Tahtinen et al, Nat Immunol 23:532, 2022) due to the activation of the intracellular inflammasomes.

[0009] The particle size of the nucleic acid delivery system is one of the most critical aspects influencing both biological performance and manufacturability. To ensure effective delivery, lipid nanoparticles (LNPs) must navigate size-dependent physiological barriers and clearance mechanisms upon administration. Nanoparticles smaller than 30 nm are rapidly eliminated by renal filtration, while those larger than 50 nm are increasingly cleared by the reticuloendothelial system (RES), particularly by the liver and spleen. Particles exceeding 200 nm are prone to splenic accumulation and enhanced opsonization, leading to phagocytosis by immune cells (Baek et al., ACS nano, 79(15), 14605-14626, 2025). Optimizing particle size between 50 and 200 nm supports systemic circulation, enhances uptake by target cells, and reduces off-target clearance. For example, LNPs around 80 nm show improved hepatic gene silencing due to efficient passage through liver sinusoidal fenestrations and preferential interaction with hepatocytes over Kupffer cells Chen et al., Journal of Controlled Release, 235, 236-244, 2016). Particles <100 nm also accumulate preferentially in tumors and inflamed tissues via the enhanced permeability and retention (EPR) effect and more readily enter circulation after intramuscular or subcutaneous injection (Islam et al., Expert Opinion on Drug Delivery, 19(2), 199-212, 2022). Industrial manufacturing constraints further the requirement of particle sizes below 200 nm, as terminal sterilization method is required for parenteral administration (e.g. intravenous or subcutaneous). Terminal sterilization methods such as ionizing radiation or heat are unsuitable for nucleic acid- loaded LNPs due to oligonucleotide degradation and lipid instability Mehta et al., ACS Materials Au, 3(6), 600-619, 2023; DeCollibus et al., Nucleic acid therapeutics, 33(3), 159-177, 2023). As a result, sterile filtration through 220 nm membranes is the industry standard, though efficiency decreases significantly with particles near the pore size. Larger particles reduce filter recovery and throughput, particularly at higher concentrations and viscosities (Taylor et al., Journal of Membrane Science, 635, 119436, 2021; Taylor et al., Journal of Membrane Science, 647, 120264, 2022; Wu, et al., International Journal of Pharmaceutics, 675, 125520, 2025). Accordingly, producing nucleic acids delivery system with a size between 50 and 200 nm is essential for achieving effective nucleic acid delivery while ensuring scalable and robust manufacturing process.BRIEF SUMMARY

[0010] The present disclosure provides an endosomal escape peptide between 15 and 50 amino acids in length comprising at least two amino acid subsequences selected from the group consistingof EALAHH (SEQ ID NO: 1336), DALAHH (SEQ ID NO: 1337), EALAHW (SEQ ID NO: 1338), DALAHY (SEQ ID NO: 1339), DALAHG (SEQ ID NO: 1340), and DALAHW (SEQ ID NO: 1341), wherein one optional non-polar amino acid can be intercalated between the subsequences, and wherein one tryptophan is located at the N-terminus and / or C-terminus of the endosomal escape peptide. In some aspects, the endosomal escape peptide comprises the EALAHH (SEQ ID NO: 1336) subsequence and is selected from the group consisting of SEQ ID NOS: 1038,1039, 1040, 1041, 1310, 1311, 1312, 1313, 1314, 1319, 1328, 1329, 1330, 1331, 1332, 1333, and 1334. In some aspects, the endosomal escape peptide comprises the DALAHH (SEQ ID NO: 1337) subsequence and is selected from the group consisting of SEQ ID NOS: 1311, 1328, 91329, 1330, and 1331. In some aspects, the endosomal escape peptide comprises the EALAHW (SEQ ID NO: 1338) subsequence and is selected from the group consisting of SEQ ID NOS: 1038, 1039,1040, 1041, 1310, 1311, 1312, 1313, 1314, 1319, 1328, 1329, 1330, 1331, 1332, 1333, and 1334. In some aspects, the endosomal escape peptide comprises the DALAHY (SEQ ID NO: 1339) subsequence and has the amino acid sequence set forth in SEQ ID NO: 1329. In some aspects, the endosomal escape peptide comprises the DALAHG (SEQ ID NO: 1340) subsequence and has the amino acid sequence set forth in SEQ ID NO: 1328. In some aspects, the endosomal escape peptide comprises the DALAHW (SEQ ID NO: 1341) subsequence and has the amino acid sequence set forth in of SEQ ID NO: 1330.

[0011] In some aspects, the one optional non-polar amino acid intercalated between the subsequences is selected from the group consisting of alanine, valine, leucine, isoleucine, methionine, and cysteine. In some aspects, more than one non-polar amino acid can be intercalated between the subsequences disclosed herein. In some aspects, two non-polar amino acids are intercalated. In some aspects, three non-polar amino acids are intercalated. In some aspects, four non-polar amino acids are intercalated. In some aspects, the endosomal escape peptide further comprises an N-terminal or C-terminal cysteine.

[0012] In some aspects, the endosomal escape peptide is conjugated to a lipid, thereby yielding a lipopeptide. In some aspects, the endosomal escape peptide is conjugated to the lipid via a click chemistry reaction. In some aspects, the endosomal escape peptide is conjugated to the lipid via a maleimide moiety. In some aspects, the endosomal escape peptide is conjugated to the lipid via an amide linkage, via an ester linkage, via a hemisuccinate linker, through an amide-amide bond, or via a keto linker such as a disulfide or hydrazine linkage.

[0013] In some aspects, the lipid is selected from the group consisting of stearic acid, palmitic acid, oleic acid, cholesterol, sitosterol, DSPE, DSPE-PEG, DPPE, DOPE, a monoacylglycerol, adiacylglycerol (e.g., myristoyl diglyceride, palmitoyl diglyceride and stearyl diglyceride), phosphatidylcholine, phosphatidylglycerol, a ceramide, a sphingolipids, and any combination thereof.

[0014] The present disclosure also provides an anionic lipid nanoparticle (aLNP) comprising a fusogenic lipid and endosomal escape peptide wherein the aLNP has net negative charge at neutral pH. In some aspects, the endosomal escape peptide is inserted in the lipid bilayer. In some aspects, the fusogenic lipid is DOPE. In some aspects, the fusogenic lipid is selected from the group consisting of phosphatidylethanolamines, phosphatidic acids, phosphatidylserines, monoacylglycerols, diphosphatidylglycerol (e.g., cardiolipin, DPG), diacylglycerols, ceramides, glycolipids, such as monogalactosyldiacylglycerol (MGDG), lysophospholipids, and unsaturated fatty acids. In some aspects, the molar ratio of fusogenic lipid is between about 5 mol% and about 60 mol%. In some aspects, the aLNP further comprises at least one bilayer-forming lipid. In some aspects, the at least one bilayer forming lipid comprises a neutral lipid, an anionic lipid, or a combination thereof. In some aspects, the neutral lipid is selected from the group consisting of DSPC, DPPC, SM, DMPC, DPPE, DSPE, DMPE, and POPC. In some aspects, the anionic lipid is selected from the group consisting of DSPG, DPPG, DOPG, POPG, DMPG, DSP A, DPP A, DOPA, POP A, DMPA, DSPS, DPPS, DOPS, POPS, a ganglioside, and DMPS. In some aspects, the molar ratio of the at least one bilayer forming lipid is between about 5 mol% and about 55 mol%. In some aspects, the molar ratio of the at least one bilayer forming lipid is between is between about 5 mol% and about 20 mol%. In some aspects, the molar ratio of the at least one bilayer forming lipid is between is about 20 mol% and about 55 mol%.

[0015] In some aspects, the aLNP further comprises a sterol. In some aspects, the sterol is sitosterol or cholesterol. In some aspects, the molar ratio of sterol is between about 20 mol% and about 50 mol%. In some aspects, the aLNP has a formulation shown in FIG. 16, 17, 18, 20, 21, 23, 24 or 25. In some aspects, the aLNP has (i) an average hydrodynamic diameter of about 50 nm to about 250 nm as determined by dynamic light scattering (DLS); (ii) a poly dispersity index (PDI) of about 0.01 to about 0.3 as determined by dynamic light scattering (DLS); (iii) a zeta potential of between about -100 and about -10 mV as determined by electrophoretic light scattering (ELS); or, (iv) a combination thereof. In some aspects, the aLNP has an average hydrodynamic diameter above about 30 nm and below about 200 nm as determined by dynamic light scattering (DLS).

[0016] The present disclosure also provides an aLNP comprising (i) at least one fusogenic lipid; (ii) a sterol; (iii) at least one non-fusogenic neutral or anionic lipid; and, (iv) an endosomal escape peptide between 15 and 50 amino acids in length comprising at least two amino acid subsequencesselected from the group consisting of EALAHH (SEQ ID NO: 1336), DALAHH (SEQ ID NO: 1337), EALAHW (SEQ ID NO: 1338), DALAHY (SEQ ID NO: 1339), DALAHG (SEQ ID NO: 1340), and DALAHW (SEQ ID NO: 1341), wherein the aLNP has net negative charge at neutral pH. In some aspects, the endosomal escape peptide is inserted in the lipid bilayer.

[0017] In some aspects, (i) the fusogenic lipid is selected from the group consisting of phosphatidylethanolamines, phosphatidic acids, phosphatidylserines, monoacylglycerols, glycolipids, such as monogalactosyldiacylglycerol (MGDG); lysophospholipids, and unsaturated fatty acids; (ii) the sterol is selected from the group consisting of cholesterol, stigmasterol or sitosterol; and (iii) the non-fusogenic neutral or anionic lipid is selected from the group consisting of EPC, DSPC, DPPC, POPC, DMPC, EPG, DSPG, DPPG, POPG, DMPG, DSP A, DPP A, POP A,DMPA, DSPS, DPPS, POPS, DMPS, sphingomyelin or a ganglioside.

[0018] In some aspects, the aLNP formulation is selected from the group consisting of DOPE:DSPC:DSPG:Sitosterol:EPP, DOPE:DSPC:DSPG:Cholesterol:EPP, DOPE:SM:DSPG:Sitosterol:EPP, DOPE:SM:DSPG:Cholesterol:EPP, DOPE :D SPC :DPPG: Sitosterol :EPP, DOPE:DSPC:DPPG:Cholesterol:EPP, DOPE: SM:DPPG: Sitosterol :EPP, DOPE:SM:DPPG:Cholesterol:EPP DOPE:SM:EEP, DOPE :DPPC :DPPG: Sitosterol :EPP, DOPE:DPPC:DPPG: Cholesterol :EPP, DOPE:DPPC:DSPG:Sitosterol:EPP, DOPE:DPPC:DSPG:Cholesterol:EPP, DOPE: SM:GM3 : Sitosterol :EPP, DOPE: SM:GM3: Cholesterol :EPP DOPE:SM, DOPE:DSPC:GM3:Sitosterol:EPP, DOPE:DSPC:GM3:Cholesterol:EPP, DOPE:DPPC :GM3 : Sitosterol :EPP, DOPE:DPPC:GM3:Cholesterol:EPP, DOPE :D SPC :D SPG: GM3 : Sitosterol :EPP, DOPE:DSPC:DSPG:GM3:Cholesterol:EPP, DOPE:SM:DSPG:GM3:Sitosterol:EPP, DOPE:SM:DSPG:GM3:Cholesterol:EPP, DOPE :D SPC :DPPG: GM3 : Sitosterol :EPP, DOPE:DSPC:DPPG:GM3:Cholesterol:EPP, DOPE:SM:DPPG:GM3:Sitosterol:EPP, DOPE:SM:DPPG:GM3:Cholesterol:EPP, DOPE:POPC:POPG: Sitosterol :EPP, DOPE:POPC:POPG: Cholesterol :EPP,DOPE: SM:DSPG: Sitosterol :EPP, and DOPE:SM:DSPG:Cholesterol:EPP, wherein EEP is an endosomal escape peptide.

[0019] In some aspects, the aLNP formulation is selected from the group consisting of the lipid formulations of the aLNP disclosed in FIGS. 16, 17, 18, 20, 21,23, 24, 25, and 26.

[0020] In some aspects, the aLNP comprises a payload. In some aspects, the payload is selected from the group consisting of a nucleic acid, a protein, a small molecule, a diagnostic reagent, or a combination thereof. In some aspects, the nucleic acid payload comprises a DNA, RNA, DNAanalogue, RNA analogue, or a combination thereof. In some aspects, the RNA comprises an mRNA, an ASO, a shRNA, a siRNA, a miRNA, or a combination thereof. In some aspects, the payload is an anti-neoplastic agent selected from the group consisting of (i) a cell-based anti- neoplastic agent; (ii) a lymphocyte-based anti -neoplastic agent selected from the group consisting of B cells, aPT cells, yST cells, NK cells, NKT cells, autologous tumor-infiltrating lymphocytes (TILs), autologous NK cells, CAR-T cells, CAR-B cells, CAR-NK cells, CAR-NKT cells, and any combination thereof; (iii) a myeloid-based anti -neoplastic agent selected from the group consisting of a dendritic cell-based anti -neoplastic agent, a macrophage-based anti-neoplastic agent, a neutrophil based anti-neoplastic agent, or any combination thereof; (iv) an antibody elected from the group consisting of an anti-CTLA4 antibody, an anti-PDl antibody, an anti-PD-Ll antibody, and any combination thereof; (v) an immune checkpoint inhibitor; (vi) a small molecule drug selected from the group consisting of an alkylating agent, an antibiotic, an anti-metabolite, a hormonal antagonist, a photosensitizer, a protein kinase inhibitor, a poly (ADP -ribose) polymerase inhibitor, a taxane, a topoisomerase inhibitor, and any combination thereof; (vii) a radiation therapy; (viii) a cytokine selected from the group consisting of IL-2, , IL-12, IL-15, IL-21, and any combination thereof; (ix) growth factor selected from the CSF family, Flt3L, and any combination thereof; (x) a steroidal or a non-steroidal anti-inflammatory drug; and, (xi) any combination thereof.

[0021] The present disclosure also provides a pharmaceutical composition comprising an aLNP of the present disclosure, i.e., an aLNP comprising (i) at least one fusogenic lipid; (ii) a sterol; (iii) at least one non-fusogenic neutral or anionic lipid; and, (iv) an endosomal escape peptide of the present disclosure, and a pharmaceutically acceptable excipient. Also provided is a method of treating a disease or condition in a subject in need thereof comprising administering an aLNP of the present disclosure, or a pharmaceutical composition of the present disclosure to the subject. In some aspects, the disease or condition is selected from the group consisting of cancer, infection, chronic inflammation, genetic disease, and autoimmune disease. Also provided is a diagnostic method comprising contacting a tissue sample of a subject with an aLNP of the present disclosure, or the pharmaceutical composition of the present disclosure, wherein the aLNP comprises a detectable moiety. Also provided is a diagnostic system comprising an aLNP of the present disclosure or the pharmaceutical composition of the present disclosure, wherein the aLNP comprises a detectable moiety.

[0022] The present disclosure provides a composition comprising an anionic lipid nanoparticle (aLNP), wherein the aLNP comprises (i) an anionic lipid bilayer surrounding a core, that in certaincases is an aqueous core, wherein the lipid bilayer comprises at least one anionic lipid and / or at least one neutral lipid; and, (ii) a payload comprising a nucleic acid; and, optionally, (iii) at least one endosomal escape peptide attached to the surface of the aLNP, wherein the endosomal escape peptide is selected from the group consisting of GALA, a GALA functional variant or functional fragment thereof, an endosomal escape peptide selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 1118 or SEQ ID NO: 1310 to SEQ ID NO: 1335, and a combination thereof; and, wherein at least part of the nucleic acid is embedded in the hydrophobic core of the lipid bilayer.

[0023] In some aspects, at least 20 wt.% of the nucleic acid is embedded in the hydrophobic core of the lipid bilayer. In some aspects, less than 5 wt.% of the nucleic acid is exposed on the outside of the aLNP. In some aspects, the aLNP comprises less than 20 mol.% of cationic lipid, cationic polymer, multivalent cation, or a combination thereof relative to total mol lipid of the aLNP. In some aspects, the aLNP comprises at least 5 mol.% of sterol or a combination thereof relative to total mol lipid of the aLNP. In some aspects, the aLNP comprises at least about 0.5 mol%, at least about 5 mol%, or at least about 15 mol% of at least one anionic lipid, relative to total mol% lipid of the aLNP. In some aspects, the aLNP comprises at least about 1 mol%, at least about 5 mol%, or at least about 15 mol% of at least one neutral lipid, relative to total mol lipid of the aLNP.

[0024] In some aspects, the aqueous core comprises at least 50 vol% aqueous solvent, relative to total core volume. In some aspects, the aLNP does not contain cationic lipid and / or comprise less than about 1 mol% cationic lipid relative to total mol lipid of the aLNP. In some aspects, the aLNP does not contain cationic polymer and / or comprises less than about 1 mol% cationic polymer relative to total mol lipid of the aLNP. In some aspects, the aLNP does not contain multivalent cations and / or comprises at most 1 mol% multivalent cation relative to total mol lipid of the aLNP. In some aspects, the aLNP has an average hydrodynamic diameter between about 50 nm to about 300 nm as determined by dynamic light scattering (DLS). In some aspects, the aLNP has a poly dispersity index (PDI) between about 0.01 and 0.3 as determined by DLS. In some aspects, the aLNP has a zeta potential between about -100 mV and about -10 mV as determined by electrophoretic light scattering (ELS).

[0025] In some aspects, the nucleic acid payload comprises a DNA, RNA, DNA analogue, RNA analogue, or a combination thereof. In some aspects, the RNA is an mRNA. In some aspects, the nucleic acid payload comprises an immunologic adjuvant and / or a toll-like-receptor (TLR) agonist. In some aspects, the TLR agonist comprises a TLR3 agonist, a TLR7 / 8 agonist, a TLR9 agonist, or a combination thereof. In some aspects, the TLR3 agonist comprises poly (EC), poly (C:G), poly (A:U), or a combination thereof.

[0026] In some aspects, the aLNP comprises a neutral lipid, an anionic lipid, and a sterol. In some aspects, the aLNP comprises a phosphatidylcholine, phosphatidylglycerol and a sterol. In some aspects, the aLNP comprises EPC, EPG, and cholesterol. In some aspects, in particular for delivery of mRNA or therapeutic oligonucleotides (e.g., siRNA or ASO) to the cytosol, the aLNP comprises at least one bilayer forming lipid, e.g., DSPC, SM, DPPC, DSPG, DPPG, at least one fusogenic lipid, e.g. DOPE and at least one sterol, e.g sitosterol. In some aspects, the aLNP comprises between about 0.5 mol% and about 40 mol% of at least one anionic lipid, relative to total mol lipid of the aLNP, and wherein the aLNP is an immunostimulatory composition. In some aspects, the aLNP comprises between about 0.5 and about 80 mol% of at least one anionic lipid, relative to total mol lipid of the aLNP, and wherein (i) the aLNP composition is an immunosilent composition or (ii) the aLNP composition is non-immunogenic. In some aspects, the non- immunogenic aLNP composition does not result in activation of dendritic cells, activation of macrophages, activation of T cells, secretion of IFN-alpha, or a combination thereof.

[0027] In some aspects, the composition is in combination with an antigen, an immunologic adjuvant, a vaccine, or a combination thereof. In some aspects, the composition is in combination with an anti-neoplastic agent. In some aspects, the anti -neoplastic agent is (i) a cell-based anti- neoplastic agent; (ii) a lymphocyte-based anti-neoplastic agent; (iii) myeloid-based anti-neoplastic agent; (iv) an antibody; (v) an immune checkpoint inhibitor; (vi) a small molecule drug; (vii) radiation therapy; (viii) a cytokine preferably chosen from IL-2, IL- 12, IL- 15 and IL-21; (ix) growth factor preferably chosen from CSF family and Flt3L; and / or (x) a steroidal or a nonsteroidal anti-inflammatory drug.

[0028] In some aspects, the lymphocyte-based anti-neoplastic agent is selected from the group consisting of B cells, aPT cells, yST cells, NK cells, NKT cells, autologous tumor-infiltrating lymphocytes (TILs), autologous NK cells, CAR-T cells, CAR-B cells, CAR-NK cells, CAR-NKT cells, and any combination thereof. In some aspects, the myeloid-based anti -neoplastic agent is selected from the group consisting of a dendritic cell-based anti -neoplastic agent, a macrophagebased anti-neoplastic agent, a neutrophil-based anti-neoplastic agent, or any combination thereof. In some aspects, the immune checkpoint inhibitor is an antibody. In some aspects, the antibody is selected from the group consisting of an anti-CTLA4 antibody, an anti-PDl antibody, an anti-PD- L1 antibody, and any combination thereof. In some aspects, the small molecule drug is selected from the group consisting of an alkylating agent, an antibiotic, an anti-metabolite, a hormonal antagonist, a photosensitizer, a protein kinase inhibitor, a poly (ADP-ribose) polymerase inhibitor, a taxane, a topoisomerase inhibitor, and any combination thereof. In some aspects, the cytokine isselected from the group consisting of IL-2, IL-12, IL-15, IL-21, and any combination thereof. In some aspects, the growth factor is selected from the group consisting of a CSF family growth factor, a Flt3L growth factor, and any combination thereof.

[0029] The present disclosure also provides a pharmaceutical composition comprising a composition comprising an aLNP disclosed herein and a pharmaceutically acceptable excipient. In some aspects, the pharmaceutical composition is formulated for intravenous administration. In some aspects, the pharmaceutical composition is formulated for intravenous administration via infusion. In some aspects, the pharmaceutical composition is formulated for intravenous administration via bolus injection. In some aspects, the pharmaceutical composition is formulated for subcutaneous administration.

[0030] The present disclosure also provides a method to treat or prevent a disease or condition in a subject in need thereof comprising administering a composition comprising an aLNP disclosed herein or a pharmaceutical composition comprising an aLNP to the subject. In some aspects, the disease or condition is selected from the group consisting of cancer, infection (e.g., a viral infection), chronic inflammation, genetic disease, and autoimmune disease.

[0031] Also provided is a method for preparing an aLNP disclosed herein, wherein the method comprises the following steps: (a) providing an organic solution comprising at least one anionic lipid and / or at least one neutral lipid; (b) providing an aqueous solution comprising a nucleic acid payload; and, (c) combining the solution of (a) and the solution of (b) using laminar flow mixing or chaotic flow mixing, thereby producing the aLNP. In some aspects, the flow rate in the laminar flow mixing is between 24: 1 and 1 :10. In some aspects, the lipid concentration in the organic solution of step (a) is between about 0.1 mM and about 50 mM.

[0032] In some aspects, the organic solution of step (a) is an ethanol solution. In some aspects, the ethanol solution is at least about 80% ethanol (v / v), at least about 90% ethanol (v / v), at least about 97.5% ethanol (v / v), or at least about 99% ethanol (v / v). In some aspects, the nucleic acid concentration in the aqueous solution of step (b) is between about 10 pg / ml and about 5000 pg / ml. In some aspects, the organic solution of step (a) comprises at least about 20 mol% of at least one anionic and / or at least one neutral lipid, relative to total mol lipid in the organic solution. In some aspects, the organic solution of step (a) comprises less than about 10 mol% of at least one cationic lipid, relative to total mol lipid in the organic solution. In some aspects, the organic solution of step (a) comprises at least about 5 mol% of at least one sterol, relative to the total mol lipid in the organic solution. In some aspects, the organic solution of step (a) is free of cationic lipids orcomprises less than about 1 mol.% of at least one cationic lipid relative to total mol lipid in the organic solution.

[0033] In some aspects, the method does not comprise the use of a cationic lipid and / or a cationic polymer. In some aspects, the method is optimized for micro-scale aLNP production, wherein the micro-scale production results in the product of between about 1 pl and about 10 ml of aLNP in solution. In some aspects, the method is optimized for medium-scale aLNP production, wherein the medium-scale production results in the product of between 10 ml and 1,000 ml of aLNP in solution. In some aspects, the method is optimized for large-scale aLNP production, wherein the large-scale production results in the product of between 1,000 ml and 100,000 ml of aLNP in solution.

[0034] In some aspects, the method is implemented using a small-scale, medium-scale, or large- scale microfluidics or a jet impingement system. In some aspects, the method is implemented using microfluidic large-scale integration (mLSI). In some aspects, the mLSI or jet impingment method uses chaotic flow mixing.

[0035] The present disclosure also provides a diagnostic method comprising using a composition comprising an aLNP disclosed herein, or a pharmaceutical composition comprising an aLNP disclosed herein, wherein the aLNP comprises a detectable moiety. Also provided is a diagnostic system comprising an aLNP disclosed herein, or a pharmaceutical composition comprising an aLNP disclosed herein, wherein the aLNP comprises a detectable moiety.

[0036] Also provided are kits comprising the aLNP of the present disclosure. In some aspects, the kit comprises an aLNP disclosed herein, or a pharmaceutical composition comprising an aLNP disclosed herein, and instructions for use. In some aspects, the kit comprises a set of lipids for the manufacture of an aLNP according to the methods disclosed herein, a microfluidics chip or jet impingement system, and optionally instructions for use. In some aspects, the set of lipids comprises a plurality of anionic lipids, neutral lipids, sterols, and combinations thereof to optimize the formulation of the aLNP. In some aspects the kit comprises the endosomal escape peptides disclosed herein, and, optionally, instructions and / or reagents for conjugation of endosomal escape peptides to the aLNP of the present disclosure or incorporation of the endosomal escape lipopeptides disclosed herein to the aLNP of the present disclosure.

[0037] In some aspects, the endosomal escape peptide is inserted in the bilayer of the aLNP as a lipopeptide. In certain embodiments, the lipid moiety is covalently attached to the N-terminal amine of the peptide through an amide bond; suitable lipid moieties include saturated fatty acids (e.g., palmitic acid, C16:0, or stearic acid, C18:0), unsaturated fatty acids (e.g., oleic acid, Cl 8: 1),sterols such as cholesterol, or phosphatidylethanolamine derivatives such as DOPE or DPPE joined through a hemisuccinate spacer to form sequential amide bonds. In some aspects, the lipid moiety is instead conjugated to the C-terminal carboxyl group or to side-chain functionalities (e.g., serine, threonine, or tyrosine) via amide, ester, or ether linkages. In some aspects, cleavable linkers such as disulfides, hydrazones, or enzymatically degradable moieties are employed to enable environmentally responsive release. In some aspects, the endosomal escape peptide is attached to the outer surface of the bilayer of the aLNP via a click chemistry reaction. In some aspects, the click chemistry reaction is a thiol-maleimide, an amino-N-hydroxysuccinimide reaction or a strain- promoted azide-alkyne cycloaddition. In some aspects, the endosomal escape peptide is attached to the outer surface of the bilayer of the aLNP via a redox reaction. In some aspects, the redox reaction is the formation of a disulfide bond between a thiol group in the endosomal escape peptide and a thiol group on the outer surface of the bilayer of the aLNP. In some aspects the lipid moiety is phosphatidylethanolamine (e.g. DOPE, DSPE, or DPPE), a fatty acid (e.g. stearic acid, palmitic acid or oleic acid) or a sterol (e.g. cholesterol or sitosterol).

[0038] The present disclosure also provides a composition comprising an aLNP, wherein the aLNP comprises (i) an anionic lipid bilayer surrounding an aqueous core, wherein the lipid bilayer comprises (a) a fusogenic lipid; (b) an sterol; (c) a neutral lipid; and / or, (d) an anionic lipid; and, (ii) a payload comprising a nucleic acid selected from the group consisting of an mRNA, a gRNA, a siRNA, an antisense oligonucleotide, or a combination thereof; and, optionally, (iii) at least one endosomal escape peptide attached to the surface of the aLNP, wherein the endosomal escape peptide is selected from the group consisting of GALA, a GALA functional variant or functional fragment thereof, an endosomal escape peptide selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 1118 or SEQ ID NO: 1310 to SEQ ID NO: 1335, and a combination thereof; and, wherein at least part of the nucleic acid of (ii) is embedded in the hydrophobic core of the lipid bilayer.

[0039] In some aspects, the sterol is cholesterol, the fusogenic lipid is DOPE, the neutral lipid is a lipid forming a solid crystalline phase, and the anionic lipid is a ganglioside. In some aspects, the lipid forming a solid crystalline phase is sphingomyelin.

[0040] In some aspects, the sterol is sitosterol, the fusogenic lipid is DOPE, the neutral lipid is a lipid forming a solid crystalline phase, and the anionic lipid is a phosphatidylglycerol. In some aspects, the lipid forming a solid crystalline phase is a phosphatidylcholine.BRIEF DESCRIPTION OF THE DRAWINGS / FIGURES

[0041] FIG. 1 shows the set-up of microfluidics chips employed for RNA (poly (EC)) encapsulation in aLNP. In Design A (Panel A), lipids dissolved in ethanol flow in the central channel of a microfluidic chip while the RNA dissolved in RNase-free purified water flows in the external channels. A co-flowing mixing induced the formation of RNA containing aLNP. In Design B (Panel B), two chips were combined for a two-step encapsulation process. In Chip N° 1, RNA (poly (I:C)) dissolved in RNase-free purified water flowed in the central side of the first mixing channel while lipids dissolved in ethanol flowed in the external side of the first mixing channel. A co-flowing mixing occurred, and the resulting mixture entered Chip N°2. The flow rate of the additional water flows influenced aLNP characteristics. The five layers of flowing particles were mixed in a laminar flow, inducing the formation of RNA-containing aLNP. Later, samples were collected from an output tube and diluted with RNase-free purified water to an ethanol concentration of less that about 20%. Ethanol and free RNA were removed using a tangential or centrifugal filtration system.

[0042] FIG. 2 shows the application of a Design of Experiment (DoE) approach for the development of a microfluidic process for the encapsulation of RNA in aLNP using Design A or B. (I) Flow rates, lipid and RNA concentration in the solvents were the variables selected in the DoE’s. (II) Mean particle size, poly dispersity (Pdl), and RNA encapsulation efficiency of the RNA-containing aLNP were studied in this DoE. (Ill) A central composite design was selected as the experimental design for building a response surface model (RSM). (IV) The DoE was designed using appropriate software (DESIGN-EXPERT™). (V) The DoE was executed for Design A (n=30) and Design B (n=53), and the data obtained was analyzed, allowing the building of RSMs. (VI) Response surface models were used to predict the optimal process parameters. (VII) Five independent batches were produced for the model validation. The grey area is an indication of the predicted interval by the RSMs of mean size, PDI and encapsulation efficiency. Mean of three analytical replicates ± SEM.

[0043] FIG. 3 shows the impact of mixing geometry and flow rate for mean size (Panel A), poly dispersity (Panel B) and encapsulation efficiency (Panel C). 500 pg / mL of RNA dissolved in RNase free water were mixed with an organic solution containing lipids (5 mM) [EPC :EPG: Cholesterol (3: 1 :2 molar ratio)] at flow rate ratio 1 : 1 and 0.25, 0.75 or 1.5 ml / min total flow rate employing laminar and chaotic mixing microfluidics chips following the Design Bscheme. Errors bars represent the standard deviation calculated from analytical triplicates from one independent experiment.

[0044] FIG. 4 shows the physical characterization of encapsulated RNA and size stability of aLNP [EPC :EPG: Cholesterol (3: 1 :2)] containing RNA under incubation with human plasma. Encapsulated poly (EC) was extracted from anionic lipid nanoparticles (aLNP) by precipitation with isopropanol 100%. The clean pellet of RNA was resuspended in PBS and injected in a size exclusion chromatography (SEC) column (TSKGEL® G-DNA-PW) running in PBS (flow rate= 0.5 mL / min, 25°C). Additionally, free poly(I:C) and standard DNA ruler (Gene Ruler Ikb plus, Thermosci entific) samples were treated similarly and injected into the SEC column. A UV detector at X=260 nm was used for RNA detection (Panel A) and a multi-angle light scattering detector for molecular weight determination (Panel B). Molecular weight analysis confirmed that the poly (I: C) employed was a polymer with wide molecular weight distribution. Encapsulated poly (EC) had a similar retention time (Panel A) and molecular weight as compared to free poly (EC), indicating that microfluidics technology successfully encapsulated nucleic acids without changing the original molecular weight distribution. The stability of empty and RNA-containing aLNP in human plasma was studied using asymmetrical flow field-flow fractionation (AF4). aLNP (2 mM of lipids) were incubated with PBS or 20% human plasma at 37° C for 2 hours and then analyzed by AF4. The radius of gyration (Rg) and light scattering intensity of the particles were determined using MALS (Panel C), and protein corona formation was determined using a fluorescence detector (X«m=280 nm, Xex=340 nm) (Panel D). Empty and RNA-containing aLNP did not showed particle aggregation in human plasma, as required for intravenous (i.v.) injection. The aLNP protected encapsulated poly (I:C) from RNA degradation. Free and encapsulated poly (I:C) were incubated with or without RNase in reaction buffer or reaction buffer supplemented with human plasma (20% v / v) at 37° C for 2 hours. Later, samples were run on a 1.0% agarose gel (Panel E).

[0045] FIG. 5 shows TEM images of empty (Panel A) and RNA containing (Panel B) aLNP [EPC :EPG: Cholesterol (3: 1 :2)] showing the conservation of lipid bilayers after incorporating nucleic acids. The contrast of the aLNP changed after nucleic acids encapsulation. Uranyl salts stain phosphate groups of phospholipids and RNA molecules, and was used to increase particle contrast. 10 pL of diluted particle suspension (2 mM phospholipids) were placed in a previous glow discharged Formvar / Carbon coated copper grid and contrasted with uranyl oxalate (pH 7), and then contrasted-embedded in a mixture of 2% methyl cellulose / 4% uranyl acetate (pH 4). Later, images were taken using a Tecnai 12 TEM microscope.

[0046] FIG. 6 shows SANS data (symbols) for empty (open grey rectangles) and poly (LC) (open black circles) containing aLNP resuspended in 27%, 50%, 68% and 100% D2O water, with the solid lines corresponding to the best fit using the sphere core-shell model (Panel A), scattering length density (SLD) profiles as a function of the distance to the center of the lipid nanoparticles corresponding to the fits of the data in A (Panel B); general polarization measurements in a temperature interval from 10 to 80° C (0,1° C change per measurement) of empty aLNP, aLNP incubated with free poly (LC) and poly (LC) containing aLNP (Panel C); and differential scanning calorimetry measurements in a temperature interval from 10 to 100 C (0,5° C change per minute) of free poly (LC), empty aLNP, empty aLNP incubated with free poly (LC) and poly (LC) containing aLNP (Panel D). Empty and poly (I:C)-containing aLNP (Empty AL and pIC-aLNP) share a shell-core sphere structure (Panels A, and B). The shell (bilayer) of the aLNP was composed of lipids, while the core was aqueous. Poly (LC) containing aLNP have a higher scattering neutron density (SLD) in the shell than empty nanoparticles. Additionally, the scattering profile of the RNA-containing aLNP does not change at 68% D2O, the contrast match for soluble RNA. This indicates that the RNA is embedded in the aLNP bilayer rather than soluble in the aqueous core. Furthermore, the encapsulation of poly (LC) changes the fluidity properties of the bilayer of the aLNP at temperatures higher than 40 °C, an indication of the presence of RNA within the hydrophobic core of the bilayer (Panel C). Additionally, RNA-containing aLNP, but not empty aLNP have an exothermic event at 74 °C, indicating that poly (LC) crystallizes when it is encapsulated in the hydrophobic core of an aLNP bilayer.

[0047] FIG. 7 shows the effect of the lipid composition on the characteristics of aLNP containing RNA. RNA-aLNP formulations with different molar ratios of EPC, EPG and Cholesterol were prepared in a design mixture experiment (n=17) (F). Particle mean size (Panel A), PDI (Panel B), zeta potential (Panel C), RNA encapsulation efficiency (Panel D) and TLR3 activation (%) vs free poly (LC) by HEK 293T TLR3+cells (Panel E) were analyzed for each formulation. RSMs were built for each parameter. Indicated is mean ± SEM of analytical replicates (n=3).

[0048] FIG. 8 shows the effect of Design A microfluidics process parameters on the particle characteristics of aLNP containing poly (LC). RNA-aLNP formulations were prepared with different aqueous RNA phase flow rate, ethanolic lipid phase flow rate, lipid concentration, and RNA concentration in a response surface designed experiment (n=30) (Panel D). Particle mean size (Panel A), PDI (Panel B) and RNA encapsulation efficiency (Panel C) were analyzed for each formulation. RSMs were built for each parameter. Indicated is mean ± SEM of analytical replicates (n=3). N.D.= non detectable. N.M.= not measurable.

[0049] FIG. 9 shows the effect of Design B microfluidics process parameters on the particle characteristics of aLNP containing poly (EC). RNA-aLNP formulations were prepared with different aqueous RNA phase flow rate, ethanolic lipid phase flow rate, water phase flow rate, lipid concentration, and RNA concentration in a response surface designed experiment (n=53) (Panel D). Particle mean size (Panel A), PDI (Panel B) and RNA encapsulation efficiency (Panel C) were analyzed for each formulation. RSMs were built for each parameter. Indicated is mean ± SEM of analytical replicates (n=3).

[0050] FIG. 10 shows that increasing poly (EC) (pIC) / lipid weight ratio decreases the scattering intensity of aLNP (AL) in 100% D2O (Panel A) and increases the scattering neutron density in the shell (Panel B). The scattering patterns of pIC encapsulated in aLNP and pIC externally added to preformed empty nanoparticles are different (Panel C). When the pIC is externally added to and incubated with preformed empty nanoparticles, there is an increase of scattering at low Q (0.1 to 1 A'1) which indicates an increase in the background. The increase in background can be attributed to more hydrogen in the D2O solvent following the addition of pIC, indicating that pIC is localized in the solvent rather than in the aLNP. No appreciable increase in the background was observed in case of aLNP with encapsulated pIC, indicating that pIC was localized in the aLNP rather than in the solvent. Comparison of the scattering patterns of aLNP (AL) and classic LNPs before (empty LNPs) and after the encapsulation of pIC (Panel D). The encapsulation of pIC in classic LNPs but not in aLNP modifies the scattering profile, indicating a rearrangement of the internal LNP structure. The rearrangement observed in the classic LNP can be explained by the electrostatic interactions between the ionizable cationic lipid and the anionic pIC, which do not occur in the aLNP (pIC-aLNP). Additionally, aLNP (pIC-aLNP) have a distinctive structure compared to pIC- classic LNP. Symbols represent SANS data and solid lines correspond to the best fit using the sphere core-shell model (Panel A), (Panel B) and (Panel D).

[0051] FIG. 11 illustrates the strength of the interaction between RNA incubated with preformed empty DMPC-containing aLNP and RNA encapsulated in DMPC-containing aLNP studied using asymmetrical flow field-flow fractionation (AF4). Encapsulation of RNA enables a strong interaction of RNA with DMPC-containing aLNP while the interaction of RNA incubated with DMPC-containing aLNP is weak and reversible. DMPC-containing aLNP and / or poly (EC) (pIC) were incubated with RNAse free water. Later, Ribogreen dye was supplemented for the fluorescence detection of RNA and samples were analyzed by AF4. RNA was determined using a fluorescence detector (kem=495 nm, kex=520 nm) (left axis) and the light scattering intensity of the particles were determined using MALS (right axis). Indicated are (Panel A) soluble pIC, (Panel B)preformed empty DMPC-containing aLNP, (Panel C) preformed empty DMPC-containing aLNP incubated with externally added RNA and (Panel D) DMPC-containing aLNP with encapsulated RNA. Melting temperatures of DMPC-containing aLNP were obtained by differential scanning calorimetry measurements, showing a significant decrease of the melting temperature only when RNA is encapsulated in the DMPC-containing aLNP, indicating an interaction of RNA within the hydrophobic core of the bilayer of DMPC-containing aLNP. n=3 analytical replicates.

[0052] FIG. 12 shows a schematic representation (Panel A) and a table (Panel B) comparing the main characteristics between classic liposomes, anionic liposomes, classic LNPs, and an exemplary aLNP of the present disclosure.

[0053] FIG. 13 shows levels of interleukin-6 (IL-6) (panel A), interferon-a (panel B) and interferon-P (panel C) in mice after administration of an non-immunogenic mRNA encapsulated in an aLNP comprising the endosomal escape lipopeptide OA-545 (n=4 for PBS group, n=2 mRNA-aLNP-OA-545). ns: not significant, *p < 0.05, **p <0.01, ***p < 0.005 and ****p < 0.0001.

[0054] FIG. 14 shows that aLNP containing poly (EC) (pIC-aLNP) have strong anti-tumoral effects when administered intravenously both as monotherapy and in combination with an a-PD- L1 antibody in a colon carcinoma model.

[0055] FIG. 15 shows that pIC-containing aLNP (pIC-aLNP) have strong anti-tumoral effects as monotherapy in an orthotopic hepatic liver cancer model.

[0056] FIG. 16 shows that incorporating an endosomal escape lipopeptide on an aLNP's surface with a high fusogenic lipid content is essential for achieving stable nanoparticles with high mRNA transfection efficiency. The figure also illustrates various solvents enabling self-incorporation of the endosomal escape lipopeptide during nanoparticle formulation (Panel A). Data represent mean ± SD from three analytical replicates (n = 3). Additionally, the mRNA transfection efficiency is expressed as the percentage of GFP-positive cells in HEK 293 and Raw 264.7 cell lines. Data from Panel B and C represent mean ± SEM from three analytical replicates (n = 3).

[0057] FIG. 17 shows the formation and particle characteristics of aLNP with various examples of bilayer forming lipids, method of incorporation of the endosomal escape lipopeptide, endosomal escape lipopeptide with different sequences, and lipid anchors. Data from Panel A represent mean ± SD from three analytical replicates (n = 3).

[0058] FIG. 18 shows the stability of selected nanoparticle formulations after 1 and 3 months of storage at 5 °C, demonstrating the maintenance of key physicochemical properties over time andthe association of the endosomal escape lipopeptide in the SNP. Data from Panel A and B represent mean ± SD from three analytical replicates (n = 3).

[0059] FIG. 19 shows mRNA transfection efficiency, expressed as the percentage of GFP positive cells, in HEK 293 and Raw 264.7 cell lines for the formulations described in FIG. 17. Data from Panel A and B represent mean ± SEM from three analytical replicates (n = 3).

[0060] FIG. 20 shows the formation and particle characteristics of fusogenic aLNP with various examples of bilayer forming lipids endosomal escape lipopeptide soluble in ethanol and the unsuccessful formation of aLNP when fusogenic lipids and anionic lipids are incorporated without endosomal escape lipopeptide.

[0061] FIG. 21 shows the formation and particle characteristics of aLNP with fusogenic lipid (DOPE) and endosomal escape lipopeptide with various examples of endosomal escape lipopeptide soluble in ethanol and the unsuccessful formation of aLNP without endosomal escape lipopeptide even if endosomal escape lipids forming lipid-bilayers are used. Data from Panel A represent mean ± SD from three analytical replicates (n = 3)

[0062] FIG. 22 shows the mRNA transfection efficiency, expressed as the percentage of GFP positive cells, in HEK 293 and Raw 264.7 cells lines for the formulations described in FIG. 21. Data from Panel A and B represent mean ± SEM from three analytical replicates (n = 3).

[0063] FIG. 23 shows the formation and particle characteristics of aLNPs formulated with varying lipid anchors of the endosomal escape lipopeptide (stearyl, cholesterol, DSPE-MAL, and DSPE-PEG2000-MAL). Data from Panel A represent mean ± SD from three analytical replicates (n = 3). Additionally, it shows the mRNA transfection efficiency, expressed as the percentage of GFP-positive cells, in HEK 293 and Raw 264.7 cells. Data from Panel B and C represent mean ± SEM from three analytical replicates (n = 3).

[0064] FIG. 24 shows the formation and particle characteristics of aLNPs formulated with varying molar percentages (25-45 mol%) of bilayer-forming lipids in combination with a sterol, a fusogenic lipid, and with or without the endosomal escape lipopeptide ID1039 (SEQ ID NO: 1039). It also includes formulations lacking fusogenic lipids, with or without the endosomal escape lipopeptide. Data from Panel A represent mean ± SD from three analytical replicates (n = 3). Additionally, the mRNA transfection efficiency, measured as the percentage of GFP-positive RAW 264.7 cells, is reported. Data from Panel B represent mean ± SEM from three analytical replicates (n = 3).

[0065] FIG. 25 shows the formation and particle characteristics of aLNPs formulated with varying molar percentages (10-20 mol%) of bilayer-forming lipids, in combination with a steroland a fusogenic lipid, and with the endosomal escape lipopeptide ID1039 (SEQ ID NO: 1039). Data from Panel A represent mean ± SD from three analytical replicates (n = 3). Additionally, the mRNA transfection efficiency, measured as the percentage of GFP positive in HEK 293 and RAW 264.7 cells, is reported. Data from Panel B and C represent mean ± SEM from three analytical replicates (n = 3).

[0066] FIG. 26 shows the formation and particle characteristics of aLNPs formulated with 35% of bilayer-forming lipids (DSPC and DSPG), in combination with a sterol (sitosterol) and a fusogenic lipid (DOPE) and with (25B) or without (25 A) the endosomal escape lipopeptide ID 1039 (SEQ ID NO: 1039). Additionally, it shows the formation and particle characteristics of an LNP containing bilayer-forming lipids (EPC and EPG) and a sterol without any endosomal escape lipopeptide (25C). In this experiment, batch size was increased from 75 pg to 225 pg, reflecting an increase of mRNA encapsulation for samples 25 B and 25 C but not 25 A. Data from Panel A represent mean ± SD from three analytical replicates (n = 3).

[0067] FIG. 27 shows Cryo-TEM images of aLNP containing mRNA with different compositions. Panel A shows a representative image of EPC :EPG: Cholesterol (50: 17:33), showing small vesicles below 200 nm with single or multiple lipid bilayers. Panel B and C show representative images of DOPE :DSPC:D SPG: Sitosterol (28:30:5:37), showing giant vesicles above 200 nm with single or multiple lipid bilayers. Panel D shows a representative image of DOPE:DSPC:DSPG:Sitosterol:Stearyl-ID1039 (23:30:5:37:5), revealing small vesicles with diameters below 200 nm, with a single lipid bilayer. Notably, perturbations observed on the bilayer surface are consistent with the presence of endosomal escape lipopeptides embedded within the membrane and partially exposed on its outer surface. Samples were vitrified using a ThermoFisher VitRobot Mark IV at 6.5°C and 100% humidity. Quantifoil Cu R2 / 2 200 mesh grids were glow discharged (ELMO device, 2.8mA, 40s, 3.6* 104mbar). A sample volume of 3pL was applied with blot force 4 and blot time 4 seconds before plunge freezing. Microscopy Parameters Data was collected on a ThermoFisher Glacios 200kV microscope with Falcon 4i camera at 92,000* magnification (1.5A pixel size). Total electron dose was 30e7A2with -3pm defocus.

[0068] FIG. 28 shows that an aLNP containing a fusogenic lipid is stable in PBS and relevant biological media (50% fetal bovine serum (FBS)) when a endosomal escape lipopeptide (stearyl- ID-1039) is incorporated in the formulation. The stability of aLNP was studied using asymmetrical flow field-flow fractionation (AF4). aLNP (2 mM of lipids) were dispersed in PBS or incubated with 50% v / v FBS at 37° C for 1.5 hours and then 40 pl of sample was analyzed with AF4. The detection of proteins and aLNPs was performed with three in-line detectors: dynamic lightscattering (DLS, top graph), absorbance at 280 nm (middle graph) and multi-angle light scattering (MALS) at 90° (bottom graph). DLS determined the hydrodynamic radius of the aLNPs, and the apparent particle concentration was inferred via the derived count rate (DCR) in DLS and the light scattering intensity in MALS. The proteins were detected by recording the absorbance at 280 nm. The smaller particles exit first from the separation channel, and therefore, the 280 nm absorbance peak at approximately 20 minutes (middle graph) corresponds to proteins in serum, while aLNPs with a wide size distribution elute between 30 and 80 minutes, as observed in the top and bottom graphs. Panel A illustrates the PBS and 50% FBS control run; Panel B illustrates the run of the aLNP composed of DOPE:DSPC:DSPG: Sitosterol (28:30:5:37), and Panel C illustrates the run of the aLNP composed of DOPE:DSPC:DSPG:Sitosterol:Stearyl-ID1039 (23:30:5:37:5)

[0069] FIG 29 demonstrates effective in vivo transfection mediated by aLNP incorporating a specific endosomal escape lipopeptide (cholesteryl-ID1039), following subcutaneous administration of 10 pg of mRNA encoding luciferase, except for sample 28B, which was 5 pg due to the low mRNA encapsulation efficiency. The transfection efficiency achieved with the aLNP formulation was comparable to that of classical LNPs (cLNPs), but without the associated reduction in body weight commonly observed with cLNPs. No in vivo transfection was observed when the aLNP Panel A shows whole-body bioluminescence signal intensity (photons / second) measured 24 hours post-administration. Panel B displays the corresponding change in body weight over the same 24-hour period, indicating improved tolerability of the aLNP formulations. Sample 28A corresponds to an aLNP formulation without fusogenic lipid comprising EPC :EPG: Cholesterol (50: 17:33). Sample 28A corresponds to an aLNP formulation without fusogenic lipid comprising EPC :EPG: Cholesterol (50: 17:33). DLin-MC3-DMA, DSPC, cholesterol, and DSPE-PEG2000. Sample 28B corresponds to an aLNP formulation with a fusogenic lipid but without a endosomal escape lipopeptide, comprising DOPE :DSPC:D SPG: Sitosterol (28:30:5:37). Sample 28C corresponds to an aLNP formulation with a fusogenic lipid and with a endosomal escape lipopeptide, comprising DOPE:DSPC:DSPG:Sitosterol:Stearyl-ID1029 (23:30:5:37:5)). Sample 28D corresponds to an aLNP formulation with a fusogenic lipid and with a endosomal escape lipopeptide, comprising DOPE:DSPC:DSPG:Sitosterol:Stearyl-ID1039 (23:30:5:37:5)). Sample 28E corresponds to an aLNP formulation with a fusogenic lipid and with a endosomal escape lipopeptide, comprising DOPE:DSPC:DSPG:Sitosterol:Cholesteryl-ID1039 (23:30:5:37:5)). cLNP corresponds to a classical LNP, comprising DLin-MC3-DMA, DSPC, DSPE-PEG2000, and cholesterol(50: 10: 1.5:38:5)). Data represents mean ± SEM from three (control group) or four animals (treatments groups) (n = 3 or 4). No bracket: not significant, *p < 0.05.DETAILED DESCRIPTION

[0070] The present disclosure provides anionic nanoparticles (aLNP) comprising, for example, a neutral lipid and / or an anionic lipid, a structural lipid, a fusogenic lipid, an optional cationic lipid, and an optional derivatizable lipid, wherein the aLNP have been optimized for endosomal escape by incorporating novel endosomal escape peptide.

[0071] In general, the aLNP disclosed herein comprise a lipid bilayer forming lipid, a fusogenic lipid, and an endosomal escape peptide wherein the endosomal escape peptide structurally stabilizes the aLNP. These non-cationic aLNP can be used for delivery of therapeutic or diagnostic agents or adjuvants without inducing inflammation. The aLNP of the present disclosure comprise at least one lipid bilayer, an aqueous core, and, optionally, one or more endosomal escape peptides, targeting moieties, half-live extenders or combination thereof attached to the surface of the aLNP.

[0072] Classic LNP used to encapsulate therapeutic nucleic acids, e.g., those used to encapsulate mRNAs encoding vaccines such as SARS-Cov2 (COVID) vaccines, comprise high molar ratios of ionizable cationic lipids. These positively charged lipids interact with the negatively charged nucleic acids, yielding stable cationic lipid nanoparticles. These classical lipid nanoparticles have a solid core. In contrast, the anionic lipid nanoparticles (aLNP) of the present disclosure generally have an aqueous core, and unexpectedly, given the negative charge of the lipid component, they are stable when a negatively charged nucleic acid payload is incorporated.

[0073] The ionizable cationic lipids in classic LNPs are major contributors to the development of an immune response, which can cause inflammation and / or neutralization or reduction of the therapeutic efficacy of the payload (e.g., via neutralizing antibodies). The absence of ionizable cationic lipids, or the presence of small amounts that still preserve the anionic charge of the LNP, in the aLNP of the present disclosure, and their particular structural properties (e.g., shielding a payload within the hydrophobic core of the lipid bilayer) allow the aLNP to reach their target without triggering an immune response. These characteristics (i) permit using lower dosages, which in turn help prevent and immune response, (ii) reduce costs since smaller amounts of payload can be used, and (iii) reduce side effects since lower amounts of payload are needed and the occurrence of immune responses is also reduced.

[0074] One drawback of LNP without ionizable cationic lipids is reduced endosomal escape compared with classic LNP, in which endosomal escape is mediated by the ionizable cationic lipid component. aLNP that lack fusogenic lipids are stable, but they do not have endosomal escape properties. The addition of a fusogenic lipid such as DOPE enables endosomal escape in vitro. However, the stability of these sLNP is low, and the yield during manufacturing is also low. While lipids that adopt cubic and hexagonal HII phases such as DOPE enhance endosomal escape properties, they are detrimental to the stability of the aLNP during manufacturing and storage, and negatively affect the plasma half-life and bioavailability of the aLNP. DOPE and other fusogenic lipids like GMO confer a negative spontaneous curvature to membranes containing these lipids, resulting in the formation of nonlamellar self-assembled phases (inverted hexagonal or gyroid cubic) yielding unstable particles unsuitable for industrial production. Surprisingly, the incorporation of endosomal escape peptides having specific physicochemical and structural properties not only confers peptide-mediated endosomal escape properties to the aLNP: the endosomal escape peptides stabilize the fusogenic lipid-containing LNP, enabling transfection in vitro and in vivo. The resulting aLNP are anionic LNP (aLNP) in which endosomal escape is mediated by two components (i) the fusogenic lipid components (e.g., DOPE or GMO), and (ii) endosomal escape peptides, which can be tuned up without the need of an ionizable cationic lipid component. In some aspects in which the presence of an ionizable cationic lipid component may be desirable, the aLPN may comprise three tunable components: (i) a fusogenic lipid component (e.g., DOPE or GMO), (ii) a endosomal escape peptide component, and (iii) an ionizable cationic lipid component, which could be tuned up to module the fusogenicity, endosomal escape, and / or immunogenicity of the LNP. In summary, the present disclosure provides a tunable platform that can yield LNP with different degrees of fusogenicity, immugenicity, and endosomal escape, and can be used to transport a variety of cargos in several available compartments (e.g., in the lumen of the particle, between bilayers, inside bilayers, on the surface of the particle).

[0075] To enhance endosomal escape, the optimized aLNP of the present disclosure comprise endosomal escape peptides covalently attached to the external surface of the lipid bilayer. In some aspects, the endosomal escape peptides can be lipopeptides that are preformed before the assembly of the aLNP. In other aspects, the endosomal escape peptides are covalently attached to the formed aLNP using, e.g., click chemistry. In one specific example, an aLNP having a derivatizable lipid comprising a thiol group can be reacted with an endosomal escape peptide comprising a maleimide group. In one specific example, an aLNP having a derivatizable lipid comprising a maleimide group can be reacted with an endosomal escape peptide comprising a thiol group. Also providedin this disclosure are novel endosomal escape peptides and libraries of endosomal escape peptides to optimize the endosomal escape of the aLNP disclosed herein.

[0076] Also provided is a method to incorporate an endosomal escape lipopeptide in the aLNP. In some aspects, the endosomal escape lipopeptide is incorporated in the aqueous phase during the manufacturing process. In other aspects, the endosomal escape lipopeptide is incorporated in the organic phase, in combination with the lipids, during the manufacturing process.

[0077] The present disclosure also provides methods of manufacturing aLNP at micro-scale, small-scale, and large-scale, pharmaceutical compositions, methods of treatment, and kits for the encapsulation of payloads in aLNP and their optimization.

[0078] In order that the present description can be more readily understood, certain terms are first defined below. Additional definitions are set forth throughout the detailed description.Definitions

[0079] It is to be noted that the term "a" or "an" entity refers to one or more of that entity; for example, "a nucleotide sequence," is understood to represent one or more nucleotide sequences. As such, the terms "a" (or "an"), "one or more," and "at least one" can be used interchangeably herein.

[0080] Furthermore, "and / or" where used herein is to be taken as specific disclosure of each of the two specified features or components with or without the other. Thus, the term "and / or" as used in a phrase such as "A and / or B" herein is intended to include "A and B," "A or B," "A" (alone), and "B" (alone). Likewise, the term "and / or" as used in a phrase such as "A, B, and / or C" is intended to encompass each of the following aspects: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0081] It is understood that wherever aspects are described herein with the language "comprising," otherwise analogous aspects described in terms of "consisting of and / or "consisting essentially of' are also provided.

[0082] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure is related. For example, the Concise Dictionary of Biomedicine and Molecular Biology, Juo, Pei- Show, 2nd ed., 2002, CRC Press; The Dictionary of Cell and Molecular Biology, 3rd ed., 1999, Academic Press; and the Oxford Dictionary of Biochemistry and Molecular Biology, Revised, 2000, Oxford University Press, provide one of skill with a general dictionary of many of the terms used in this disclosure.

[0083] Units, prefixes, and symbols are denoted in their Systeme International de Unites (SI) accepted form. Numeric ranges are inclusive of the numbers defining the range. Unless otherwise indicated, nucleotide sequences are written left to right in 5' to 3' orientation. Amino acid sequences are written left to right in amino to carboxy orientation. The headings provided herein are not limitations of the various aspects of the disclosure, which can be had by reference to the specification as a whole. Accordingly, the terms defined immediately below are more fully defined by reference to the specification in its entirety.

[0084] About'. The term "about" is used herein to mean approximately, roughly, around, or in the regions of. When the term "about" is used in conjunction with a numerical range, it modifies that range by extending the boundaries above and below the numerical values set forth. In general, the term "about" can modify a numerical value above and below the stated value by a variance of, e.g., 15 percent, up or down (higher or lower). Thus, in some aspects, about is interchangeable with ± 15%. As described herein, any numerical range, concentration range, percentage range, ratio range or integer range is to be understood to include the value of any integer within the recited range and, when appropriate, fractions thereof (such as one-tenth and one-hundredth of an integer), unless otherwise indicated.

[0085] Antibody. The term "antibody" includes, without limitation, a glycoprotein immunoglobulin which binds specifically to an antigen and comprises at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. In the context of the present disclosure, antibodies can be used, for example, as therapeutic agents (payloads), a targeting agents directing the aLNP to specific cell types or tissues, or as combination therapies in which the antibody is coadministered with an aLNP of the present disclosure comprising, e.g., a therapeutic payload or an immunomodulatory payload (e.g., a vaccine adjuvant).

[0086] The term "monoclonal antibody," as used herein, refers to an antibody that is produced by a single clone of B-cells and binds to the same epitope. The term "antibody" includes, by way of example, monoclonal and polyclonal antibodies; chimeric and humanized antibodies; human or non-human antibodies; wholly synthetic antibodies; and single chain antibodies. A non-human antibody can be humanized by recombinant methods to reduce its immunogenicity in man.

[0087] An "antigen binding portion" of an antibody refers to one or more fragments or portions of an antibody that retain the ability to bind specifically to the antigen bound by the whole antibody. It has been shown that the antigen binding function of an antibody can be performed by fragments or portions of a full-length antibody.

[0088] As used herein, the term "variable region" typically refers to a portion of an antibody, generally, a portion of a light or heavy chain, typically about the amino-terminal 110 to 120 amino acids, or 110 to 125 amino acids in the mature heavy chain and about 90 to 115 amino acids in the mature light chain, which differ extensively in sequence among antibodies and are used in the binding and specificity of a particular antibody for its particular antigen. The variability in sequence is concentrated in those regions called Complementarity Determining Regions (CDRs) while the more highly conserved regions in the variable domain are called framework regions (FR).

[0089] The terms "complementarity determining region" or "CDR", as used herein, refer to each of the regions of an antibody variable domain which are hypervariable in sequence and / or form structurally defined loops (hypervariable loops) and / or contain the antigen-contacting residues. Antibodies can comprise six CDRs, e.g., three in the VH and three in the VL.

[0090] The terms "VL", "VL region," and "VL domain" are used herein interchangeably to refer to the light chain variable region of an antigen binding polypeptide, antigen binding polypeptide complex, antibody or antigen binding fragment thereof.

[0091] The terms "VH", "VH region," and "VH domain" are used herein interchangeably to refer to the heavy chain variable region of an antigen binding polypeptide, antigen binding polypeptide complex, antibody or antigen binding fragment thereof.

[0092] In some aspects, an antibody is a payload within an aLNP of the present disclosure. In some aspects, the antibody payload is a polypeptide or combination thereof. In some aspects, the antibody payload is a polynucleotide or combination thereof encoding an antibody. In some aspects, an antibody is a targeting moiety attached to the external surface of an aLNP of the present disclosure.

[0093] Base'. As used herein, the term "base" and grammatical variants thereof includes purines and pyrimidines, which further include natural compounds adenine, thymine, guanine, cytosine, uracil, inosine, natural analogs, and synthetic derivatives of purines and pyrimidines, which include, but are not limited to, modifications which place new reactive groups such as, but not limited to, amines, alcohols, thiols, carboxylates, and alkylhalides.

[0094] Chimeric Antigen Receptor'. As used herein, the term “Chimeric Antigen Receptor” or alternatively “CAR” refers to a recombinant polypeptide construct comprising at least an extracellular antigen binding domain, a transmembrane domain, and a cytoplasmic signaling domain comprising a functional signaling domain derived from a stimulatory molecule. In some aspects, the payload of an aLNP of the present disclosure comprises a CAR. In some aspects, the CAR is inserted in the lipid bilayer of the aLNP (see FIG. 12 for a schematic representation of anexemplary aLNP compared to a ‘classic’ LNP). In other aspects, the CAR is encoded by a polynucleotide encapsulated in an aLNP of the present disclosure.

[0095] Identity. As used herein, the terms "identity" and "sequence identity" are used interchangeably and refer to the overall monomer conservation between polymeric molecules, e.g., between polypeptide molecules or polynucleotide molecules (e.g. DNA molecules and / or RNA molecules). The term "identical" without any additional qualifiers, e.g., protein A is identical to protein B, implies the sequences are 100% identical (100% sequence identity). Describing two sequences as, e.g., "70% identical," is equivalent to describing them as having, e.g., "70% sequence identity."

[0096] Calculation of the percent of sequence identity of two polypeptide sequences, for example, can be performed by aligning the two amino acid sequences for optimal comparison purposes (e.g, gaps can be introduced in one or both of a first and a second polypeptide sequences for optimal alignment and non-identical sequences can be disregarded for comparison purposes). In certain aspects, the length of a sequence aligned for comparison purposes is at least about 70%, at least about 80%, at least about 90%, at least about 95%, or about 100% of the length of the reference sequence. The amino acids at corresponding amino acid positions are then compared.

[0097] When a position in the first sequence is occupied by the same amino acid as the corresponding position in the second sequence, then the molecules are identical at that position. The percentage of sequence 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 percentage of sequence identity between two sequences can be accomplished using a mathematical algorithm.

[0098] Suitable software programs are available from various sources, and for alignment of both protein and nucleotide sequences. Suitable programs are, e.g., Needle, Stretcher, MAFFT, Clustal (ClustalW, Clustal X or Clustal Omega), MUSCLE, etc. Different regions within a single polynucleotide or polypeptide target sequence that aligns with a polynucleotide or polypeptide reference sequence can each have their own percentage of sequence identity. It is noted that the percentage of sequence identity value is rounded to the nearest tenth. For example, 80.11, 80.12, 80.13, and 80.14 are rounded down to 80.1, while 80.15, 80.16, 80.17, 80.18, and 80.19 are rounded up to 80.2. It also is noted that the length value will always be an integer.

[0099] In certain aspects, the percentage of sequence identity (%ID) or of a first amino acid sequence (or nucleic acid sequence) to a second amino acid sequence (or nucleic acid sequence) iscalculated as %ID = 100 x (Y / Z), where Y is the number of amino acid residues (or nucleobases) scored as identical matches in the alignment of the first and second sequences (as aligned by visual inspection or a particular sequence alignment program) and Z is the total number of residues in the second sequence. If the length of a first sequence is longer than the second sequence, the percentage of sequence identity of the first sequence to the second sequence will be higher than the percentage of sequence identity of the second sequence to the first sequence.

[0100] Isolated. A lipid, polypeptide, antibody, or polynucleotide which is "isolated" is a lipid, polypeptide, antibody, or polynucleotide which is in a form not found in nature. Isolated lipids, polypeptides, antibodies, or polynucleotides include those which have been purified to a degree that they are no longer in a form in which they are found in nature. In some aspects, a lipid, polypeptide, antibody, or polynucleotide which is isolated is substantially pure. As used herein, "substantially pure" refers to material which is at least 90% pure (i.e., free from contaminants), at least 95% pure, at least 98% pure, or at least 99% pure

[0101] Nanoparticle-. As used herein, the term “nanoparticle” refers to lipidic particles having a particle size on the nanometer scale, less than 1 micrometer. For example, the nanoparticle can have a particle size up to about 100 nm. In another example, the nanoparticle can have a particle size up to about 200 nm. In another example, the nanoparticle can have a particle size up to about 300 nm. In another example, the nanoparticle can have a particle size up to about 400 nm. In another example, the nanoparticle can have a particle size up to about 500 nm. For clinical purposes, for example to be filtered using 200 nm filters, nanoparticles can preferably have sizes (diameters) below 200 nanometers. Accordingly, in some aspects, the nanoparticles have sizes below 500 nm, below 400 nm, below 300 nm, below 200 nm, or below 100 nm. In some aspects, the nanoparticles (e.g., aLNP) have an average size (diameter) of about 200 nm (e.g., 200 nm ± 10 nm, or 200 nm ± 20 nm, or 200 nm ± 30 nm). In some aspects, the nanoparticles (e.g., aLNP) have an average size (diameter) of about 100 nm (e.g., 100 nm ± 10 nm, or 100 nm ± 20 nm, or 100 nm ± 30 nm). In some aspects, the nanoparticles (e.g., aLNP) have an average size (diameter) of about 150 nm (e.g., 150 nm ± 10 nm, or 150 nm ± 20 nm, or 150 nm ± 30 nm). In some aspects, the nanoparticles (e.g., aLNP) have an average size (diameter) of about 70 nm (e.g., 70 nm ± 10 nm, or 70 nm ± 20 nm, or 70 nm ± 30 nm).

[0102] As used herein, “nanoparticle” refers to a number of nanoparticles, including, but not limited to, nanoclusters, nanovesicles, micelles, lamaellae shaped particles, polymersomes, dendrimers, liposomes, and other nano-size particles of various other small fabrications that are known to those in the art. The shapes and compositions of nanoparticles can be guided duringcondensation of atoms by selectively favoring growth of particular crystal facets to produce spheres, rods, wires, discs, cages, core-shell structures and many other shapes.

[0103] Lipid nanoparticle (LNP)'. As used herein, the term “lipid nanoparticle” is used interchangeably with the abbreviation “LNP” and refers to a microscopic lipid formulation that can be used to deliver an active agent or therapeutic agent, such as a nucleic acid (e.g., an mRNA, dsDNA), to a target site of interest (e.g., an immune cell). Lipid nanoparticles typically have a size of less than about 1000 nm in at least one dimension.

[0104] Classic LNP'. A “classic lipid nanoparticle” or “classic LNP” is a spherical lipid nanoparticle composed of pH-responsive lipids or cationic lipids bearing tertiary or quaternary amines to encapsulate a polyanionic nucleic acid (e.g., an mRNA), neutral helper lipids such as zwitterionic lipids and / or sterol lipids to stabilize the lipid bilayer of the lipid nanoparticle, and a polyethylene glycol (PEG)-lipid to improve the colloidal stability in biological environments. In general, the lipid content of classic LNP comprises about 40% to about 60% of ionizable cationic lipids. The ionizable cationic lipids form inverse micelles in complex with oligonucleotide cargos, and are critical LNP components for in vivo delivery. Classic LNPs also comprise phospholipids in a range of about 8% to about 12% (contribute to particle structure and efficacy of membrane fusion; neutral o zwitterionic phospholipids are commonly used), PEGylated lipids in a range of about 1% to about 2% (“stealth lipids” that prevent serum protein adsorption and nanoparticle aggregation, increase in vivo circulation time, and be functionalized for targeted delivery), and sterol lipids in a range of about 30% to 50% (provide structural integrity and aid in membrane fusion to the target). Encapsulation of the nucleic acid cargo relies on the electrostatic interaction between the positively charged ionizable cationic lipids and the negatively charged nucleic acids, which takes place in the lumen of inverted micelles in the core of the LNP.

[0105] Anionic Lipid Nanoparticle (aLNP)'. As used herein the terms “aLNP,” “anionic LNP,” and grammatical variants thereof refer to lipid nanoparticles having a structure comprising at least one classic lipid bilayer (e.g., one or two concentric lipid bilayers) and, generally (when the concentrations of fusogenic lipids used are low), an aqueous core, wherein the particle is anionic. In some aspects, e.g., when high concentrations of fusogenic lipids such as DOPE are used, the core of the aLNP can comprise inverse micelles. These non-cationic nanoparticles are characterized by the absence of ionizable cationic lipids, or if they are present, the molar ratio of ionizable cationic lipid to total lipid content is below 20 %, e.g., less than about 20%, less than about 15%, less than about 10%, less than about 5%, or less than about 1%. The aLNP of the present disclosure are capable to encapsulate therapeutic nucleic acids (e.g., an mRNA), at leastpartially, in the hydrophobic core of the lipid bilayer. In some aspects, at least 20 wt% of the nucleic acid payload (e.g., an mRNA) is inserted in the hydrophobic core of the outer bilayer of the aLNP. Structural and functional differences between classic LNP and classic liposomes and the aLNP of the present disclosure are exemplified in FIG. 12. See International Application No. PCT / EP2023 / 086994, which is herein incorporated by reference in its entirety. In some aspects, the aLNP comprise a single bilayer with an aqueous core, i.e., the aLNP is an “unilamellar aLNP.” In other aspects, an aLNP of the present disclosure can be multilamellar, i.e., the aLNP comprises multiple concentric bilayer, with an aqueous layer between each pair of concentric bilayers, the aLNP is a “multilamellar aLNP.” In one particular aspect, the aLNP comprises two concentric bilayers. In some aspects, an endosomal escape peptide (e.g., a lipopeptide) can be located on the external surface of the aLNP as observed in FIG. 12. In some aspects, the endosomal escape peptide (e.g., a lipopeptide) located on the external surface of the aLNP contributes to (i) the structural integrity of the membrane aLNP and / or (ii) the fusogenicity of the aLNP to its target.

[0106] Nucleic acid. The term "nucleic acid" as used herein means a polymer containing at least two nucleotides, preferably at least two deoxyribonucleotides and / or ribonucleotides, in either single- or double-stranded form. The term thus includes DNA, RNA and DNA / RNA hybrids. DNA can be in the form of e.g. antisense molecules, plasmid DNA, pre-condensed DNA, a PCR product, vectors (Pl, PAC, BAC, YAC, and artificial chromosomes), expression cassettes, chimeric sequences, chromosomal DNA, or derivatives and combinations of these groups. RNA can be in the form of siRNA, asymmetrical interfering RNA (aiRNA), microRNA (miRNA), IncRNA, gRNA, mRNA, tRNA, rRNA, tRNA, viral RNA (vRNA), and combinations thereof. The term "nucleic acid” includes nucleic acids containing nucleotide analogs or modified backbone residue(s) or linkage(s), which are synthetic, naturally occurring, or non-naturally occurring, and which can have similar or different binding properties as the reference nucleic acid not having said modifications or differences. Examples of such analogs include, without limitation, cyclic dinucleotides, phosphorothioates, phosphoramidates, methyl phosphonates, chiral-methyl phosphonates, 2'-O-methyl ribonucleotides, and peptide-nucleic acids (PNAs). Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences as well as the sequence explicitly indicated. Specifically, degenerate codon substitutions can be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and / or deoxyinosine residues (Batzer et al., Nucleic Acid Res., 19:5081 (1991 ); Ohtsuka et al., J. Biol. Chem., 260:2605-2608 (1985 );Rossolini et al., Mol. Cell. Probes, 8:91-98 (1994 )). The “nucleic acid” of the current disclosure can be naturally occurring or not naturally occurring (i.e. a “nucleic acid analogue"). Accordingly, the term “nucleic acid” as used herein can be replaced by “nucleic acid and / or nucleic acid analogue”. Where reference is made to “one or more” nucleic acids, this can refer to one or more different types or forms of nucleic acid, for example chosen from the above, or can refer to one or more nucleic acid with different constituent residues or different sequence. For example, one or more can refer to at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10 nucleic acids. Thus, the term “one nucleic acid” can refer to one (type of) nucleic acid which can be present as multiple (identical) nucleic acid molecules. The one or more nucleic acid can be non-coding nucleic acid. In a specific aspect, the nucleic acid is an immunologic adjuvant, e.g., Poly I:C.

[0107] Nucleic acid analogue'. The “nucleic acid analogue” can for example be a polymer of nucleotides (e.g. at least two nucleotides) having at least one alteration in one or more of the phosphate backbone, pentose sugar, and one of four nucleobases. The “nucleic acid analogue” can for example be synthetic, i.e. produced by chemical synthesis method. The “nucleic acid analogue” can be a molecule structurally similar to naturally occurring nucleic acid, but having an alteration in the backbone of the molecule, for example in the case of peptide nucleic acid (PNA), Morpholino and locked nucleic acid (LNA), as well as glycol nucleic acid (GNA), threose nucleic acid (TNA) and hexitol nucleic acids (HNA) and lipid nucleic acid. The term “RNA analogue” as used herein can for example refer to a nucleic acid analogue wherein the pentose sugar is ribose.

[0108] Protein'. The terms "polypeptide," "peptide," and "protein" are used interchangeably herein to refer to polymers of amino acids of any length. The polymer can be linear or branched, it can comprise modified amino acids, and it can be interrupted by non-amino acids. The terms also encompass an amino acid polymer that has been modified naturally or by intervention; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeling component. Also included within the definition are, for example, polypeptides containing one or more analogs of an amino acid (including, for example, unnatural amino acids, etc.), as well as other modifications known in the art. It is understood that, because the polypeptides of this disclosure are based upon antibodies, in some aspects, the polypeptides can occur as single chains or associated chains.

[0109] Vector: A “vector” is a composition of matter which comprises an isolated nucleic acid and which can be used to deliver the isolated nucleic acid to the interior of a cell. Numerous vectors are known in the art including, but not limited to, linear polynucleotides, polynucleotidesassociated with ionic or amphiphilic compounds, plasmids, and viruses. Examples of viral vectors include, but are not limited to, adenoviral vectors, adeno-associated virus vectors, retroviral vectors, and the like.

[0110] Lipid. As used herein, the term “lipid” refers to a group of organic compounds that include, but are not limited to, esters of fatty acids and are characterized by being insoluble in water, but soluble in many organic solvents. Lipids are usually divided into at least three classes: (1) “simple lipids,” which include fats and oils as well as waxes; (2) “compound lipids,” which include phospholipids and glycolipids; and (3) “derived lipids” such as steroids. The selection of the individual lipid components of the lipid formulation is made to optimize delivery of a payload (e.g., a nucleic acid) to a target cell. As used herein, the phrase “lipid formulation” refers to a formulation comprising one or more lipids (e.g., anionic lipids, neutral lipids, structural lipids, fusogenic lipids, lipid conjugates, and the like).

[0111] Lipid bilayer '. As used herein, the term "lipid bilayer" refers to a two-layered arrangement of lipid molecules. The two-layered arrangements can be at least partially enabled by amphiphilic lipids and accordingly contain a polar and an apolar region. The polar region typically consist of a phosphate group, an acidic group and / or tertiary or quaternary ammonium salts and can either have a net negative (anionic), neutral or positive (cationic) surface charge at physiological pH, depending on the composition of the lipid head groups. The apolar region typically consists of one or more fatty acid chains with at least 8 carbons and / or cholesterol. The lipids constituting the vesicular bilayer membranes can be organized such that the apolar hydrocarbon "tails" are oriented toward the center of the bilayer while the polar "heads" orient towards the in- and outside aqueous phase, respectively. If there is more than one lipid bilayer in the particle of the disclosure, each lipid bilayer is preferably composed of two lipid monolayers, each of which has a hydrophobic "tail" region and a hydrophilic polar "head" region. In some aspects, the lipid tail can comprise unsaturated bonds. In some aspects, the lipid tail can be branched. Where reference is made to lipid(s) herein, preferably lipid(s) capable of forming one or more lipid bilayers is meant. The term “one or more lipid bilayer” can refer to (at least) 1, 2, 3 lipid bilay er(s).

[0112] Antineoplastic agent'. As used herein, the term “anti -neoplastic agent” refers to a bioactive compound that can be used for the treatment of malignancies, e.g., a cancer such as a tumor. In some aspects, the antineoplastic agent comprises a radiotherapeutic agent, a chemotherapeutic agent, an antibody, a T-cell receptor or immune cell (e.g., as a CAR T-cell), an immune checkpoint inhibitor (e.g., an anti-CTLA-4, an anti-PDl, and / or anti-PD-Ll antibody). In some aspects, the antibody can be monospecific, bispecific, or multispecific. In some aspects, theantibody can be monovalent, bivalent, trivalent, or tetravalent. The immune cell can be an immune cell administered as part of immunotherapy. In some aspects, the anti-neoplastic agent can comprise (i) a cell-based anti -neoplastic agent; (ii) a lymphocyte-based anti -neoplastic agent, preferably chosen from B cells, aPT cells, yST cells, NK cells, NKT cells, autologous tumorinfiltrating lymphocytes (TILs), autologous NK cells, CAR-T cells, CAR-B cells, CAR-NK cells, CAR-NKT cells; (iii) a myeloid-based anti -neoplastic agent, preferably chosen from dendritic cellbased anti-neoplastic agent, macrophage-based anti-neoplastic agent or a neutrophil based anti- neoplastic agent\; (iv) an antibody, or an immune checkpoint inhibitor, wherein the immune checkpoint inhibitor is preferably an antibody, e.g., anti-CTLA4 antibody, anti-PDl antibody and / or anti-PD-Ll antibody; (v) a small molecule drug preferably chosen from alkylating agent, antibiotic, anti-metabolite, hormonal antagonist, photosensitizer, protein kinase inhibitor, poly (ADP -ribose) polymerase inhibitor, taxane and / or topoisomerase inhibitor; (vi) radiation therapy; (vii) a cytokine preferably chosen from IL-2, IL- 12, IL- 15 and IL-21; (viii) a growth factor preferably chosen from CSF family, and Flt3L; (ix) a steroidal or a non-steroidal anti-inflammatory drug; or (ix) any combination thereof.

[0113] Immune cell. As used herein, the term “immune cell” refers to any cell belonging to the immune system, preferably chosen from a lymphocyte, granulocyte, myeloid cell, T cell (e.g. T helper cell, T helper 17 cell, follicular helper T cell, cytotoxic T cell, gamma delta T cell), monocyte, macrophage, NK cell, basophil, dendritic cell (e.g. myeloid dendritic cell, plasmacytoid dendritic cell), neutrophil, eosinophil, basophil, mast cell, B cell, or plasma cell, among others. An immune cell according to the present disclosure can also be an engineered immune cell as taught herein, e.g., a CAR T-cell. In some aspects, , the immune cell according to the present disclosure is an engineered cytotoxic T cell, B cell, or NK cell such as a CAR T or CAR NK cell.

[0114] Vaccine'. As used herein, the term “vaccine” refers to a preparation that contains one or more antigens, e.g. used for the purpose of the prevention or treatment of infections, cancer, autoimmune disease or allergy.

[0115] Cationic polymer'. As used herein, the term “cationic polymer” refers to a polymer bearing a net positive charge, preferably in aqueous medium at pH 7.4 (or at pH 3-9, 5-8, or 6.0- 7.5). In some aspects, the cationic polymer is poly-L-lysine, polyamidoamine, poly [2- (N, N- dimethylamino) ethylmethacrylate], chitosan, poly-L- ornithine, cyclodextrin, histone, collagen, dextran, polyethyleneimine (PEI), or a combination thereof.

[0116] Free of. As used herein, the term “free of’ in the context of a molecule, substance or compound in an aLNP of presence disclosure means that the aLNP is “essentially free” or“substantially free” from the molecule, substance or compound, e.g., the amount of molecule, substance or compound is not measurable according to a standard analytic technique in the field. In the context of the present disclosure “essentially free” and “substantially free” are interchangeable. In some aspect, “free of’ can mean that the molecule, substance or compound is present in an amount less than about 0.001 wt%, less than about 0.0001 wt%, or less than 0.00001 wt%.

[0117] Aqueous core'. As used herein, the term “aqueous core” refers to aqueous medium surrounded by the one or more lipid bilayers of an aLNP of the present disclosure. In the case of a unilamellar aLNP of the present disclosure, the term “lumen” can be used refer to the compartment enveloped by the lipid bilayer. The aLNP of the present disclosure, when generated using low concentrations of fusogenic lipids, comprises an aqueous core. This features is a tunable feature that can be altered by the inclusion of higher amounts of fusogenic lipids, converting the aLNP of the present disclose from the aqueous core type (i.e., an aLNP with an aqueous core) to the inverted lamellar core type (i.e., an aLNP with a core comprising inverted micelles).

[0118] Encapsulate'. The terms “encapsulate,” “encapsulation,” and grammatical variants thereof mean that at least part of the payload, e.g., one or more nucleic acids (e.g., mRNA), is enclosed or trapped within the outer bilayer of an aLNP of the present disclosure. In some aspects, part or all of the payload (e.g., a nucleic acid such as an mRNA) is not in direct contact with the aqueous medium surrounding the aLNP. In some aspects, the payload can be encapsulated between the bilayers of the aLNP. In some aspects, the payload can be encapsulated in the aqueous core of the aLNP. In some aspects, the payload can be encapsulated in micelles in the core of the aLNP.

[0119] In some aspects, a payload can be encapsulated:(i) in the outer bilayer of an aLNP;(ii) in the / an inner bilayer of an aLNP;(iii) in the aqueous compartment between two bilayers of an aLNP;(iv) in the aqueous core of an aLNP;(v) in the micelles in the core of an aLNP; or,(iv) in a combination thereof.

[0120] aLNP of the present disclosure'. As used herein, the term “aLNP of the present disclosure” refers to an anionic nanoparticle (aLNP) characterized by being highly tunable in its physicochemical and payload bearing characteristics by combining (i) a bilayer forming lipid or a combination thereof, (ii) a fusogenic lipid, and (iii) an endosomal escape peptide. When comprising a nucleic acid payload, the nucleic acid can be inserted in a bilayer of the aLNP.1. Anionic Nanoparticles (aLNP)

[0121] The present disclosure provides anionic nanoparticles (aLNP) comprising, for example, neutral lipid and / or an anionic lipid, and a structural lipid (e.g., an sterol), wherein the aLNP have been optimized for endosomal escape by including in its composition a fusogenic lipid and a liposomal escape peptide. As illustrated, for example, in FIG. 12, the aLNP of the present disclosure are nano-sized lipid particles comprising a bilayer structure similar to that of a classic liposome, and in some aspects have an aqueous core. In contrast, a classic LNP has a fatty core. Optionally, the aLNP disclosed herein can comprise low concentrations of ionizable cationic lipids (in amounts low enough to not alter the anionic nature of the LNP) and derivatizable lipids (e.g., to covalently incorporate targeting or diagnostic molecules).

[0122] The aLNP disclosed herein are a highly tunable platform combining (i) one or more bilayer forming lipids, (ii) one or more fusogenic lipids, (iii) one or more endosomal escape peptides, and (iv) an optional (small) amount of an ionizable cationic lipid.

[0123] In one specific aspects, the endosomal escape peptide is between 15 and 50 amino acids in length an comprises at least two amino acid subsequences selected from the group consisting of EALAHH (SEQ ID NO: 1336), DALAHH (SEQ ID NO: 1337), EALAHW (SEQ ID NO: 1338), DALAHY (SEQ ID NO: 1339), DALAHG (SEQ ID NO: 1340), and DALAHW (SEQ ID NO: 1341), wherein one optional non-polar amino acid can be intercalated between the subsequences, and wherein one tryptophan is located at the N-terminus and / or C-terminus of the endosomal escape peptide.

[0124] In some aspects, the endosomal escape peptide comprises the EALAHH (SEQ ID NO: 1336) subsequence and is selected from the group consisting of SEQ ID NOS: 1038, 1039, 1040, 1041, 1310, 1311, 1312, 1313, 1314, 1319, 1328, 1329, 1330, 1331, 1332, 1333, and 1334. In some aspects, the endosomal escape peptide comprises the DALAHH (SEQ ID NO: 1337) subsequence and is selected from the group consisting of SEQ ID NOS: 1311, 1328, 91329, 1330, and 1331. In some aspects, the endosomal escape peptide comprises the EALAHW (SEQ ID NO: 1338) subsequence and is selected from the group consisting of SEQ ID NOS: 1038, 1039, 1040, 1041, 1310, 1311, 1312, 1313, 1314, 1319, 1328, 1329, 1330, 1331, 1332, 1333, and 1334. In some aspects, the endosomal escape peptide comprises the DALAHY (SEQ ID NO: 1339) subsequence and has the amino acid sequence set forth in SEQ ID NO: 1329. In some aspects, the endosomal escape peptide comprises the DALAHG (SEQ ID NO: 1340) subsequence and has the amino acid sequence set forth in SEQ ID NO: 1328. In some aspects, the endosomal escape peptidecomprising the DALAHW (SEQ ID NO: 1341) subsequence and has the amino acid sequence set forth in of SEQ ID NO: 1330.

[0125] In some aspects, the one optional non-polar amino acid intercalated between the subsequences is selected from the group consisting of alanine, valine, leucine, isoleucine, methionine, and cysteine. In some aspects, more than one non-polar amino acid can be intercalated between the subsequences disclosed herein. In some aspects, two non-polar amino acids are intercalated. In some aspects, three non-polar amino acids are intercalated. In some aspects, four non-polar amino acids are intercalated. In some aspects, the endosomal escape peptide further comprises an N-terminal or C-terminal cysteine.

[0126] In some aspects, the endosomal escape peptide is conjugated to a lipid, thereby yielding a lipopeptide. In some aspects, the endosomal escape peptide is conjugated to the lipid via a click chemistry reaction. In some aspects, the endosomal escape peptide is conjugated to the lipid via a maleimide moiety. In some aspects, the endosomal escape peptide is conjugated to the lipid via an amide linkage, via an ester linkage, via a hemisuccinate linker, through an amide-amide bond, or via a keto linker such as a disulfide or hydrazone.

[0127] As used herein, the term “bilayer forming lipid” refers to a lipid with a cylinder shape and a packaging parameter around 1, that is capable of forming a lipid bilayer with a hydrophobic interior and a hydrophilic exterior, comprising, for example, phospholipids (e.g. saturated phosphatidylcholines, phosphatidylglycerol), sphingolipids, glycolipids, alkylphospholipids, ether lipids, and plasmalogens. One type of bilayer-forming lipid may be used or a mixture of two or more types.

[0128] As used herein, the term “fusogenic lipid” refers to a lipid that can induce membrane fusion, destabilization or disruption, thereby facilitating the intracellular delivery of nucleic acids in the cytosol or nucleous. A fusogenic lipid may be neutral or anionic and may exhibit a packing parameter greater than or less than 1, depending on the molecular geometry and the lipid environment. Cone-shaped lipids, which typically have a packing parameter (P) greater than 1, are capable of inducing negative membrane curvature and promoting lamellar-to-nonlamellar phase transitions, such as the formation of inverted hexagonal (HII) or inverted micellar structures, particularly under acidic conditions and high concentration of calcium encountered in the endosomal compartment. Inverted-cone lipids, which typically have a packing parameter less than 1, may also contribute to membrane fusion or destabilization by inducing curvature stress or interfacial tension, particularly when co-formulated with cone-shaped or cylindrical lipids. Examples of fusogenic lipids include, but are not limited to, phosphatidylethanolamines, such asdioleoylphosphatidylethanolamine (DOPE) and palmitoyloleoylphosphatidylethanolamine (POPE); phosphatidic acids, such as dioleoylphosphatidic acid (DOPA); phosphatidylglycerols, such as di oleoylphosphatidylglycerol (DOPG); phosphatidylserines, such as 1,2-dioleoyl-sn- glycero-3-phospho-L-serine (DOPS); diacylglycerols, such as 1,2-dioleoyl-sn-glycerol; monoacylglycerols, such as glyceryl monooleate (monoolein); glycolipids, such as monogalactosyldiacylglycerol (MGDG); bis(monoacylglycero)phosphate (BMP) stereoisomers, including BMP-S,R and BMP-S,S; lysophospholipids containing a single hydrocarbon tail, such as lysophosphatidic acid (LPA) and lysophosphatidylethanolamine (LPE); ceramides and related sphingolipid analogues, such as erythro-C8-ceramide; and unsaturated fatty acids, such as oleic acid. Additionaly, acidic cholesterol esters (e.g. cholesteryl hemissucinate) can be employed as at neutral pH, self-assemble in to lipid bilayers but at acidic pH, adopt a cone-shape structure and promotes the fusion of the membranes,

[0129] Classic LNPs used to deliver RNA therapeutics generally have high percentages of ionizable cationic lipids. The positively charged lipids interact with the negative charges of nucleic acids, and additionally mediate endosomal escape. Classic LNPs have a fatty core and are neutral charged at pH 7 and positive charged at endosomal acidic pH. In contrast, the aLNP of the present disclosure are anionic, and therapeutic nucleic acid payloads (e.g., mRNAs) are at least partially inserted in the hydrophobic core of the lipid bilayer, not exposed to the environment outside of the particle. Advantageously, the aLNP of the present disclosure provide several compartments that can be used to carry different payloads (e.g., therapeutic, diagnostic, marker, or combinations thereof) and / or targeting molecules. For example, mRNA and hydrophobic therapeutic molecules (e.g., anticancer drugs) can be inserted in the hydrophobic core of the membrane; other therapeutic agents that are hydrophilic can be encapsulated in the aqueous core of an aqueous core-type aLNP (low fusogenic lipid content aLNP); therapeutic agents can also be attached to the internal surface of the bilayer, facing the aqueous core; and different molecules (e.g., targeting moieties, therapeutic moieties, detectable moieties, functional moieties, or combinations thereof) can be attached to the external surface of the aLNP. In other cases, therapeutic agents that are encapsulated in micelles of an inverted micelle-type aLNP (high fusogenic lipid content aLNP).

[0130] At the time the present application was filed, the prevailing opinion regarding the presence of negative charge on lipid nanoparticles for nucleic acid delivery was that is was an undesirable characteristic. For example, Carrasco et al. (2021) Communications Biology 4:956, teaches away from using negatively charged lipid nanoparticles due to undesirable off-target effects, explaining that “more negatively charged LNPs exhibit higher off-target systemicexpression of mRNA in the liver following IM administration.” This undesirable systemic off- target expression of mRNA-LNP vaccines could be minimized through appropriate design of the ionizable lipid and LNP.” See also Gyanani and Goswami (2023) Pharmaceutics 15(4): 1184 (“since the cell surface is a negatively charged phospholipid bilayer membrane with embedded biomolecules in a fluid mosaic structure, cell membranes pose a challenge for anionic NPs, as they are repelled by the cell surface leading to the cargo being unable to reach the site of action. Additionally, the key challenge with anionic liposomes is the poor encapsulation of genetic material”).

[0131] In a classic LNP, endosomal escape is in great part mediated by the presence of ionizable cationic lipids. Ionizable lipids acquire a positive charge in a low pH endosomal environment and interact electrostatically with the anionic lipids present in the endosomal membrane. Such interactions generate cationic-anionic ion pairs, which in turn allow for the transition from the lamellar phase to the inverted hexagonal phase, triggering delivery of cargo into the cytoplasm. In other words, the presence of ionizable cationic lipids is an absolute necessity in classic LNP in order to deliver their cargo to the cytoplasm.

[0132] In contrast, the endosomal escape of aLNP of the present disclosure is mediated by the combination of neutral or anionic fusogenic lipids and endosomal escape peptides attached to their external surface. These peptides can be part of a lipid-protein construct inserted in the aLNP bilayer when the aLNP is formed, or they can be subsequently attached to the surface of an aLNP after it has been formed, for example, by covalent derivatization of a component of the aLNP. Thus, in some aspects, the endosomal escape peptide comprises a thiol reactive group that can react with a maleimide group present in a lipid component of the aLNP. In other aspects, the endosomal escape peptide comprises a maleimide reactive group that can react with a thiol group present in a lipid component of the aLNP. Alternative chemistries known in the art can be used to attach the endosomal escape peptides disclosed herein to aLNPs of the present disclosure without undue experimentation.

[0133] In some aspects, the lipidic composition of an aLNP of the present disclosure can be represented according to the formulaN:A:S:[C]:[D]:[P] whereinN is a Neutral lipid (e.g., egg phosphatidylcholine, EPC, DPPC, POPG, DOPC, DSPC, DOPE, Sphingomyelin, or a combination thereof);A is an Anionic lipid (e.g., egg phosphatidylglycerol, EPG, DPPG, POPG, DOPG, DSPG, GM3, GM1, DOPA, DOPS, or a combination); wherein either N or A is a fusogenic lipid; wherein either N or A is a bilayer forming lipid;S is a Structural lipid (e.g., cholesterol or sitosterol);[C] is an optional cationic lipid, e.g., an ionizable cationic lipid, which can be present, e.g., at a molar ratio lower than the A component ([C] mol% < A mol%) and in any case below 20 mol%;[D] is an optimal derivatizable anionic lipid (e.g., Lipid-SH or Lipid-maleimide), peptide-lipid (Lipid-Endosomal escape peptide), or a derivatized lipid (e.g., Lipid-Maleimide-Endosomal escape peptide); and,[P] is a polymer-derived lipid, e.g., a neutral or anionic lipid comprising a polymeric moiety, e.g., a PEG-lipid.

[0134] In some aspects, the anionic (A) or neutral (N) lipid is not DOPG, DOPE, or DSPG.

[0135] In some aspects, the lipidic composition of an aLNP of the present disclosure can be represented according to the formulaN:[A]:S:[C]:[D]:[P] whereinN is a Neutral lipid or combination thereof, wherein a neutral lipid is a fusogenic lipid (e.g., in a specific aspect, a first neutral lipid is DSPC, DPPC or SM and the second neutral lipid is the fusogenic lipid DOPE);A is an optional Anionic lipid;S is a Structural lipid (e.g., cholesterol or sitosterol);[C] is an optional cationic lipid, e.g., an ionizable cationic lipid, which can be present, e.g., at a molar ratio lower than the A component ( [C] mol% < A mol%) and in any case below 20 mol%;[D] is an optimal derivatizable anionic lipid (e.g., Lipid-SH or Lipid-maleimide), peptide-lipid (Lipid-Endosomal escape peptide), or a derivatized lipid (e.g., Lipid-Maleimide-Endosomal escape peptide); and,[P] is a polymer-derived lipid, e.g., a neutral or anionic lipid comprising a polymeric moiety, e.g., a PEG-lipid.

[0136] In some aspects, the lipidic composition of an aLNP of the present disclosure can be represented according to the formula[N]:A:S:[C]:[D]:[P] whereinN is an optional Neutral lipid;A is an Anionic lipid or combination thereof, wherein an anionic lipid is a fusogenic lipid or a bilayer forming lipid;S is a Structural lipid (e.g., cholesterol or sitosterol);[C] is an optional cationic lipid, e.g., an ionizable cationic lipid, which can be present, e.g., at a molar ratio lower than the A component ( [C] mol% < A mol%) and in any case below 20 mol%;[D] is an optimal derivatizable anionic lipid (e.g., Lipid-SH or Lipid-maleimide), peptide-lipid (Lipid-Endosomal escape peptide), or a derivatized lipid (e.g., Lipid-Maleimide-Endosomal escape peptide); and,[P] is a polymer-derived lipid, e.g., a neutral or anionic lipid comprising a polymeric moiety, e.g., a PEG-lipid.

[0137] In some aspects, the aLNP comprises a first neutral lipid (e.g., DSPC), a second neutral lipid wherein the second neutral lipid is a fusogenic lipid (e.g., DOPE), a structural lipid (e.g., an sterol such as sitosterol), and an endosomal escape peptide. In some aspects, the aLNP comprises a first neutral lipid (e.g., DSPC or SM), a second neutral lipid wherein the second neutral lipid is a fusogenic lipid (e.g., DOPE), an anionic lipid (e.g. DSPG or DPPG) a structural lipid (e.g., an sterol such as sitosterol), and an endosomal escape peptide. In some aspects, the aLNP has a molar lipid ratio DOPE:DSPC: sitosterol (53:4:37). In some aspects, the aLNP as a molar ratio DOPE :DSPC: sitosterol :endosomal_escape_peptide (53:5:37:5). endosomal_escape_peptide can be abbreviated in the formulas disclosed in the present application as EPP as used also to describe a lipopeptide.

[0138] In some aspects, the aLNP has a molar lipid ratio DOPE:DSPC:DSPG:sitosterol (23:30:5:37). In some aspects, the aLNP as a molar ratio DOPE:DSPC:DSPG:sitosterol:endosomal_escape_peptide (23:30:5:37:5). In some aspects, the aLNP has a molar lipid ratio DOPE:SM:DSPG: sitosterol (23:30:5:37). In some aspects, the aLNP as a molar ratio DOPE:SM:DSPG:sitosterol:endosomal_escape_peptide (23:30:5:37:5). In some aspects, the aLNP has a molar lipid ratio DOPE:DPPC:DPPG: sitosterol (23:30:5:37). In some aspects, the aLNP as a molar ratio DOPE:DPPC:DPPG: sitosterol :endosomal_escape_peptide (23:30:5:37:5). In some aspects, the aLNP as a molar ratio DOPE :D SPC :D SPG: sitosterol : endosomal_escape_peptide (13:40:5:37:5).

[0139] In some aspects, the aLNP as a molar ratio DOPE:DSPC:DSPG:sitosterol:endosomal_escape_peptide (33:20:5:37:5). In some aspect, the endomal escape peptide is a stearyl conjugate. In some aspect, the endosomal escape peptide is acholesterol conjugate. In some aspect, the endosomal escape peptide is a phoshopolipid conjugate. In some aspect, the endosomal escape peptide is a DSPE conjugate. In some aspect, the endosomal escape peptide is DPPE. In some aspects, the aLNP comprises a formulation set forth in FIG. 16, 17, 18, 20, 21, 23, 24, 25, 26, 29.

[0140] In some aspects, the anionic lipid content (A) is between 0.5 mol% and 15% mol, e.g., about 0.5%, about 1%, about 5%, about 10%, or about 15%, relative to the total mol lipid content of the aLNP; and / or, the neutral lipid content (N) is between 0.5 mol% and 15% mol, e.g., about 0.5%, about 1%, about 5%, about 10%, or about 15%, relative to the total mol lipid content of the aLNP. Increases in the ratio of anionic lipid (A) to neutral lipid (N) result in an increase in the anionic surface of the aLNP and decrease in the particle size.

[0141] Increased anionic lipid (A) ratios to total lipid enhance colloid stability, and aLNP formulations without anionic lipid or anionic lipopeptide aggregate after a few days of storage.

[0142] In some aspects, the aLNP of the present disclosure comprises at least about 10 mol%, at least about 15 mol%, at least about 20 mol%, at least about 25 mol%, at least about 30 mol%, at least about 35 mol%, at least about 40 mol%, at least about 45 mol%, at least about 50 mol%, at least about 55 mol%, at least about 60 mol%, at least about 65 mol%, at least about 70 mol%, at least about 75 mol%, at least about 80 mol%, at least about 85 mol%, about least about 90 mol%, at least about 95 mol%, at least about 99 mol%, or about 100 mol% of anionic lipid plus neutral lipid content (A + N) relative to the total mol lipid of the aLNP.

[0143] In some aspects, the aLNP of the present disclosure comprises about 10 mol%, about 15 mol%, about 20 mol%, about 25 mol%, about 30 mol%, about 35 mol%, about 40 mol%, about 45 mol%, about 50 mol%, about 55 mol%, about 60 mol%, about 65 mol%, about 70 mol%, about 75 mol%, about 80 mol%, about 85 mol%, about 90 mol%, about 95 mol%, about 99 mol%, or about 100 mol% of anionic lipid plus neutral lipid content (A + N) relative to the total mol lipid of the aLNP.

[0144] In some aspects, the aLNP of the present disclosure comprises between about 10 mol% and about 15 mol%, between about 15 mol% and about 20 mol%, between about 20 mol% and about 25 mol%, between about 25 mol% and about 30 mol%, between about 30 mol% and about 35 mol%, between about 35 mol% and about 40 mol%, between about 40 mol% and about 45 mol%, between about 45 mol% and about 50 mol%, between about 50 mol% and about 55 mol%, between about 55 mol% and about 60 mol%, between about 60 mol% and about 65 mol%, between about 65 mol% and about 70 mol%, between about 70 mol% and about 75 mol%, between about 75 mol% and about 80 mol%, between about 80 mol% and about 85 mol%, between about 85mol% and about 90 mol%, between about 90 mol% and about 95 mol%, between about 95 mol% and about 99 mol%, between about 99 mol% and about 100 mol% of anionic lipid plus neutral lipid content (A + N) relative to the total mol lipid of the aLNP.

[0145] In some aspects, the aLNP comprises between about 0.5 mol% and about 80 mol% of one or more anionic lipids relative to total mol lipid present in the aLNP. In some aspects, the aLNP comprises between about 5 mol% and about 40 mol% of one or more anionic lipids relative to total mol lipid present in the aLNP. In some aspects, the aLNP comprises between about 5 mol% and about 30 mol% of one or more anionic lipids relative to total mol lipid present in the aLNP.

[0146] In some aspects, the aLNP comprises between about 1 mol% and about 99.5 mol% of one or more neutral lipids relative to total mol lipid present in the aLNP. In some aspects, the aLNP comprises between about 20 mol% and about 90 mol% of one or more neutral lipids relative to total mol lipid present in the aLNP. In some aspects, the aLNP comprises between about 60 mol% and about 90 mol% of one or more neutral lipids relative to total mol lipid present in the aLNP.

[0147] In some aspects, at least one anionic lipid and / or at least one neutral lipid in an aLNP of the present disclosure is a phospholipid or a sphingolipid.

[0148] In some aspects, the aLNP of the present disclosure comprises (i) between about 0.5 mol% and about 40 mol% of at least one anionic lipid, relative to total mol lipid of the aLNP; (ii) between about 5 mol% and about 35 mol% of at least one anionic lipid, relative to total mol lipid of the aLNP; or, (iii) between about 10 mol% and about 30 mol% of at least one anionic lipid, relative to total mol lipid of the aLNP, when the aLNP is an immunostimulatory composition which, e.g., leads to activation of dendritic cells, activation of macrophages activation of T cells and / or secretion of IFN-alpha.

[0149] In some aspects, the aLNP of the disclosure are immunostimulatory, i.e., they can enable the activation of nucleic acid sensing receptors (e.g., TLR, RLR, or STING), can induce the maturation of immune cells (e.g., macrophages, dendritic cells, or T-cells), or a combination thereof.

[0150] In some aspects, the aLNP of the present disclosure comprises (i) between about 0.5 mol% and about 80 mol% of at least one anionic lipid, relative to total mol lipid of the aLNP; (ii) between about 3 mol% and about 40 mol% of at least one anionic lipid, relative to total mol lipid of the aLNP; or, (iii) between about 5 mol% and about 30 mol% of at least one anionic lipid, relative to total mol lipid of the aLNP, when the aLNP is an immunosilent composition, i.e., it does not lead to immunostimulation such as, e.g., activation of dendritic cells, activation of macrophages, activation of T cells and / or secretion of IFN-alpha

[0151] In some aspects, the aLNP is immunosilent or immunotolerant, i.e., it can reduce the activation of nucleic acid sensing receptors (e.g., TLR, RLR, or STING) and / or can avoid the maturation of immune cells (e.g., macrophages, dendritic cells, or T-cells). In some aspects, the aLNP is non-inflammatory and / or non-immunogenic, i.e., they do not (substantially) result in activation of dendritic cells, activation of macrophages, activation of T cells and / or secretion of IFN-alpha.

[0152] In some aspects, the aLNP comprises less than about 25%, less than about 20 mol%, less than about 19 mol%, less than about 18 mol%, less than about 17 mol%, less than about 16 mol%, less than about 15 mol%, less than about 14 mol%, less than about 13 mol%, less than about 12 mol%, less than about 11 mol%, less than about 10 mol%, less than about 9 mol%, less than about 8 mol%, less than about 7 mol%, less than about 6 mol%, less than about 5 mol%, less than about 4 mol%, less than about 3 mol%, less than about 2 mol%, less than about 1 mol%, less than about 0.9 mol%, less than about 0.8 mol%, less than about 0.7 mol%, less than about 0.6 mol%, less than about 0.5 mol%, less than about 0.4 mol%, less than about 0.3 mol%, less than about 0.2 mol%, less than about 0.1 mol%, of about 0 mol% of one or more cationic lipids relative to total mol lipid present in the aLNP.

[0153] In some aspects, the aLNP comprises less than about 25%, less than about 20 mol%, less than about 19 mol%, less than about 18 mol%, less than about 17 mol%, less than about 16 mol%, less than about 15 mol%, less than about 14 mol%, less than about 13 mol%, less than about 12 mol%, less than about 11 mol%, less than about 10 mol%, less than about 9 mol%, less than about 8 mol%, less than about 7 mol%, less than about 6 mol%, less than about 5 mol%, less than about 4 mol%, less than about 3 mol%, less than about 2 mol%, less than about 1 mol%, less than about 0.9 mol%, less than about 0.8 mol%, less than about 0.7 mol%, less than about 0.6 mol%, less than about 0.5 mol%, less than about 0.4 mol%, less than about 0.3 mol%, less than about 0.2 mol%, less than about 0.1 mol%, of about 0 mol% of one or more cationic lipids, cationic polymers, multivalent cations, or any combination thereof relative to total mol lipid present in the aLNP.

[0154] In some aspects, the aLNP comprises at least about 5 mol%, at least about 10 mol%, at least about 15 mol%, at least about 20 mol%, at least about 25 mol%, at least about 30 mol%, at least about 35 mol%, at least about 40 mol%, at least about 45 mol%, at least about 50 mol%, at least about 55 mol%, at least about 60 mol%, at least about 65 mol%, at least about 70 mol%, at least about 75 mol%, at least about 80 mol%, at least about 85 mol%, at least about 90 mol%, or at least about 95 mol% of at least one sterol, e.g., cholesterol, relative to total mol lipid (including sterol) of the aLNP.

[0155] In some aspects, the aLNP comprises less than about 95 mol%, less than about 90 mol%, less than about 85 mol%, less than about 80 mol%, less than about 75 mol%, less than about 70 mol%, less than about 65 mol%, less than about 60 mol%, less than about 55 mol%, less than about 50 mol%, less than about 45 mol%, less than about 40 mol%, less than about 35 mol%, less than about 30 mol%, less than about 25 mol%, less than about 20 mol%, less than about 15 mol%, less than about 10 mol%, or less than about 5 mol% of at least one sterol, e.g., cholesterol, relative to total mol lipid (including sterol) of the aLNP.

[0156] In some aspects, the aLNP comprises between about 5 mol% and about 50 mol% sterol, e.g., cholesterol, relative to total of mol lipid (including sterol) of the aLNP. In some aspects, the aLNP comprises between about 10 mol% and about 40 mol% sterol, e.g., cholesterol, relative to total of mol lipid (including sterol) of the aLNP. In some aspects, the aLNP comprises between about 30 mol% and about 35 mol% sterol, e.g., cholesterol, relative to total of mol lipid (including sterol) of the aLNP.

[0157] In some aspects, the aLNP is free of or substantially free of cationic lipids. In some aspects, the aLNP of the present disclosure comprise less than about 1 mol%, less than about 0.1 mol%, less than about 0.01 mol%, less than about 0.001 mol%, less than about 0.0001 mol%, or about 0 mol% of (at least one) cationic lipid, cationic polymer, or multivalent cationic ion relative to the total mol lipid of the aLNP. In some aspects, the aLNP is free of polymers (other than the nucleic acid payload), e.g., cationic polymers. In some aspects, the aLNP comprises less than about 1 mol%, less than about 0.1 mol%, less than about 0.01 mol%, less than about 0.001 mol %, less than about 0.0001 mol%, or about 0 mol% of polymer (other than the nucleic acid payload), e.g., cationic polymer, with respect to the total mol lipid of the aLNP.

[0158] In some aspects, the aLNP of the present disclosure (i) does not contain cationic lipid and / or comprise less than about 1 mol%, less than about 2 mol%, less than about 3 mol%, less than about 4 mol%, or less than about 5 mol% of cationic lipid relative to total mol lipid of the aLNP; (ii) does not contain cationic polymer and / or comprise less than about 1 mol%, less than about 2 mol%, less than about 3 mol%, less than about 4 mol%, or less than about 5 mol% of cationic lipid relative to total mol lipid of the aLNP; (iii) does not contain multivalent cation and / or comprise less than about 1 mol%, less than about 2 mol%, less than about 3 mol%, less than about 4 mol%, or less than about 5 mol% of cationic lipid relative to total mol lipid of the aLNP; or, (iv) any combination thereof.

[0159] The phospholipid content of the aLNP of the present disclosure can be determined using any of the methods known in the art, e.g., the Rouser assay (Rouser et al. Lipids 5, 494-496 (1970))or liquid chromatography (LC) couple with charge aerosol detector (CAD) or coupled evaporative light scattering detector (ELSD) . In some aspects, the aLNP has a net negative charge in aqueous medium at physiologically acceptable pH (e.g., pH 7.0-7.8, preferably at ~7.4), e.g., in O.lx PBS.

[0160] In some aspects, net negative charge refers to a zeta potential of less than about -2 mV, measured as described below. In some aspects, net negative charge refers to a zeta potential of less than about -10 mV. In some aspects, net negative charge refers to a zeta potential of less than about -50 mV. In some aspects, the zeta potential is less than about -50 mV, less than about -45 mV, less than about -40 mV, less than about -35 mV, less than about -30 mV, less than about -25 mV, less than about -20 mV, less than about -15 mV, less than about -10 mV, less than about -9 mV, less than about -8 mV, less than about -7 mV, less than about -6 mV, less than about -5 mV, less than about -4 mV, less than about -3 mV, or less than about -2 mV. In some aspects, the zeta potential is about -50 mV, about -45 mV, about -40 mV, about -35 mV, about -30 mV, about -25 mV, about -20 mV, about -15 mV, about -10 mV, about -9 mV, about -8 mV, about -7 mV, about -6 mV, about -5 mV, about -4 mV, about -3 mV, or about -2 mV. In some aspects, the zeta potential is between about -2 mV and about -10 mV, between about -10 mV and about -20 mV, between about -20 mV and about -30 mV, between about -30 mV and about -40 mV, or about -40 mV and about -50 mV, between about -5 mV and about -15 mV, between about -15 mV and about -25 mV, between about -25 mV and about -35 mV, between about -25 mV and about -35 mV, between about -35 mV and about -45 mV, between about -2 mV and about -20 mV, between about -10 mV and about -30 mV, between about -15 mV and about -35 mV, between about -20 mV and about - 40 mV, between about -25 mV and about -45 mV, between about -30 mV and about -50 mV, between about -2 mV and about -25 mV, between about -2 mV and about -35 mV, between about -2 mV and about -50 mV, between about -10 mV and about -50 mV, between about -15 mV and about -50 mV, or between about -20 mV and about -50 mV.

[0161] In some aspects, the aLNP has a size (diameter) between about 50 nm and about 300 nm. In some aspects, the aLNP has a size (diameter) of about 25 nm, about 50 nm, about 75 nm, about 100 nm, about 125 nm, about 150 nm, about 175 nm, about 200 nm, about 225 nm, about 250 nm, about 275 nm, about 300 nm, about 325 nm, about 350 nm, about 375 nm, about 400 nm, about 425 nm, about 450 nm, about 475, or about 500 nm. In some aspects, the aLNP has a size (diameter) of less than about 25 nm, less than about 50 nm, less than about 75 nm, less than about 100 nm, less than about 125 nm, less than about 150 nm, less than about 175 nm, less than about 200 nm, less than about 225 nm, less than about 250 nm, less than about 275 nm, less than about 300 nm, less than about 325 nm, less than about 350 nm, less than about 375 nm, less than about 400 nm,less than about 425 nm, less than about 450 nm, less than about 475, or less than about 500 nm. In some aspects, the aLNP has a size (diameter) between about 25 nm and about 50 nm, between about 50 nm and about 75 nm, between about 75 nm and about 100 nm, between about 100 nm and about 125 nm, between about 125 nm and about 150 nm, between about 150 nm and about 175 nm, between about 175 nm and about 200 nm, between about 200 nm and about 225 nm, between about 225 nm and about 250 nm, between about 250 nm and about 275 nm, between about 275 nm and about 300 nm, between about 300 nm and about 325 nm, between about 325 nm and about 350 nm, between about 350 nm and about 375 nm, between about 375 nm and about 400 nm, between about 400 nm and about 425 nm, between about 425 nm and about 450 nm, between about 450 nm and about 475 nm, between about 475 nm and about 500 nm, between about 50 nm and about 100 nm, between about 100 nm and about 150 nm, between about 150 nm and about 200 nm, between about 200 nm and about 250 nm, between about 250 nm and about 300 nm, between about 300 nm and about 350 nm, between about 350 nm and about 400 nm, between about 400 nm and about 450 nm, between about 450 and 500 nm, between about 50 nm and about 150 nm, between about 100 nm and about 200 nm, between about 150 nm and about 250 nm, between about 200 nm and about 300 nm, between about 250 nm and about 350 nm, between about 300 nm and about 400 nm, between about 350 nm and about 450 nm, between about 400 nm and about 500 nm, between 50 nm and about 250 nm, between about 50 nm and about 300 nm, between about 100 nm and about 400 nm, between about 100 nm and about 500 nm, or between 150 nm and about 350 nm.

[0162] In some aspects, the aLNP of the present disclosure encapsulates one or more nucleic acids (e.g., mRNA), wherein at least about 1, at least about 2, at least about 3, at least about 4, at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, at least about 10, at least about 15, or at least about 20 wt.% of the one or more nucleic acids in the aLNP payload is encapsulated within the outer bilayer of the aLNP. In some aspects, the aLNP of the present disclosure encapsulates one or more nucleic acids (e.g., mRNA), wherein about 1, about 2, at about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 15, or about 20 wt.% of the one or more nucleic acids in the aLNP payload is encapsulated within the outer bilayer of the aLNP.

[0163] In some aspects, at least about 1, at least about 2, at least about 3, at least about 4, at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, at least about 10, at least about 15, or at least about 20 wt.% or all of the one or more nucleic acids in the aLNP payload is comprised in the hydrophobic region of the one or more lipid bilayer of an aLNP, preferably in theform of nucleic acid-lipid complexes (e.g., wherein hydrophobic moieties of the one or more nucleic acid in the aLNP payload interact with lipid tails of the one or more lipid bilayer).

[0164] In some aspects, at least about 1, at least about 2, at least about 3, at least about 4, at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, at least about 10, at least about 15, or at least about 20 wt.% or all of the one or more nucleic acid in the aLNP payload is not in contact with aqueous medium (e.g. as comprised in the aqueous core of the aLNP or surrounding the aLNP).

[0165] In some aspects, less than about 1, less than about 2, less than about 3, less than about 4, less than about 5, less than about 6, less than about 7, less than about 8, less than about 9, less than about 10, less than about 15, or less than about 20 wt.% of the one or more nucleic acid in the aLNP payload is exposed on (or adsorbed to) the outside of the aLNP. In some aspects, less than about 1, less than about 2, less than about 3, less than about 4, less than about 5, less than about 6, less than about 7, less than about 8, less than about 9, less than about 10, less than about 15, or less than about 20 wt.% of the one or more nucleic acid in the aLNP payload is comprised in the (aqueous) core of the aLNP.

[0166] In some aspects, at least about 1, at least about 2, at least about 3, at least about 4, at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, at least about 10, at least about 15, at least about 20, at least about 25, at least about 30, at least about 35, at least about 40, at least about 45, at least about 50, at least about 55, at least about 60, at least about 65, at least about 70, at least about 75, at least about 80, at least about 85, at least about 90, at least about 91, at least about 92, at least about 93, at least about 94, at least about 95, at least about 96, at least about 97, at least about 98, or at least about 99 wt.% of the one or more nucleic acid in the aLNP payload is present in the one or more lipid bilayer, relative to total weight of the one or more nucleic acid of the aLNP.

[0167] In some aspects, about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 55, about 60, about 65, about 70, about 75, about 80, about 85, about 90, about 91, about 92, about 93, about 94, about 95, about 96, about 97, about 98, or about 99 wt.% of the one or more nucleic acid in the aLNP payload is present in the one or more lipid bilayer, relative to total weight of the one or more nucleic acid of the aLNP.

[0168] In some aspects, the one or more nucleic acid in the aLNP payload is comprised in the particles or in the one or more lipid bilayer thereof in the form of nucleic acid- lipid complexes, preferably stabilized by hydrophobic interaction(s) between lipid and nonpolar moieties of thenucleic acid. In addition or alternatively, the one or more nucleic acid in the aLNP payload is comprised / encapsulated in the aLNP or in the one or more lipid bilayer thereof, wherein the encapsulation is not or substantially not by electrostatic interaction(s).

[0169] In some aspects, the nucleic acid payload in an aLNP of the present disclosure can have any length of, for example, at least about 10 nucleotides, at least about 15 nucleotides, at least about 20 nucleotides, at least about 25 nucleotides, at least about 30 nucleotides, at least about 35 nucleotides, at least about 40 nucleotides, at least about 50 nucleotides, at least about 60 nucleotides, at least about 70 nucleotides, at least about 80 nucleotides, at least about 90 nucleotides, at least about 100 nucleotides, at least about 200 nucleotides, at least about 300 nucleotides, at least about 400 nucleotides, at least about 500 nucleotides, at least about 600 nucleotides, at least about 700 nucleotides, at least about 800 nucleotides, at least about 900 nucleotides, at least about 1000 nucleotides (1 kb), at least about 2 kb, at least about 10 kb, at least about 20 kb, at least about 30 kb, at least about 40 kb, at least about 50 kb, at least about 60 kb, at least about 70 kb, at least about 80 kb, at least about 90 kb, at least about 100 kb.

[0170] In some aspects, the nucleic acid payload in an aLNP of the present disclosure can have a length of, for example, less than 1000 nucleotides. In some aspects, the nucleic acid payload in an aLNP of the present disclosure can have a length of, for example, less than 100 nucleotides. In some aspects, the nucleic acid payload in an aLNP of the present disclosure can have a length of, for example, about 10 nucleotides to about 100 kb. In some aspects, the nucleic acid payload in an aLNP of the present disclosure can have a length of, for example, about 0.1 kb to about 20 kb.

[0171] In some aspects, the one or more lipid bilayer of the present aLNP is / are arranged at least about 30, at least about 35, at least about 40, at least about 45, at least about 50, at least about 55, at least about 60, at least about 65, at least about 70, at least about 75, at least about 80, at least about 85, at least about 90, or at least about 95% in a lamellar phase structure. In some aspects, the one or more lipid bilayer of the present aLNP is / are arranged about 30, about 35, about 40, about 45, about 50, about 55, about 60, about 65, about 70, about 75, about 80, about 85, about 90, or about 95% in a lamellar phase structure. A person skilled in the art can characterize the assembly of the lipid phases and overall structure of the aLNP using electron microscopy, nuclear magnetic resonance (NMR), small angle X-ray scattering (SAXS) and / or small angle neutron scattering (SANS).

[0172] In some aspects, the aLNP as disclosed herein preferably has a core-shell structure (with a lipid shell) and / or a core encapsulated by the one or more lipid bilayer, e.g. determined by SANS, and / or wherein the core comprises at least about 30, at least about 40, at least about 50, at leastabout 60, at least about 70, at least about 80, at least about 90 vol.% aqueous medium / aqueous solvent, relative to total core volume. The aqueous medium / solvent content in the core compartment can be determined by SANS solvent contrast variation technique, using the calculated neutron SLD values of the components and their volume fraction. In addition and / or alternatively, the aqueous compartment comprises at most about 25, at most about 20, at most about 15, at most about 10, at most about 5, at most about 4, at most about 2, at most about 1 wt.% nucleic acid, relative to total nucleic acid payload of the aLNP.

[0173] In some aspects, at least about 5, at least about 10, at least about 15, at least about 20, at least about 25, 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 99 wt.% (e.g. 100 wt.%) of the one or more nucleic acid in the aLNP is present in the one or more lipid bilayer, relative to the total weight of nucleic acid payload in the aLNP. Presence of nucleic acid in the one or more lipid bilayer can be evident from absence of nucleic acid in the (aqueous) core. In addition or alternatively, presence of nucleic acid in the one or more lipid bilayer can change the neutron contrast of the anionic lipid nanoparticle shell, the bilayer fluidity properties and / or the thermal properties of the nucleic acid.

[0174] In some aspects, the aLNP of the present disclosure comprises the following characteristics (i) the aLNP has a net negative charge, e.g., with a zero potential between -2 mV and -50 mV; (ii) the aLNP is nanosized, e.g., with an average size (diameter) between about 50 nm and about 300 nm; (iii) the aLNP comprises a classic lipid bilayer; (iv) the aLNP comprises an aqueous core; (v) the aLNP is unilamellar; (vi) the aLNP does not comprise a cationic lipid or cationic polymer, e.g., an ionizable cationic lipid, or comprises at most 20 mol% of a cationic molecule, such as cationic lipid, cationic polymer or multivalent cationic ion (e.g. Ca2+), relative to total mol lipid of the aLNP; (vii) the aLNP can encapsulate a nucleic acid payload (e.g., an mRNA) at least partially (e.g., at least 20% of the nucleic acid payload) within the hydrophobic core of the lipid bilayer; and, (viii) the aLNP comprises at least one endosomal escape peptide covalently attached to its external surface or attached as a lipopeptide.

[0175] In some aspects, the aLNP of the present disclosure comprises (i) lipids selected from the group consisting of glycerolphospholipids, glycosphingolipids, phospholipids, cholesterol, and combinations thereof; (ii) a lipid bilayer comprising lipids conferring a net anionic charge; (iii) one or more payloads, e.g., a nucleic acid payload embedded in one of the bilayers; (iv) at least one fusion protein, peptide, fragment thereof, or combination thereof coupled (e.g., covalently linked) to the outer surface of the aLNP, wherein the fusion protein, peptide, fragment thereof, orcombination thereof mediate the fusion of the aLNP with endosomal membranes and / or stabilization of the particle and can also contribute to the negative charge of the aLNP; (v) no (ionizable) cationic lipids, peptides, polymers, or cations; (vi) a size between about 50 nm and about 300 nm or between about 50 nm and about 250 nm; (vii) a poly dispersity index (PDI) between about 0.01 and about 0.3; and, (viii) a zero potential below -10 mV, e.g., between -100 mV and -10 mV.

[0176] The aLNP of the present disclosure comprise at least one endosomal escape peptide attached to the surface of the aLNP. In some aspects, the endosomal escape peptide is in a lipopeptide form, i.e., the endosomal escape peptide is conjugated to a lipid moiety that is inserted in the external face of the lipid bilayer of the aLNP.

[0177] In some aspects, the endosomal escape peptide is a viral hemagglutinin (HA) fusion protein. In some aspects, the endosomal escape peptide is a GALA peptide or a variant thereof. GALA is a endosomal escape peptide having the sequence of SEQ ID NO: 1029 with is negatively charged at pH 7, but at the lower endosomal pH its glutamic acid residues become protonated causing the adoption of a helical structure and subsequent interaction of the peptide with the endosome (lysosome) membrane. See Li & Szoka (2004) Advanced drug delivery reviews, 56(7), 967-985, which is herein incorporated by reference in its entirety. The isoelectric point of glutamic acid is 3.22, which means that protonation is only completed at the very acid lysosomal compartment, where the degradation of the cargo might have already occurred. To induce endosomal escape earlier in the endocytic pathway, secondary structures can be alternatively formed by salt bridges. This is achieved by the inclusion of histidines (pK=6) in the peptide sequence. At the pH of early endosomes (pH 6-6.5), all histidines are positively charged, while almost all glutamic acid or aspartic acid are negatively charged, leading to the interaction of these opposite charged residues. In some aspects, the endosomal escape peptide attached to an aLNP of the present disclosure has an amino acid sequence set forth in SEQ ID NO: 1 to SEQ ID NO: 1118 or SEQ ID NO : 1310 to SEQ ID NO : 1335

[0178] In some aspects, the endosomal escape peptide comprises less than about 25%, less than about 20 mol%, less than about 19 mol%, less than about 18 mol%, less than about 17 mol%, less than about 16 mol%, less than about 15 mol%, less than about 14 mol%, less than about 13 mol%, less than about 12 mol%, less than about 11 mol%, less than about 10 mol%, less than about 9 mol%, less than about 8 mol%, less than about 7 mol%, less than about 6 mol%, less than about 5 mol%, less than about 4 mol%, less than about 3 mol%, less than about 2 mol%, less than about 1 mol%, less than about 0.9 mol%, less than about 0.8 mol%, less than about 0.7 mol%, less thanabout 0.6 mol%, less than about 0.5 mol%, less than about 0.4 mol%, less than about 0.3 mol%, less than about 0.2 mol%, less than about 0.1 mol%, of about 0 mol% relative to total mol lipid present in the aLNP. In some aspects, the endosomal escape peptide compromises about 0.5% mol to 15% mol relative to total mol lipid present in the aLNP. In some aspects, the endosomal escape peptide compromises about 2.5% mol to 10% mol relative to total mol lipid present in the aLNP.

[0179] In some aspects, the endosomal escape peptide is attached to the outer bilayer of an aLNP of the present disclosure through a lipidic anchor molecule, i.e., in some aspects, the endosomal escape peptide is part of a lipopeptide (“endosomal escape lipopeptide”). In some aspects, the lipidic anchor molecule is attached to the C-terminus of the endosomal escape peptide. In some aspects, the lipidic anchor molecule is attached to N-terminus of the endosomal escape peptide. In some aspects, the lipid anchor molecule is attached to the side chain of an amino acid in the endosomal escape peptide. In some aspects, the lipid anchor molecule is attached to the thiol group of a cysteine in the endosomal escape peptide (e.g., via maleimide chemistry). In some aspects, the lipid anchor molecule is attached to the amino group of the side chain of a lysine in the endosomal escape peptide (e.g., via N-hydroxy succinimide chemistry) or in the hydroxyl group of the side chain of a serine, threonine, or tyrosine (e.g. via ester or amides linkages).

[0180] In some aspects, the endosomal escape peptide has a net negative charge at physiological pH.

[0181] In some aspects, the endosomal escape peptide is a peptide conforming to the following rules:(i) a length between 15 and 100 amino acids;(ii) one or more aromatic amino acid residues such as tryptophan, threonine or tyrosine, preferentially tryptophan, as one of the five amino acids closest to the C- and / or N-terminal of the sequence, to promote the anchoring of the endosomal escape peptide in the lipid bilayers of the aLNP and endosome membrane;(iii) an (amphiphilic) peptide subsequence, containing at least 30% of hydrophobic residues (e.g. valine, alanine, leucine or isoleucine), and at least 2 anionic amino acids (e.g. glutamic acid or aspartic acid);(iv) at least 2 histidines are included in the sequence;(v) at least one salt bridge between protonated histidine residues and anionic glutamic or aspartic residues to promote the formation of the secondary structure and the neutralization of the anionic charges.

[0182] In some aspect, the endosomal escape peptide (e.g., a variant of anendosomal escape peptide or lipopeptide having an amino acid sequence set forth in SEQ ID NO: 1 to SEQ ID NO: 1118, or SEQ ID NO: 1310 to SEQ ID NO: 1335) comprises one or more aromatic amino acid residue such as tryptophan, threonine or tyrosine, preferentially tryptophan, as one of the five amino acids closest to the C- and / or N-terminal of the sequence, to promote the anchoring of the endosomal escape peptide (e.g., an endosomal escape peptide or lipopeptide having an amino acid sequence set forth in SEQ ID NO: 1 to SEQ ID NO1118 or SEQ ID NO: 1310 to SEQ ID NO: 1335) in the lipid bilayers of the aLNP and endosome membrane.

[0183] In some aspects, the endosomal escape peptide (e.g., an endosomal escape peptide or lipopeptide having an amino acid sequence set forth in SEQ ID NO: 1 to 1118 or SEQ ID NO: 1310 to SEQ ID NO: 1335) comprises an amphiphilic peptide, containing at least 40% of hydrophobic residues (e.g. valine, alanine, leucine or isoleucine), and at least 2 anionic amino acids (e.g. glutamic acid or aspartic acid). In certain aspects, at least 2 histidines are included in the sequence. In certain aspects, the salt bridge between protonated histidine residues and anionic glutamic or aspartic residues promote the formation of the secondary structure and the neutralization of the anionic charges of the endosomal escape peptide (e.g., an endosomal escape peptide or lipopeptide having an amino acid sequence set forth in SEQ ID NO: 1 to SEQ ID NO: 1118 or SEQ ID NO: 1310 to SEQ ID NO: 1335) at the acidic pH present in endosomes.

[0184] In some aspects, the endosomal escape peptide (e.g., an endosomal escape peptide or lipopeptide having an amino acid sequence set forth in SEQ ID NO: 1 to SEQ ID NO: 1118 or SEQ ID NO: 1310 to SEQ ID NO: 1335) is not required for an effective encapsulation of the nucleic acid payload of the aLNP (e.g., an RNA such as an mRNA). In some aspect, the encapsulation efficiency is at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, or at least about 70%. In some aspect, the encapsulation efficiency is about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, or about 70%. In some aspect, the encapsulation efficiency is between about 20% and about 30%, between about 25% and about 35%, between about 30% and about 40%, between about 35% and about 45%, between about 40% and about 50%, between about 45% and about 55%, between about 50% and about 60%, between about 55% and about 65%, between about 60% and about 70%, between about 20% and about 40%, between about 30% and about 50%, between about 40% and about 60%, between about 50% and about 70%, between about 20% and about 50%, between about 30% and about 60%, between about 40% and about 70%, between about 20% and about60%, between about 30% and about 70%, or between about 20% and about 70%. In some aspects, the encapsulation efficiency is higher than about 70%; thus, in some aspects, the encapsulation efficiency is about 75%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100%.

[0185] In some aspects, the endosomal escape peptide (e.g., an endosomal escape peptide or lipopeptide having an amino acid sequence set forth in SEQ ID NO: 1 to SEQ ID NO: 1118 or SEQ ID NO: 1310 to SEQ ID NO: 1335) comprises a peptide sequence between 5 amino acids and 50 amino acids in length. In some aspects, the endosomal escape peptide (e.g., an endosomal escape peptide or lipopeptide having an amino acid sequence set forth in SEQ ID NO: 1 to SEQ ID NO: 1118 to SEQ ID NO: 1335) comprises a peptide sequence of less than 50 amino acids in length. In some aspects, the endosomal escape peptide (e.g., an endosomal escape lipopeptide) comprises a peptide sequence of less than 40 amino acids in length. In some aspects, the endosomal escape peptide (e.g., an endosomal escape lipopeptide) comprises a peptide sequence of less than 35 amino acids in length. In some aspects, the endosomal escape peptide (e.g., an endosomal escape lipopeptide) comprises a peptide sequence of less than 20 amino acids in length. In some aspects, the endosomal escape peptide or the endosomal escape peptide portion of an endosomal escape lipopeptide has a length of 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 amino acids. In some aspects, the endosomal escape peptide or the endosomal escape peptide portion of an endosomal escape lipopeptide has a length of about 5 to about 10, about 10 to about 15, about 15 to about 20, about 20 to about 25, about 25 to about 30, about 30 to about 35, about 35 to about 40, about 40 to about 45, about 45 to about 50, about 10 to about 20, about 20 to about 30, about 30 to about 40, about 40 to about 50, about 10 to about 20, about 20 to about 40, about 40 to about 50, about 10 to about 40, about 20 to about 50, about 5 to about 50, or about 10 to about 50 amino acids.

[0186] Exemplary endosomal escape peptides that can be incorporated to an aLNP of the present disclosure comprise peptides having the amino sequences set forth in SEQ ID NOS: 1034, 1041, 1038, 1054, 1051, 1050, 1049, 60, 364, or 610. In some aspects, the endosomal escape peptide has the amino acid sequence set forth in SEQ ID NO: 1034. In some aspects, the endosomal escape peptide has the amino acid sequence set forth in SEQ ID NO: 1041. In some aspects, the endosomal escape peptide has the amino acid sequence set forth in SEQ ID NO: 1038. In some aspects, the endosomal escape peptide has the amino acid sequence set forth in SEQ ID NO: 1054. In some aspects, the endosomal escape peptide has the amino acid sequence set forth in SEQ ID NO: 1051.In some aspects, the endosomal escape peptide has the amino acid sequence set forth in SEQ ID NO: 1050. In some aspects, the endosomal escape peptide has the amino acid sequence set forth in SEQ ID NO: 1049 In some aspects, the endosomal escape peptide has the amino acid sequence set forth in SEQ ID NO: 60. In some aspects, the endosomal escape peptide has the amino acid sequence set forth in SEQ ID NO: 364. In some aspects, the endosomal escape peptide has the amino acid sequence set forth in SEQ ID NO: 610.

[0187] In some aspects, the endosomal escape peptide is a functional fragment or functional variant of an endosomal escape peptide sequence disclosed herein. In the context of the present disclosure, a “functional fragment” is a sequence derived from an endosomal escape peptide sequence disclosed herein wherein the peptide has a C-terminal truncation, N-terminal terminal truncation, internal truncation, or a combination thereof, wherein the resulting peptide is still capable of functioning as an endosomal escape peptide as disclosed herein. In the context of the present disclosure, a “functional variant” is a sequence derived from an endosomal escape peptide sequence disclosed herein wherein the peptide has a mutation or combination thereof, e.g., amino acid substitutions (conservative and / or non-conservative), insertions, or deletions, wherein the resulting peptide is still capable of functioning as an endosomal escape peptide as disclosed herein. In some aspects, the endosomal escape peptide has about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity with respect to the sequence of an endosomal escape peptide disclosed herein, wherein the resulting peptide is still capable of functioning as an endosomal escape peptide as disclosed herein.

[0188] In some aspects, the endosomal escape lipopeptide is anionic at physiological pH (approx. pH 7.4) and has pH-dependent membrane interaction activity and fusogenicity. In some aspects, the peptide portion of the endosomal escape lipopeptide assumes a random coil conformation at physiological pH with reduced capacity to induce the lysis of the membrane of red blood cells. In some aspects, when the aLNP is at the acidic endosomal pH (approx. pH 4-6.5) the peptide portion of the endosomal escape lipopeptide assumes a secondary structure such as an alpha-helix, beta sheet, or combination thereof, that promotes the insertion of the peptide into the endosome membrane and the fusion of the aLNP of the present disclosure with the endosome membrane, therefore releasing the aLNP payload.

[0189] Instead of disrupting the membrane of a target cell or endosomal membrane like cationic peptides (e.g., arginine- and / or lysine-rich peptides) or ionizable cationic lipids, the endosomal escape lipopeptides disclosed herein use a different mechanism. The endosomal escape lipopeptides disclosed herein bring the aLNP bilayer into close proximity with endosomalmembranes, promoting their fusion, which is an otherwise unfavorable process due to charge repulsion. One advantage of the aLNP system disclosed herein is that the integrity of the endosomal membrane is preserved, aided by the use of cone shaped lipid. This is an important factor to consider since damage to endosomal compartments is known to cause the activation of inflammatory pathways and apoptosis (Meyer & Kravic (2024) Annual Review of Biochemistry, 93). In contrast, encapsulating a nucleic acid payload (e.g., an mRNA) in the hydrophobic core of the outer bilayer of an aLNP of the present disclosure, wherein the aLNP comprises an endosomal escape lipopeptide anchored to the external surface of its out bilayer, results in a highly biocompatible and stable structure having an enhanced uptake by cells in the body compared to classic LNP or classic liposomes.

[0190] The highly anionic nucleic acid payloads of aLNP of the present disclosure, the negative charge of the bilayers due to the presence of neutral and / or anionic lipids, the absence of cationic components compensating the negative charges, and the presence of anionic endosomal escape lipopeptides, and the use of a fusogenic peptide to stabilize a nanoparticle enriched with cone shape lipids, present such repulsive electrostatic forces that a person of ordinary skill in the art would assume that they would prevent the formation of an organized nanosized lipid particle and efficient encapsulation of the nucleic acid payload. Considering the prevalence of negative charges in the aLNP delivery system disclosed herein, a person of ordinary skill in the art would not have considered the assembly of the disclosed aLNP to be possible. The person of ordinary skill in the art would have considered that the energy barrier posed by the prevalence of negative charges would prevent self-assembly of the aLNP.

[0191] Not only was the assembly of the aLNP unexpected; it was also unexpected (i) that the assembled aLNP were highly stable, (ii) that the aLNP were capable of encapsulating a high load of nucleic acid, (iii) that the negatively charged nucleic acid would be stably embedded in the hydrophobic core of the aLNP bilayer, (iv) that the aLNP would have a controlled mean particle size distribution, (v) that the aLNP would have a controlled polydispersity, (vi) that the aLNP would be highly stable during storage, (vii) that the aLNP would be highly stable after injection in the body of a subject, that the aLNP would result in high transfection efficient comparable to classical LNP containing 50% mol of ionizable cationic lipids, without any observable toxicity. For example, the aLNP of the present disclosure are physically stable during storage for 5 months, at 5°C or -20°C. Structural characterization of the aLNP of the present disclosure shows that the nucleic acid payload (e.g., an RNA such as an mRNA) is mainly embedded within the hydrophobic core of the lipid bilayer of the aLNP.

[0192] Encapsulation of nucleic acids in anionic or neutral liposomes was considered very inefficient according to the art at the time the present application was filed. Such encapsulation is generally conducted by condensation of the nucleic acids with cationic molecules. The use of multivalent cationic ions such as Ca2+to encapsulate nucleic acids in anionic liposomes leads to the formation of aggregates and significantly increases of the mean size of the liposomes to values significantly higher than about 220 nm. This is a disadvantage if the liposomes containing nucleic acids are administered as an injection, as it is then required to deploy a sterile filtration step in the manufacturing process. Sterile filtration is done by passing the formulation through a 0.22 pm rated sterilizing-grade filters. Any particles present that are greater than 220 nm can cause significant reduction in the filter throughput or eventually clog the filters, negatively affecting the manufacturability of the formulation. See Bailey et al. (2000) Biochim Biophys Acta. 1468:239- 52; Patil et al. (2004) AAPS J 6: 13-22; and Kapoor et al. (2012) International Journal of Pharmaceutics 432: 80-90; which are herein incorporated by reference in their entireties. An advantage of the aLNP of the present disclosure is that efficient encapsulation of the nucleic acids is possible without the need of a condensation step requiring cationic components in the delivery system. Another advantage is that the aLNP of the present disclosure can have a mean size below 220 nm, making it suitable for a sterile filtration step. Additionally, in contrast to prior art thin film method, the present microfluidics method used in the disclosure allows the upscaling of the production.

[0193] aLNP of the present disclosure prepared with low percentages of fusogenic lipids (e.g., 5, 10, 15, 20, 25, 30, 35%, 40 or 50%) have an aqueous core and one or more lipid bilayers, each one of which can contain the nucleic acid payload (e.g., an RNA such as an mRNA) embedded in the hydrophobic core of each bilayer. In contrast, a classic LNP has a solid core containing RNA, cationic lipids, and cholesterol with low water content (27% water volume vs total core volume) and the lipids organized in inverted hexagonal phases (Hu) as compared to regular liposome particles (Arteta et al 2018, PNAS 115(15):E3351 -E3360). The localization of the nucleic acids in the hydrophobic core of the lipid bilayers of the aLNP of the present disclosure, a location devoid of water, explains the stable preservation of nucleic acid content and biological activity during storage. The nucleic acid payload of the aLNP of the present disclosure (i) remains intact, (ii) does not aggregate, (iii) is not released (leaked) from the aLNP, and (iv) is protected from nuclease degradation when incubated with human plasma at 37° C.

[0194] In some aspects, the aLNP of the present disclosure does not comprise a SARS-Cov2 vaccine. In some aspects, the aLNP of the present disclosure does not comprise a SARS-Cov2 antigen, or a polynucleotide (e.g., mRNA or vector) encoding a SARS-Cov2 antigen.

[0195] In some aspects, the aLNP of the present disclosure is highly stable and can be stored between pH 6.5 and pH 7.5 without premature fusion of the bilayers.

[0196] In some aspects, the aLNP composition of the present disclosure is in liquid form. In some aspects, the aLNP composition of the present disclosure is in solid form. In some aspects, the aLNP composition of the present disclosure is in a powder form. In some aspects, the aLNP composition of the present disclosure is a lyophilized composition. In some aspects, the aLNP composition of the present disclosure is a frozen composition.

[0197] The present disclosure also provides a manufacturing process in which all the components of the aLNP are incorporated via a unique manufacturing step. In this process, the lipids forming the anionic bilayer and the endosomal escape lipopeptide are dissolved in a water miscible organic solvent (e.g., at least 80% ethanol) and the nucleic acid payload (e.g., an mRNA) is dissolved in nuclease free purified water or water for injectables, and the two components are mixed under controlled conditions, e.g., using a microfluidics device or a jet impingement system that enables the formation of the aLNP. In some aspects, an aLNP of the present disclosure can be formed by mixing under laminar flow conditions, e.g. using a relatively low flow rate (e.g. between about 25 pL / min and about 5,000 pL / min). In other aspects, e.g., in an industrial setup, an aLNP of the present disclosure can be formed by mixing under a chaotic regimen. In some aspects, the initial mixing of organic and aqueous solutions is followed by the mixing of one or more additional aqueous solutions (e.g. RNase free aqueous medium) or organic solutions, e.g., using a relatively low flow rate (e.g., about 10 pL / min to about 20,000 pL / min, about 25 pL / min to about 5,000 pL / min, or about 25 to about 500 pL / min). In some aspects, the one or more additional aqueous solutions can comprise one or more water-soluble lipopeptides. This increase formulation reproducibility and allows for better control of particle size. See Design B in in FIG. 1 (two chip system).

[0198] Advantageously, the aLNP formulations (lipidic compositions in combination with the novel endosomal escape peptides disclosed herein) and manufacturing methods disclosed herein yield aLNP below 200 nm in size and with high encapsulation efficiencies. The distribution of the LNP to different organs is limited by the endothelial fenestrations. In healthy blood vessels, the size of the fenestrations between endothelial cells is approximately 10 nm. On the other hand, specialized organs such as the kidney, liver and spleen, exhibit larger intercellular gaps of 70-90,150, and 200-500 nm, respectively. Therefore, LNP smaller than 30 nm are rapidly excreted by the kidneys while LNP larger than 50 nm are cleared from the circulation via reticuloendothelial system (RES) in the liver and spleen. Those LNP with size bigger than 200 nm accumulate mainly in the spleen (Baek et al., ACS nano, 79(15), 14605-14626, 2025). Thus, LNPs size must be modulated to reduce the clearance of LNPs and enhance the potency of gene therapies. This has been shown for example for hepatic delivery in a mouse model, for which the highest gene silencing was achieved following intravenous administration of siRNA-LNPs of 80 nm as these NPs can distribute more easily through the sinusoidal capillaries in the liver and interact preferentially with the hepatocytes over phagocytic Kupfer cells (Chen etal., Journal of Controlled Release, 235, 236-244, 2016). Furthermore, particles larger than 200 nm have been shown to induce more opsonization by complement proteins and subsequently trigger phagocytosis by RES cells in the blood or by tissue-resident macrophages and dendritic cells (Harashima et al., Pharmaceutical research, 11, 402-406, 1994; Owens III et al., International Journal of Pharmaceutics, 307(1), 93-102, 2006). In the case of intramuscular injection and subcutaneous injection, smaller particles have been also shown to reach the blood circulation more easily (Kong, et al., Journal of Nanobiotechnology, 22(1), 553. 2024; Chen etal., Journal of Controlled Release, 196, 106-112, 2014). Lastly, LNPs with a diameter smaller than 100 nm can accumulate preferentially in tumour and inflammation areas due to the enhance permeability and retention effect (Islam etal., Expert Opinion on Drug Delivery, 19(2), 199-212, 2022). Therefore, a particle size range between with 50 nm and 200 nm is essential for an efficient delivery of nucleic acids with non-viral NPs. Another factor for the preference for particles with size lower than 200 nm is that facilitates the successful manufacturing of the LNPs at industrial scale. The last step during manufacturing is the sterilization of the product, for which terminal sterilization with ionizing radiation or high temperature (e.g., dry heat or steam sterilization) assure the highest sterility. However, the sensitive nature of nucleic acid-containing LNPs hampers the use of the above- mentioned techniques. Ionizing radiation can induce degradation of the oligonucleotides and lipid oxidation, while high temperature sterilization might cause denaturation of the double-stranded molecules and thermal stress to the LNPs (Mehta et al., ACS Materials Au, 3(6), 600-619, 2023; DeCollibus et al., Nucleic acid therapeutics, 33(3), 159-177, 2023). Therefore, sterilization of LNPs is limited to sterile filtration, where particles are pushed through a membrane with a nominal pore size of 200 nm to eliminate any possible microbial contamination. However, this method can cause significant yield loss depending on the size and concentration of the LNPs. The particle transmission through the membrane strongly correlates with the ratio of particle diameter to porefilter diameter, and thus 200 nm particles are the upper size limit to achieve high recovery of LNPs without inducing shear stress. Moreover, increasing the mRNA-LNP concentration with subsequent increase in product viscosity has been shown to reduce filter capacity by lowering the flux (Taylor et aL, Journal of Membrane Science, 635, 119436, 2021; Taylor et al., Journal of Membrane Science, 647, 120264, 2022; Wu, et al., International Journal of Pharmaceutics, 675, 125520, 2025). The aLNP formulations and manufacturing methods disclosed yield LNP within the 30 nm to the 200 nm size range, have high mRNA encapsulation efficiencies, are capable of transfections efficacies comparable to those of classic LNP comprising ionizable cationic lipids, and can achieve the same transfection as classic LNPs without the negative effects associated to their use.1.A Lipid components of aLNP l.A.i Structural lipids

[0199] In some aspects, the anionic lipid nanoparticles (aLNP) of the preset disclosure comprise a structural lipid, e.g., a sterol such as cholesterol, sitosterol, stigmasterol or a combination thereof. As used herein, the term “structural lipid” refers to sterols and to lipids containing sterol moieties. Incorporation of structural lipids in the LNP can help mitigate aggregation of other lipids in the particle, especially when bilayer-forming lipids and cone-shape lipids are combined..

[0200] The “sterol” as used herein refers a steroid which has a hydroxyl group at position C-3 and has a skeleton derived from cholestane. Herein, the term “a skeleton derived from cholestane” refers to a skeleton wherein an unsaturated bond is introduced into the cholestane skeleton. In some aspects, the sterol used in the aLNP of the present disclosure is a cholesterol or a cholesterol derivative. As used herein, “cholesterol derivative”, e.g. refers to at least one selected from the group consisting of cholesterol, sitosterol (e.g. p-sitosterol), ergosterol, stigmasterol, 4,22- stigmastadien-3-on, stigmasterol acetate, lanosterol, and cycloartenol, or any combination thereof. In addition or alternatively, sterols in the context of the current disclosure can be one or more of ergosterol, ergocalciferol, steroidal saponin, vitamin D, campesterol, desmosterol, beta.- cholestanol, and estradiol. The sterol can be derived from and / or naturally found in a plant and / or animal.

[0201] In some aspects, the structural lipid used in an aLNP of the present disclosure is selected from the group consisting of cholesterol, beta-cholesterol, ergosterol, 7-dehydrocholesterol, 24S- hydroxycholesterol, lanosterol, cycloartenol, fucosterol, saringosterol, campesterol, P-sitosterol, sitostanol, coprostanol, avenasterol, stigmasterol, and any combination thereof. Other sterols suitable for use as structural lipids in the aLNP of the present disclosure comprise cholesterolsulfate, desmosterol-d6, lathosterol-d7, desmosterol, dihydrolanosterol, zymosterol, lathosterol, zymosterol-d5, 14-demethyl-lanosterol, 14-demethyl-lanosterol-d6, 8(9)-dehydrocholesterol, 8(14)-dehydrocholesterol, diosgenin, DHEA sulfate, DHEA, lanosterol-d6, dihydrolanosterol-d7, campesterol-d6, lanosterol-95, dihydro FF-MAS-d6, zymostenol-d7, zymostenol, campestanol, 7- dehydrodesmosterol, pregnenolone, sitosterol-d7, dihydro T-MAS, delta 5-avenasterol, brassicasterol, dihydro FF-MAS, 24-methylene cholesterol, cholic acid derivatives, cholesteryl esters, glycosylated sterols, hopanoids, hydroxysteroid, phytosterol, zoosterol, gonane, dexamethasone, and medrogestone. In particular aspects, the aLNP of the present disclosure comprises cholesterol.

[0202] Enriching the aLNP of the present disclosure with cholesterol allows for lower polydispersity index (PDI) of the formulation and higher encapsulation efficiency. Cholesterol stabilizes aLNP containing RNA, e.g., mRNA, inducing changes in the lipid packaging in the bilayers and promoting the formation of lipid domains. Nucleic acid payloads, e.g., mRNA, induce the lipid segregation of aLNP bilayers into neutral lipid-rich areas and anionic lipid-rich areas. Including cholesterol in the bilayer stabilizes lipid segregation, avoiding the electrostatic repulsion between RNA (e.g., mRNA) and anionic lipids in the bilayer.

[0203] Enriching the aLNP of the present disclosure with sitosterol allows for higher transfection efficiencies. l.A.ii Anionic lipids

[0204] In some aspects, the aLNP of the preset disclosure comprise an anionic lipid or a combination thereof. In some aspects, the anionic lipid is a fusogenic lipid. In some aspects, the anionic lipid is a bilayer-forming lipid. The term "anionic lipid" as used herein refers to a lipid with a net negative charge in an aqueous solution at a pH between about 3 and about 9. In some aspects, the anionic lipid has a net negative charge in an aqueous solution at a pH between about 5 and about 8. In some aspects, the anionic lipid has a net negative charge in an aqueous solution at a pH between about 6.0 and about 7.5. In some aspects, the anionic lipid has a net negative charge in an aqueous solution at a pH about 7.4. In some aspects, the anionic lipids of the present disclosure have a negative charge at physiologically acceptable pH, e.g., a pH between about 7.0 and about 7.8, for example, about 7.4. In some aspects, a net negative charge means a zeta potential of less than -2 mV, e.g., less than -10 mV or less than -50 mV.

[0205] In some aspects, the anionic lipid is a phospholipid. Phospholipids can be of a symmetric or an asymmetric type. As used herein, the term "symmetric phospholipid" includes glycerophospholipids having matching fatty acid moieties and sphingolipids in which the variablefatty acid moiety and the hydrocarbon chain of the sphingosine backbone include a comparable number of carbon atoms. As used herein, the term "asymmetric phospholipid" includes lysolipids, glycerophospholipids having different fatty acid moieties (e.g., fatty acid moieties with different numbers of carbon atoms and / or unsaturations (e.g., double bonds)), and sphingolipids in which the variable fatty acid moiety and the hydrocarbon chain of the sphingosine backbone include a dissimilar number of carbon atoms (e.g., the variable fatty acid moiety include at least two more carbon atoms than the hydrocarbon chain or at least two fewer carbon atoms than the hydrocarbon chain).

[0206] In some aspects, the aLNP of the present disclosure comprises at least one anionic lipid selected from the group consisting of DSP A; DPP A; DMPA; DLPA; DOPA; DSPG; DPPG; DMPG; DLPG; DOPG; phosphatidylglycerol, e.g. egg PG (EPG); cardiolipin; diacylphosphatidylinositol; diacylphosphatidylserine; N-succinyl phosphatidylethanolamine; N- glutarylphosphatidylethanolamine; lysylphosphatidylglycerol; gangliosides (e.g. GM1, GM2, GM3); sulphogycosphingolipids; fatty acids; anionic modifying groups joined to neutral lipids; and combinations thereof. In some aspects, the anionic lipid is EPG. In some aspects, the aLNP of the present disclosure do not comprise DPPG.

[0207] In some aspects, the anionic lipid content in an aLNP of the present disclosure is less than about 5%, less than about 4%, less than about 3%, less than about 2%, less than about 1%, or less than about 0.5 mol.%, relative to total mol lipid of the particles. In some aspects, the anionic lipid content in an aLNP of the present disclosure is about 5%, about 4%, about 3%, about 2%, about 1%, or about 0.5 mol.%, relative to total mol lipid of the aLNPs. In some aspects, the anionic lipid content in an aLNP of the present disclosure is between about 5% and about 4%, between about 4% and about 3%, between about 3% and about 2%, between about 2% and about 1%, or between about 1% and about 0.5%, relative to total mol lipid of the aLNPs. l.A.iii Neutral lipids

[0208] In some aspects, the aLNP of the preset disclosure comprise a neutral lipid or a combination thereof. In some aspects, the neutral lipid is a fusogenic lipid. The term “neutral lipid” in the context of the current disclosure means a lipid which does not (substantially) carry a net charge in aqueous solution at a pH between about 5 and about 9. In some aspects, the neutral lipid does not (substantially) carry a net charge in aqueous solution at a pH between about 6 and about 8. In some aspects, the neutral lipid does not (substantially) carry a net charge in aqueous solution at a pH between about 6.5 and about 7.5. In some aspects, the neutral lipid does not (substantially) carry a net charge in aqueous solution at a pH about 7.4.

[0209] In some aspects, the neutral lipid can be selected from the group consisting of EPC; HSPC; Soy PC; DSPC; DPPC; DMPC; DLPC; DOPC; DSPE; DPPE; DMPE; DLPE; DOPE; ceramide; sphingosines; sphingomyelin; cephalin; glycolipids; MGD; DGD; SQDG; glycosphingolipids; a-galactosylceramide; beta-mannoseceramide; di or triacylglycerols; and any combination thereof. In one specific aspect, the neutral lipid is EPC. In another aspect, the neutral lipid is DPPC, In another aspect, the neutral lipid is DSPC. In another aspect, the neutral lipid is sphyngomielin. In another aspect, the neutral lipid is DSPC. In another aspect, the neutral lipid is DOPE. In addition or alternatively, the neutral lipids in an aLNP of the present disclosure can comprise a glycolipid to target the immune system.

[0210] In some aspects, the neutral lipid comprises a symmetric phosphocholine selected from the group consisting of 03:0 PC; 04:0 PC; 05:0 PC; 06:0 PC; 07:0 PC; 08:0 PC; 09:0 PC; 10:0 PC; 11 :0 PC (DUPC); DLPC; DLOPC; 13:0 PC; 14:0 PC (DMPC); 15:0 PC; 16:0 PC (DPPC); 4ME 16:0 PC; 17:0 PC; 18:0 PC (DSPC); 19:0 PC; 20:0 PC; 21 :0 PC; 22:0 PC; 23:0 PC; 24:0 PC; 14: 1 (A9-Cis) PC; 14: 1 (A9-Trans) PC; 16: 1 (A9-Cis) PC; 16: 1 (A9-Trans) PC; 18: 1 (A6-Cis) PC; 18: 1 (A9-Cis) PC (DOPC); 18:1 (A9-Trans) PC; 18:2 (Cis) PC (DLPC); 18:3 (Cis) PC (DLnPC); 20: 1 (Cis) PC; 20:4 (Cis) PC (DAPC); 22: 1 (Cis) PC; 22:6 (Cis) PC (DHAPC); 24: 1 (Cis) PC; DEPC; 18:0 di ether PC; and any combination thereof.

[0211] In some aspects, the neutral lipid comprises a symmetric phosphoethanolamine (PE) selected from the group consisting of 06:0 PE; 08:0 PE; 10:0 PE; 12:0 PE; 14:0 PE (DMPE); 15:0 PE; 16:0 PE (DPPE); 4ME 16:0 PE; 17:0 PE; 18:0 PE (DSPE); 16: 1 PE; 18: 1 (A9-Cis) PE (DOPE); 118: 1 (A9-Trans) PE; 18:2 PE (DLPE); 18:3 PE (DLnPE); 20:4 PE (DAPE); 22:6 PE (DHAPE); DEPE; and any combination thereof. l.A.iv Cationic or ionizable lipids

[0212] In some aspects, the aLNP of the present disclosure comprise a cationic lipid or ionizable lipid or a combination thereof. As used herein, the term “ionizable lipid” refers to any of a number of lipid species that carry a net positive charge at a selected pH, such as physiological pH 4, and a neutral charge at other pHs such as physiological pH 7. These compounds are capable of forming charged particles in contact with suitable counterions, such as particles that include an ionizable hydrogen atom. Ionizable lipids can be used as a component of a classic LNP to facilitate or enhance the delivery and release of a nucleic acid, e.g., an RNA, to one or more target cells (e.g., by permeating or fusing with the lipid membranes of such target cells).

[0213] In some aspects, the ionizable lipid is selected from the group consisting of cKK-E12 (3, 6-bis(4-(bis(2-hydroxydodecyl)amino)butyl)piperazine-2, 5-dione), ALC-0315, SM-102, YK-009, MC3 (DLin-MC3-DMA), KC2 (DLin-KC2-DMA), A6, OF-02, A18-Iso5-2DC18, 98N12-5, 9Alp9, C12-200, 7C1, G0-C14, L319, 304013, OF-Deg-Lin, 306-O12B, 3060iio, FTT5, and any combination thereof. cKK-E12 is an ionizable cationic lipomer that has been used in combination with other lipids in the formation of aLNPs for the delivery of mRNA. ALC-0315 is an ionizable lipid that has been used to form lipid nanoparticles for delivery of RNA. ALC-0315 is one of the components in the BNT162b2 vaccine against SARS-CoV-2 in addition to ALC-0159, DSPC, and cholesterol. SM-102 is a synthetic amino lipid that is used in combination with other lipids to form aLNPs. These are used for the delivery of mRNA-based vaccines, and in particular SM-102 forms part of the drug delivery system for the Moderna COVID-19 vaccine.

[0214] As used herein, the term “cationic lipid” refers to a lipid that is cationic or becomes cationic (protonated) as the pH is lowered below the pK of the ionizable group of the lipid, but is progressively more neutral at higher pH values. At pH values below the pK, the lipid is then able to associate with negatively charged nucleic acids. In certain aspects, the cationic lipid comprises a zwitterionic lipid that assumes a positive charge on pH decrease.

[0215] In some aspects, the cationic lipid is DOTAP or DOTMA. In some aspects, the cationic lipid comprises any of a number of lipid species that carry a net positive charge at a selective pH, such as physiological pH. Such lipids include, but are not limited to DODAC; DOTMA; DDAB; DOTAP; DC-Chol; DOSPA; DOGS; DODAP; DODMA; and, DMRIE. Additionally, a number of commercial preparations of cationic lipids are available which can be used in the aLNP of the present disclosure. These include, for example, LIPOFECTIN® (commercially available cationic liposomes comprising DOTMA and DOPE, from GIBCO / BRL); LIPOFECTAMINE® (commercially available cationic liposomes comprising DOSPA and DOPE, from GIBCO / BRL); and TRANSFECTAM® (commercially available cationic lipids comprising DOGS in ethanol from Promega Corp). The following lipids are cationic and have a positive charge at below physiological pH: 20 DODAP, DODMA, DMDMA, DLinDMA, and DLenDMA.

[0216] In some aspects, the cationic lipid is an amino lipid. Representative amino lipids include, but are not limited to DLin-DAC; DLin-MA; DLinDAP; DLin-S-DMA; DLin-2-DMAP; DLin- TMA.C1; DLin-TAP.Cl; DLin-MPZ; DLinAP; DOAP; DLin-EG-DMA; and, DLin-K-DMA. l.A.v Derivatizable lipids / Chemically modified lipids

[0217] In some aspects, the aLNP of the present disclosure comprise a derivatizable lipid that can be used, for example, to attach an endosomal escape peptide, a targeting moiety, a half-life extending moiety, or a combination thereof, to the external surface of an aLNP of the present disclosure. As used herein, the term “chemically modified lipid” refers to a lipid that has beenmodified to be derivatizable by incorporating a chemically reactive group (e.g., a maleimide group or a sulfhydryl group) that can be used to attach a biologically active moiety (e.g., an antibody) covalently (e.g., via reaction between the maleimide group and a sulfhydryl group) or non- covalently to the lipid.

[0218] Thus, in some aspects, the aLNP of the present disclosure comprises a derivatizable lipid, e.g., a PEG lipid comprising a maleimide group such as DSPE-PEG2000-maleimide, wherein the maleimide reactive group is free (i.e., prior to the reaction with an endosomal escape lipid or antibody). In some aspects, the derivatizable lipid is conjugated to a targeting molecule (e.g., an antibody that specifically binds to a receptor or cancer-specific antigen on the surface of a cancer cells, or binds to a protein specifically present in a certain cell type or tissue) thereby anchoring the targeting molecule to the surface of the aLNP.

[0219] In some aspects, where a certain class of lipids present in an aLNP of the present disclosure includes both an unmodified lipid and a chemically modified lipid and, the chemically modified lipid can be derived from the unmodified lipid. By way of example, the one or more lipids of an aLNP can include an unmodified DSPE-PEG2000 lipid and a modified DSPE-PEG2000 lipid that includes a functionalized group capable of forming a covalent bond, e.g., DMG-PEG2000- maleimide or DSPE-PEG2000-maleimide.

[0220] In some aspects, aLNP of the present disclosure can comprise a lipid comprising a maleimide group, e.g., DMG-PEG2000-maleimide or DSPE-PEG2000-maleimide, which can react with a thiol group present in a targeting molecule (e.g., an antibody) or an endosomal escape peptide. In some aspects, aLNP of the present disclosure can comprise a lipid comprising a thiol group, e.g., DMG-PEG2000-SH or DSPE-PEG2000-SH, which can react with a maleimide group present in a targeting molecule (e.g., an antibody) or an endosomal escape peptide.

[0221] In some aspects, the chemically modified lipid is selected from the group consisting of DSPE-PEG2000-maleimide, DSPE-PEG5000-maleimide, DMG-PEG2000-maleimide, DMG- PEG5000-maleimide, cholesterol-PEG2000-maleimide, cholesterol-PEG5000-maleimide, DSPE- PEG2000-SH, DSPE-PEG5000-SH, DMG-PEG2000-SH, DMG-PEG5000-SH, cholesterol- PEG2000-SH, cholesterol-PEG5000-SH, or any combination thereof.

[0222] A person of ordinary skill in the art would understand that there are numerous compounds and procedures that can be used to attach a biologically active molecule, e.g., an antibody or a peptide such as an endosomal escape peptide to the surface of a bilayer, for example, the external surface of an aLNP of the present disclosure.LA.vi Polymer-derived lipids

[0223] In some aspects, the aLNP of the present disclosure comprises a polymer-derived lipid, e.g., PEG-modified lipid, i.e., a lipid comprising a PEG moiety as disclosed herein. In some aspects, the polymer-derived lipid is a polysarcosine or polyoxazoline.

[0224] In some aspects, an aLNP of the present disclosure can comprise polysarcosine. Polysarcosine is a lipid-polypeptoid conjugate based on the endogenous amino acid sarcosine. It has been shown to have a higher protein secretion with a reduced immunostimulatory response observed when compared to systems based on polyethylene glycol (PEG). Polysarcosine (pSar) lipids are offered as alternatives to traditional PEGylated lipids used in RNA delivery vehicles. Compared to similar PEGylated LNPs, pSar LNPs have shown improved mRNA transfection potency and improved safety profiles. When formulating aLNPs, pSar lipids of varying polymeric chain lengths have been used to tune physiochemical properties such as particle size, morphology, and internal structure.

[0225] In some aspects, an aLNP of the present disclosure can comprising a polyoxazoline-lipid (POZ-lipod). POZ-lipid decorated liposomes have enhanced blood circulation, with similar blood circulation times compared to PEG decorated liposomes. POZ-lipids have similar ability to shield non-specific interactions with proteins and cells as PEG. More and more evidence arises that a large percentage of the human population has antiPEG antibodies, which can induce an allergic response upon treatment with PEGylated formulations, including COVID-19 vaccines, further stimulating research on alternative polymers that suppress non-specific interactions, including poly(2-oxazoline)s.

[0226] As used herein, the terms "PEG-modified lipid," “PEG-lipid,” or “PEGylated lipid” are used interchangeably and refer to a lipid linked (e.g., covalently attached) to at least one PEG polymer chain. In some aspects, the PEG-lipid described herein comprises a poly(ethylene) glycol (PEG) chain of about 2 kDa, about 3 kDa, about 4 kDa, about 5 kDa, about 6 kDa, about 7 kDa, about 8 kDa, about 9 kDa, about 10 kDa, about 11 kDa, about 12 kDa, about 13 kDa, about 14 kDa, about 15 kDa, about 16 kDa, about 17 kDa, about 18 kDa, about 19 kDa, about 20 kDa, about 21 kDa, about 22 kDa, about 23 kDa, about 24 kDa, or about 25 kDa. In some aspects, the PEG- lipid described herein comprises a PEG chain of about 2 kDa. In some aspects, the PEG-lipid described herein comprises a PEG chain of about 5kDa. In some aspects, the PEG-lipid described herein comprises a PEG chain of about 10 kDa. In some aspects, the PEG-lipid described herein comprises a PEG chain of about 20 kDa. In some aspects, the PEG-lipid described herein comprises a PEG chain of less than 20 kDa. In some aspects, the PEG-lipid described herein comprises a PEGchain of less than 15 kDa. In some aspects, the PEG-lipid described herein comprises a PEG chain of less than 10 kDa. In some aspects, the PEG-lipid described herein comprises a PEG chain of less than 5 kDa. In some aspects, the PEG-lipid described herein comprises a PEG chain of less than 4 kDa. In some aspects, the PEG-lipid described herein comprises a PEG chain of less than 3 kDa. In some aspects, the PEG-lipid comprises a PEG chain between about 2 kDa and about 5 kDa. In some aspects, the PEG-lipid comprises a PEG chain between about 5 kDa and about 10 kDa. In some aspects, the PEG-lipid comprises a PEG chain between about 10 kDa and about 15 kDa. In some aspects, the PEG-lipid comprises a PEG chain between about 15 kDa and about 20 kDa. In some aspects, the PEG is covalently attached to the lipid via a linker. In some aspects, the linker is an alkyl linker. In some aspects, the alkyl linker comprises an alkyl chain with a length between C6 and C20. In some aspects, the alkyl linker comprises an alkyl chain with a length of C6, C7, C8, C9, CIO, Cl l, C12, C13, C14, C15, C16, C17, C18, C19, or C20.

[0227] In some aspects, the PEG-lipid is reversibly linked to the aLNP described herein. Thus, in some aspects, the PEG moiety is gradually released in blood circulation upon administration. In some aspects, an alternative to a PEG-lipid can be used, for example, a derivatized lipid such as a derivatized ceramide (PEG-CER), including C8 PEG-2000 ceramide.

[0228] In some aspects, the PEG-lipid described herein comprises or consists of a PEG- phospholipid and / or a PEG-ceramide. In some aspects, the PEG-lipid is a PEG-modified phosphatidylethanolamine, a PEG-modified phosphatidic acid, a PEG-modified ceramide, a PEG- modified dialkylamine, a PEG-modified diacylglycerol, a PEG-modified dialkylglycerol, or a combination thereof.

[0229] In some aspects, the PEG-lipid is selected from the group consisting of mPEG-2000-DSPE; mPEG-2000-DOPE; mPEG-2000-DPPE; mPEG-2000-DMPE; mPEG-2000- DLPE; mPEG-5000-DSPE; mPEG-5000-DOPE; mPEG-5000-DPPE; mPEG-5000-DMPE; mPEG-5000-DLPE; and any combination thereof.

[0230] In some aspects, the PEG-lipid is selected from the group consisting of PEG-c-DOMG, PEG-DMG, PEG-DLPE, PEG-DMPE, PEG-DPPC, PEG-DSPE, and any combination thereof. In some aspects, the PEG-lipid is DMG-PEG2000. In some aspects, the PEG-lipid is DSPE- PEG2000. In some aspects, the PEG-lipid is a ceramide PEG derivatives such as C8 PEG2000 ceramide, C16 PEG2000 ceramide, C8 PEG5000 ceramide, C16 PEG5000 ceramide, C8 PEG750 ceramide, and C16 PEG750 ceramide. In some aspects, the PEG-lipid is a PEG derivative, such as 16:0 PEG5000 PE, 14:0 PEG5000 PE, 18:0 PEG5000 PE, 18: 1 PEG5000 PE, 16:0 PEG3000 PE, 14:0 PEG3000 PE, 18:0 PEG3000 PE, 18: 1 PEG3000 PE, 16:0 PEG2000 PE, 14:0 PEG2000 PE,18:0 PEG2000 PE, 18: 1 PEG2000 PE, 16:0 PEG1000 PE, 14:0 PEG1000 PE, 18:0 PEG1000 PE, 18: 1 PEG1000 PE, 16:0 PEG750 PE, 14:0 PEG750 PE, 18:0 PEG750 PE, 18: 1 PEG750 PE, 16:0 PEG550 PE, 14:0 PEG550 PE, 18:0 PEG550 PE, 18: 1 PEG550 PE, 16:0 PEG350 PE, 14:0 PEG350 PE, 18:0 PEG350 PE, and 18: 1 PEG350.

[0231] In some aspects, the PEG-lipid is a sterol PEG derivative such as Chol-PEG600. In some aspects, the PEG-lipid is a glycerol PEG derivative such as DMG-PEG5000, DSG-PEG5000, DPG-PEG5000, DMG-PEG3000, DSG-PEG3000, DPG-PEG3000, DMG-PEG2000, DSG- PEG2000, DPG-PEG2000, DMG-PEG1000, DSG-PEG1000, DPG-PEG1000, DMG-PEG750, DSG-PEG750, DPG-PEG750, DMG-PEG550, DSG-PEG550, DPG-PEG550, DMG-PEG350, DSG-PEG350, and DPG-PEG350. In some aspects, the PEG-lipid is a phospholipid PEG derivative such as DSPE-PEG5000, DSPE-PEG2000, DSPE-PEG1000, or DSPE-PEG550.

[0232] In some specific aspects, the PEG- lipid employed in the compositions and methods of the present disclosure is 1,2-dimyristoyl-sn-glycerol, methoxypolyethylene Glycol (2000 MW PEG), also known as DMG-PEG2000.

[0233] The addition of PEG-modified lipids to the lipid delivery vehicle can prevent complex aggregation and can also provide a means for increasing circulation lifetime and increasing the delivery of the lipid-polynucleotide composition to the target tissues, (Klibanov et al. (1990) FEBS Letters, 268 (1): 235-237), or they can be selected to rapidly exchange out of the formulation in vivo (see U.S. Pat. No. 5,885,613). Particularly useful exchangeable lipids are PEG-ceramides having shorter acyl chains (e.g., C14 or C18). In some aspects, the lipid moiety of the PEG-lipids includes those having lengths of from about Cwto about C22, such as from about Cwto about Ci6. In some aspects, the PEG-lipid is a non-diffusible PEG conjugates. Non-limiting examples of non- diffusible PEG conjugates include PEG-DSG and PEG-DSPE.

[0234] In general, the aLNP of the present disclosure does not comprise a PEG-lipid, or less than about 5 mol%, less than about 4 mol%, less than about 3 mol%, less than about 2 mol%, less than about 1 mol%, or less than 0.5 mol%, relative to total mol lipid of the aLNP. l.A.vii Other lipid components

[0235] In some aspects, the aLNP of the present disclosure can comprise additional components such as fatty acids, lysolipids, or vitamins. In some aspects, the fatty acid is a short-chain, mediumchain, or long-chain fatty acid. In some aspects, the fatty acid is a saturated fatty acid. In some aspects, the fatty acid is an unsaturated fatty acid. In some aspects, the fatty acid is a monounsaturated fatty acid. In some aspects, the fatty acid is a polyunsaturated fatty acid, such as a co-3 (omega-3) or co-6 (omega-6) fatty acid.l.A.viii Lipid Packing

[0236] Amphipathic lipids adopt specific molecular geometries in aqueous media according to the ratio of their hydrophobic-tail volume and length (v / lc) to their polar headgroup area (ao). This relationship is captured by the packing parameter P=v / a01c. Lipids with a packing value P ~ 1 are cylindrical lipids that favor planar, lamellar bilayers. Lipids with a packing value P > 1 are cone- shaped lipids that favor inverted-hexagonal (H(ii) phases) or inverted-micellar phases. Lipids with a packing value P < 1 are inverted-cone lipids that favor normal micelles. Lipids with reduced headgroup area (higher v / aoLn) form larger vesicles, less-curved bilayers, or inverted micellar phases. Cullis et al. Biochim Biophys Acta 559: 399-420, 1979). In anionic lipids, headgroup condensation (decrease in ao) can be induced, e.g., by divalent cations (e.g., Ca2+binding neutralizes charge and reduces effective ao) or by acidification (protonation below pK reduces headgroup hydration). These triggers also promote chain straightening (“condensation”), further promoting nonlamellar structures. Branching and unsaturation — especially cis-double bonds — decrease the effective chain length £nwithout altering total volume v, thereby increasing v / aoLn.Bilayer-Forming Lipids: Phosphatidylcholines (PC) and sphingomyelins (SM), exemplify neutral cylindrical or structural lipids and phosphatidylserine (PS), phosphatidylglycerol (PG) exemplify anionic cylindrical or structural lipids. Their headgroup and tail regions pack with nearly equal cross-sectional areas, exhibit ao ~ v / Lc (P ~ 1), and yielding highly stable lamellar bilayers that impart membrane integrity, low permeability, and extended shelf life under storage and physiological conditions.Fusogenic Lipids: Cone-shaped lipids such as the phosphatidylethanolamine (PE) family (e.g. DOPE) , diphosphatidylglycerol (cardiolipin, DPG), glycerolipids, e,g, diacylglycerol (DAG) and monoacylglycerol (MAG, such as monoolein), glycolipids, e.g., monogalactosyldiacylglycerol (MGDG)), the phosphatidic acid (PA) family (e.g. DOPA), the ceramide family (e.g. erythro-C8- Cer), and certain fatty acids (e.g. oleic acid, linoeic acid) possess ao < v / Ln(P > 1), inverted-cone lipids — e.g., bis(monoacylglycero)phosphate stereoisomers (BMP-S,R and BMP-S,S) — , lysophospholipids with only one lipid tail such as lyso-phosphatidic acid (LPA) and , lysophosphatidylethanolamine (LPE) likewise favor nonlamellar, inverted-micellar arrangements. Cone-shape lipids may have one o more branching and unsaturation of the lipid changes to increase the P value>l. Incorporation of cone-shaped and inverted cone-shaped species confers triggered membrane fusion (e.g., endosomal escape); however, it undermines colloidal stability of the nanoparticles, and it can leading to undesirable effects such as (i) uncontrolled fusion and aggregation; (ii) undesirable payload loss via transient defects; (iii) short shelf life from phaseseparation; or (iv) unexpected mixing of particle populations. Fusogenic lipids, like unsaturated lipids, play a key role in cell membrane fusion, facilitating the merging of cell membranes or vesicles. Unsaturated lipids, containing double bonds in their fatty acid chains like DOPE (dioleoylphosphatidylethanolamine) and GMO (glyceryl monooleate), have an inverted cone shape, promoting the formation of membranes with negative spontaneous curvature. This negative curvature can facilitate membrane fusion by increasing the HII phase transition propensity and creating beneficial packing stress. The aLNP of the present disclosure are characterized by comprising at least one fusogenic lipid, e.g., DOPE. l.B Non-lipid aLNP components

[0237] In some aspects, aLNP of the present disclosure can comprise non-lipid components decorating the external surface of the lipid bilayer, e.g., endosomal escape peptides, targeting moi eties (e.g., antibodies or antigen-binding fragments thereof), half-life extenders (generally molecules that reduce the clearance rate of the aLNP, therefore extending the time that the aLNP is in the bloodstream), don’t eat me signals, or any combination thereof. l.B.i Endosomal escape peptides

[0238] In some aspects, the aLNP of the preset disclosure comprises an endosomal escape peptide or a combination thereof attached to the external surface of the aLNP.

[0239] Endosomal escape peptides containing arginine and lysine residues are positively charged at neutral pH. Lipopeptides enriched with arginines and / or lysines can promote the fusion of lipid bilayers and the increase of the nanoparticle size during storage, making it not suitable for pharmaceutical applications. Additionally, positive charged peptides can be toxic. Furthermore, the electrostatic interactions between the cationic amino acids and the nucleic acids can be replaced by anionic physiological proteins in the body, reducing uptake by the cells in the body. Accordingly, in some aspects, the aLNP of the present disclosure do not contain arginine and / or lysine rich endosomal escape peptide, or endosomal escape peptides having a net positive charge.

[0240] In some aspects, the endosomal escape peptide attached to an aLNP of the present disclosure is a peptide having an amino acid sequence set forth in SEQ ID NO:1 to SEQ ID NO: 1118 or SEQ ID NO: 1310 to SEQ ID NO: 1335. In some aspects, an endosomal escape peptide having an amino acid sequence set forth in SEQ ID NO: 1 to SEQ ID NO: 1118 or SEQ ID NO: 1310 to SEQ ID NO: 1335 or a combination thereof can be covalently attached to a lipid molecule, resulting in an endosomal escape lipopeptide. The lipid molecule (e.g., a phospholipid) can be attached, e.g., to the C-terminus or to the N-terminus of the endosomal escape peptide to yield the lipopeptide using any conjugation method known in the art. The endosomal escape peptides ofSEQ ID NO: 1 to 1118 or SEQ ID NO: 1310 to SEQ ID NO: 1335, as well as lipopeptide variants thereof can be synthesized or manufactured using any solid-phase synthetic technique or solution methods readily available to those skilled in the art.

[0241] Examples for useful subsequences to be incorporated in the endosomal escape peptide (e.g., an endosomal escape peptide of SEQ ID NO: 1 to SEQ ID NO:1118 or SEQ ID NO: 1310 to SEQ ID NO: 1335) include ELVH (SEQ ID NO: 1192), EVLH (SEQ ID NO: 1193), ELLH (SEQ ID NO: 1194), EVVH (SEQ ID NO: 1195), ELAH (SEQ ID NO: 1196), EALH (SEQ ID NO: 1197), EAVH (SEQ ID NO: 1198), EV AH (SEQ ID NO: 1199), EIVH (SEQ ID NO: 1200), EVIH (SEQ ID NO: 1201), EILH (SEQ ID NO: 1202), ELIH (SEQ ID NO: 1203), EIAH (SEQ ID NO: 1204), EAIH (SEQ ID NO: 1205), EMVH (SEQ ID NO: 1206), EVMH (SEQ ID NO: 1207), EMLH (SEQ ID NO: 1208), ELMH (SEQ ID NO: 1209), EMAH (SEQ ID NO: 1210), EAMH (SEQ ID NO: 1211), EIMH (SEQ ID NO: 1212), EMIH (SEQ ID NO: 1213), ELHV (SEQ ID NO: 1214), EVHL (SEQ ID NO: 1215), ELHL (SEQ ID NO: 1216), EVHV (SEQ ID NO: 1217), EAHL (SEQ ID NO: 1218), EAHV (SEQ ID NO: 1219), EVHA (SEQ ID NO: 1220), EIHV (SEQ ID NO: 1221), EVHI (SEQ ID NO: 1222), EIHL (SEQ ID NO: 1223), ELHI (SEQ ID NO: 1224), EIHA (SEQ ID NO: 1225), EAHI (SEQ ID NO: 1226), EMHV (SEQ ID NO: 1227), EVHM (SEQ ID NO: 1228), EMHL (SEQ ID NO: 1229), ELH, EMH, EAH, and EIH. Such sequences can be repeated, e g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 13, 14, 15 or more times, or combined with each other. Optionally, a sequence rich in lipophilic amino acids can contain at least one proline, which can serve as a structure breaker of longer sequences of Leu, Vai, He, Ala and / or Met. Two, three or more prolines can also be incorporated, in particular in longer sequences

[0242] Examples for useful subsequences to be incorporated in the endosomal escape peptide (e.g., endosomal escape peptide of SEQ ID NO: 1 to SEQ ID NO: 1118 or SEQ ID NO: 1310 to SEQ ID NO: 1335) also include DLVH (SEQ ID NO: 1230), DVLH (SEQ ID NO: 1231), DLLH (SEQ ID NO: 1232), DVVH (SEQ ID NO: 1233), DLAH (SEQ ID NO: 1234), DALH (SEQ ID NO: 1235), DAVH (SEQ ID NO: 1236), DVAH (SEQ ID NO: 1237), DIVH (SEQ ID NO: 1238), DVIH (SEQ ID NO: 1239), DILH (SEQ ID NO: 1240), DLIH (SEQ ID NO: 1241), DIAH (SEQ ID NO: 1242), DAIH (SEQ ID NO: 1243), DMVH (SEQ ID NO: 1244), DVMH (SEQ ID NO: 1245), DMLH (SEQ ID NO: 1246), DLMH (SEQ ID NO: 1247), DMAH (SEQ ID NO: 1248), DAMH (SEQ ID NO: 1249), DIMH (SEQ ID NO: 1250), DMIH (SEQ ID NO: 1251), DLHV (SEQ ID NO: 1252), DVHL (SEQ ID NO: 1253), DLHL (SEQ ID NO: 1254), DVHV (SEQ ID NO: 1255), DAHL (SEQ ID NO: 1256), DAHV (SEQ ID NO: 1257), DVHA (SEQ ID NO: 1258), DIHV (SEQ ID NO: 1259), DVHI (SEQ ID NO: 1260), DIHL (SEQ ID NO: 1261), DLHI (SEQ ID NO: 1262), DIHA (SEQ IDNO: 1263), DAHI (SEQ ID NO: 1264), DMHV (SEQ ID NO: 1265), DVHM (SEQ ID NO: 1266), DMHL (SEQ ID NO: 1267), DLH, DMH, DAH, and DIH. Such sequences can be repeated, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 13, 14, 15 or more times, or combined with each other. Optionally, a sequence rich in lipophilic amino acids can contain at least one proline, which can serve as a structure breaker of longer sequences of Leu, Vai, He, Ala and / or Met. Two, three or more prolines can also be incorporated, in particular in longer sequences

[0243] Examples for useful subsequences to be incorporated in the endosomal escape peptide (e.g., an endosomal escape peptide of SEQ ID NO: 1 to SEQ ID NO:1118 or SEQ ID NO: 1310 to SEQ ID NO: 1335) also include EL, EA, El, EM, EV, ELV, EVL, ELL, EVV, ELVL (SEQ ID NO: 1123), EVLV (SEQ ID NO: 1124), ELLL (SEQ ID NO: 1125), EVVV (SEQ ID NO: 1126), ELVLV (SEQ ID NO: 1127), EVLVL (SEQ ID NO: 1128), ELLLL (SEQ ID NO: 1129), EVVVV (SEQ ID NO: 1130), EIA, EAI, Eli, EAA, EIAI (SEQ ID NO: 1131), EAIA (SEQ ID NO: 1132), EIII (SEQ ID NO: 1133), EAAA (SEQ ID NO: 1134), EIAIEA (SEQ ID NO: 1135), EAIAI (SEQ ID NO: 1136), EIIII (SEQ ID NO: 1137), EAAAA (SEQ ID NO: 1138), EMA, EAM, EMM, EAA, EMAM (SEQ ID NO: 1139), EAMA (SEQ ID NO: 1140), EMMM (SEQ ID NO: 1141), EAAA (SEQ ID NO: 1142), EMAMA (SEQ ID NO: 1143), EAMAM (SEQ ID NO: 1144), EMEMMM (SEQ ID NO: 1145), DL, DA, DI, DM, DV, DLV, DVL, DLL, DVV, DLVL (SEQ ID NO: 1146), DVLV (SEQ ID NO: 1147), DLLL (SEQ ID NO: 1148), DVVV (SEQ ID NO: 1149), DLVLV (SEQ ID NO: 1150), DVLVL (SEQ ID NO: 1151), DLLLL (SEQ ID NO: 1152), DVVVV (SEQ ID NO: 1153), DIA, DAI, DII, DAA, DIAI (SEQ ID NO: 1154), DAIA (SEQ ID NO: 1155), Dill (SEQ ID NO: 1156), DAAA (SEQ ID NO: 1157), DIAIA (SEQ ID NO: 1158), DAIAI (SEQ ID NO: 1159), DIIII (SEQ ID NO: 1160), DAAAA (SEQ ID NO: 1161), DMA, DAM, DMM, DAA, DMAM (SEQ ID NO: 1162), DAMA (SEQ ID NO: 1163), DMMM (SEQ ID NO: 1164), DAAA (SEQ ID NO: 1165), DMAMA (SEQ ID NO: 1166), DAMAM (SEQ ID NO: 1167), or DMDMMM (SEQ ID NO: 1168). Such sequences can be repeated, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 13, 14, 15 or more times, or combined with each other. Optionally, the sequence rich in lipophilic amino acids can contain at least one proline, which can serve as a structure breaker of longer sequences of Leu, Vai, He, Ala and / or Met. Two, three or more prolines can also be incorporated, in particular in longer sequences

[0244] Examples for useful subsequences to be incorporated in the endosomal escape peptide (e.g., an endosomal escape peptide of SEQ ID NO: 1 to SEQ ID NO:1118 or SEQ ID NO: 1310 to SEQ ID NO: 1335) also include ST, TS, SS, TT, STS, TST, SSS, TTT, STST (SEQ ID NO: 1169), TSTS (SEQ ID NO: 1170), SSSS (SEQ ID NO: 1171), TTTT (SEQ ID NO: 1172), QN, NQ, QQ,NN, QNQ, NQN, QQQ, NNN, QNQN (SEQ ID NO: 1173), NQNQ (SEQ ID NO: 1174), QQQQ (SEQ ID NO: 1175), NNNN (SEQ ID NO: 1176), SN, NS, SS, NN, SNS, NSN, SSS, NNN, SNSN (SEQ ID NO: 1177), NSNS (SEQ ID NO: 1178), SSSS (SEQ ID NO: 1179), or NNNN (SEQ ID NO: 1180).

[0245] Examples for useful subsequences to be incorporated in the endosomal escape peptide (e.g., an endosomal escape peptide of SEQ ID NO: 1 to SEQ ID NO:1118 or SEQ ID NO: 1310 to SEQ ID NO: 1335) also include LV, VL, LL, VV, LVL, VLV, LLL, VW, LVLV (SEQ ID NO: 1181), VLVL (SEQ ID NO: 1182), LLLL (SEQ ID NO: 1183), VVVV(SEQ ID NO: 1184), IA, Al, II, AA, IAI, AIA, III, AAA, IAIA (SEQ ID NO: 1185), AIAI (SEQ ID NO: 1186), IIII (SEQ ID NO: 1187), AAAA (SEQ ID NO: 1188), MA, AM, MM, AA, MAM, AMA, MMM, AAA, MAMA (SEQ ID NO: 1189), AMAM (SEQ ID NO: 1190), or MMMM (SEQ ID NO: 1191). Such sequences can be repeated, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 13, 14, 15 or more times, or combined with each other. Optionally, the sequence rich in lipophilic amino acids can contain at least one proline, which can serve as a structure breaker of longer sequences of Leu, Vai, He, Ala and / or Met. Two, three or more prolines can also be incorporated, in particular in longer sequences.

[0246] Examples for useful subsequences to be incorporated in the endosomal escape peptide (e.g., an endosomal escape peptide having a sequence set forth in SEQ ID NO:1 to SEQ ID NO: 1118 or SEQ ID NO: 1310 to SEQ ID NO: 1335) also include AHHA (SEQ ID NO: 1268), AHHE (SEQ ID NO: 1269), AHHD (SEQ ID NO: 1270), EHHE (SEQ ID NO: 1271), DHHD (SEQ ID NO: 1272), EHHD (SEQ ID NO: 1273), DHHE (SEQ ID NO: 1274), HHEE (SEQ ID NO: 1275), HHDD (SEQ ID NO: 1276), DDHH (SEQ ID NO: 1277), EEHH (SEQ ID NO: 1278), EHEH (SEQ ID NO: 1279), DHEH (SEQ ID NO: 1280), EHHH (SEQ ID NO: 1281), DHHH (SEQ ID NO: 1282), HHHE (SEQ ID NO: 1283), HHHD (SEQ ID NO: 1284), HHAE (SEQ ID NO: 1285), HHAD (SEQ ID NO: 1286), HAEA (SEQ ID NO: 1287), HADA (SEQ ID NO: 1288), LAHH (SEQ ID NO: 1289), LAHD (SEQ ID NO: 1290), LAHE (SEQ ID NO: 1291), LAEH (SEQ ID NO: 1292), LADH (SEQ ID NO: 1293), AHAD (SEQ ID NO: 1294), ADAH (SEQ ID NO: 1295), AEAH (SEQ ID NO: 1296), AHAE (SEQ ID NO: 1297), AHLD (SEQ ID NO: 1298), ADLH (SEQ ID NO: 1299), AELH (SEQ ID NO: 1300), AHLE (SEQ ID NO: 1301), LHAD (SEQ ID NO: 1302), LDAH (SEQ ID NO: 1303), LEAH (SEQ ID NO: 1304), LHAE (SEQ ID NO: 1305), LHLD (SEQ ID NO: 1306), LDLH (SEQ ID NO: 1307), LELH (SEQ ID NO: 1308), or 1309 LHLE (SEQ ID NO: Such sequences can be repeated, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 13, 14, 15 or more times, or combined with each other. Optionally, the sequence rich in lipophilic amino acids can contain at least one proline, which can serve as a structure breaker of longersequences of Leu, Vai, He, Ala and / or Met. Two, three or more prolines can also be incorporated, in particular in longer sequences. The incorporation of prolines may help the stabilization of the aLNP.

[0247] In another aspect, the endosomal escape molecule (e.g., an endosomal escape peptide of SEQ ID NO: 1 to SEQ ID NO: 1118 or SEQ ID NO: 1310 to SEQ ID NO: 1335) that can be attached to the surface of an aLNP of the present disclosure is a polymer. In some aspects, the polymer is selected from natural, synthetic or semisynthetic polymers. In some aspects, the polymer exhibits a molecular weight of about 0.5 kDa to about 20 kDa, such as from about 0.5 kDa to about 11.5 kDa, or from about 1 kDa to about 10 kDa, or from about 0.1 kDa to about 8 kDa, or from about 0.1 kDa to about 6 kDa, or from about 0.1 kDa to about 5 kDa, or from about 0.5 kDa to about 5 kDa, or from about 0.3 kDa to about 20 kDa, or from about 0.3 kDa to about 10 kDa, or from about 0.4 kDa to about 10 kDa, or from about 0.5 kDa to about 10 kDa, or from about 0.5 kDa to about 7.5 kDa, or from about 0.5 kDa to about 4 kDa, or from about 0.5 kDa to about 3 kDa, or from about 0.67 kDa to about 2.7 kDa, respectively.

[0248] In some aspects, the endosomal escape peptide is a pH-dependent fusogenic peptide. In some aspects, the endosomal escape peptide is amphipathic. In some aspects, the endosomal escape peptide is anionic and glutamic acid or aspartic acid rich, and, optionally, histidine rich. In some aspects, the endosomal escape peptide is anionic and glutamic acid or aspartic acid rich, and, is histidine rich. In some aspects, the endosomal escape peptide is about 60, about 55, about 50, about 40, about 45, about 40, about 35, about 34, about 33, about 32, about 31, about 30, about 29, about 28, about 27, about 26, about 25, about 24, about 23, about 22, about 21, or about 20 amino acid residues in length. In some aspects, the endosomal escape peptide is between about 20 amino acid residues and about 60 amino acids residues in length. In some aspects, the endosomal escape peptide is about 25 amino acid residues to about 60 amino acid residues in length. In some aspects, the endosomal escape peptide is about 30 amino acid residues to about 60 amino acid residues in length.

[0249] In some aspects, the glutamic acid (E) or aspartic acid (D) residues are evenly distributed along the length of the endosomal escape peptide; and / or the ratio of negatively charged amino acid glutamic acid (E) or aspartic acid (D) to positively charged amino acid residues arginine (R) or lysine (K) is from at least 10:9 to 9:2; and / or the ratio of hydrophilic amino acid residues to hydrophobic amino acid residues at pH 7 is at least 30:70 or 30:60 to 40:60.

[0250] In some aspects, the endosomal escape peptide comprises an amphipathic cell penetrating peptide of less than about 50 amino acid residues, less than about 40 amino acid residues, less thanor equal to about 30 amino acid residues, less than or equal to about 29 amino acid residues, comprising or consisting of at least 2 glutamic acid (E) or aspartic acid (D), at least 6 non-polar amino acids residues (e.g. Alanine (A), Leucine (L), Isoleucine (I), Valine (V) or Methionine (M)), optionally at least one aromatic amino acid (tryptophan (W), tyrosine (Y), Threonine (T), and no more than three positively charged amino acid residues arginine (R) or lysine (K).

[0251] Endosomal escape peptides are provided in U.S. Pat. No. 10,822,595, which is herein incorporated by reference in its entirety.

[0252] In some aspects, the endosomal escape peptide is not GALA. l.B.ii Targeting moieties

[0253] In some aspects, the aLNP of the present disclosure comprises one or more targeting moieties. In some aspects, the targeting moiety comprises an antibody or antigen-binding portion thereof or a ligand that specifically binds to a receptor. In some aspects, the targeting moiety can direct the aLNP to a specific receptor, antigen, cell type, tissue, organ, or physiological compartment.

[0254] The present disclosure provides cell- or tissue-specific aLNP -based delivery systems in which at least one targeting molecule is conjugated to the surface of the aLNP. The targeting molecules can specifically bind to a target present on the surface of specific cell types, e.g., T-cells or cancer cells, or tissues, e.g., endothelium, muscle tissue, CNS, or tumors. Proteins that are suitable as targets for directing payloads via the aLNP of the present disclosure to specific tissues, cell types, human cancers, etc. are disclosed, e.g., at the Human Protein Atlas, Ryboshapkina & Hamma (2019) Sci. Rep. 9:7233; or US20230203199A1 which are herein incorporated by reference in their entireties. In some aspects, a targeting molecule comprises an antibody or antigen-binding portion.

[0255] In some aspects, at least one T-cell targeting molecule in an aLNP -based delivery system comprises an antigen-binding molecule, wherein the antigen is specifically binds to a T-cell specific surface protein. In some aspects, the aLNP-based delivery system comprises a bispecific or multispecific antigen-binding molecule capable of specifically binding to at least one T-cell specific surface proteins. In other aspects, the aLNP-based delivery system comprises a set of monospecific (monovalent, bivalent, or multivalent) antigen-binding molecules capable of specifically binding to at least two T-cell specific surface proteins. In some aspects, the antigenbinding molecules used in the aLNP-based delivery system of the present disclosure are antibodies, or at least comprise an antibody.

[0256] In some aspects, the antibody is IgG, IgM, IgE, IgA or IgD. In some aspects, the IgG is IgGl, IgG2, IgG3 or IgG4. In some aspects, at least one T-cell targeting molecule in the aLNP- based delivery system of the present disclosure comprises a Fab, scFab, Fab', F(ab')2, Fv, or scFv. In some aspects, the antibody is human or humanized. l.B.iii Half-life extenders

[0257] In some aspects, the aLNP of the preset disclosure comprises one or more half-life extenders. As used herein, the term “half-life extender” refers to a molecule attached to the surface of an aLNP of the present disclosure that reduces the clearance rate of the aLNP and / or prevents or slows down the degradation of the aLNP. l.B.iv “Don’t eat me” signals

[0258] In some aspects, the aLNP of the present disclosure comprises one or more don’t eat me signals. As used herein, the term “don’t eat me signal” refers to a molecule attached to the surface of an aLNP of the present disclosure that prevents phagocytosis of the aLNP by macrophages. In some aspects, the don’t eat me signal attached to the surface of an aLNP comprises CD47, CD24, functional fragments or variants thereof, or a combination thereof.I.B.v Biodistribution modifying agents

[0259] In some aspects, the aLNP-based delivery system of the present disclosure comprises a bio-distribution modifying agent. As used herein, the term a "bio-distribution modifying agent," which refers to an agent (i.e., payload) that can modify the distribution the aLNP in vivo or in vitro (e.g., in a mixed culture of cells of different varieties).

[0260] In some aspects, the bio-distribution modifying agent is a tropism moiety. As used herein, the term "tropism moiety" refers to a molecule on the surface of an aLNP that alters and / or enhances the natural movement of the aLNP. Pharmacokinetics, biodistribution, and in particular tropism and retention in the desired tissue or anatomical location also can be accomplish by selecting the appropriate administration route (e.g., intrathecal administration or intraocular administration to improve tropism to the central nervous system).

[0261] In principle, the aLNP of the present disclosure comprising at least one tropism moiety that can direct the aLNP to a specific target cell or tissue (e.g., a cancer cell) can be administered using any suitable administration method known in the art (e.g., intravenous injection, infusion, or subcutaneous administration) since the presence of the tropism moiety (alone or in combination of a targeting moiety, an antiphagocytic “Don’t eat me” signal, and the use of a specific administration route) will induce a tropism of the aLNP towards the desired target cell or tissue.

[0262] In some aspects, the aLNP -based delivery system comprises a surface ligand covalently attached to the surface of an aLNP of the present disclosure wherein the surface ligand can increase permeation through the blood-brain barrier. In some aspects, the surface ligand is a transferrin receptor.

[0263] In some aspects, the aLNP -based delivery system comprises a surface ligand covalently attached to the surface of an aLNP of the present disclosure wherein the surface ligand is a tissue or cell-specific target ligand that increases aLNP tropism to a tissue or physiological compartment. l.C Physicochemical properties of aLNP l.C.i Size

[0264] The aLNP of the present disclosure have a particle diameter or average particle diameter of about 30 nm to about 500 nm. In some aspects, the aLNP of the present disclosure have a particle diameter or average particle diameter of about 50 nm to about 300 nm. In some aspects, the aLNP of the present disclosure have a particle diameter or average particle diameter of about more 50 nm to about 250 nm. In some aspects, the aLNP of the present disclosure have a particle diameter or average particle diameter of about 75 nm to about 200 nm. In some aspects, the aLNP of the present disclosure have a particle diameter or average particle diameter of about 75 nm to about 190 nm. In some aspects, the aLNP of the present disclosure have a particle diameter or average particle diameter of about 100 nm to about 150 nm.

[0265] In some aspects, the aLNP of the present disclosure have a particle diameter or average particle diameter below about 300 nm. In some aspects, the aLNP of the present disclosure have a particle diameter or average particle diameter below about 290 nm. In some aspects, the aLNP of the present disclosure have a particle diameter or average particle diameter below about 280 nm. In some aspects, the aLNP of the present disclosure have a particle diameter or average particle diameter below about 270 nm. In some aspects, the aLNP of the present disclosure have a particle diameter or average particle diameter below about 260 nm. In some aspects, the aLNP of the present disclosure have a particle diameter or average particle diameter below about 250 nm. In some aspects, the aLNP of the present disclosure have a particle diameter or average particle diameter below about 240 nm. In some aspects, the aLNP of the present disclosure have a particle diameter or average particle diameter below about 230 nm. In some aspects, the aLNP of the present disclosure have a particle diameter or average particle diameter below about 220 nm. In some aspects, the aLNP of the present disclosure have a particle diameter or average particle diameter below about 210 nm. In some aspects, the aLNP of the present disclosure have a particle diameter or average particle diameter below about 200 nm. In some aspects, the aLNP of thepresent disclosure have a particle diameter or average particle diameter below about 199 nm. In some aspects, the aLNP of the present disclosure have a particle diameter or average particle diameter below about 198 nm. In some aspects, the aLNP of the present disclosure have a particle diameter or average particle diameter below about 197 nm. In some aspects, the aLNP of the present disclosure have a particle diameter or average particle diameter below about 196 nm. In some aspects, the aLNP of the present disclosure have a particle diameter or average particle diameter below about 195 nm. In some aspects, the aLNP of the present disclosure have a particle diameter or average particle diameter below about 194 nm. In some aspects, the aLNP of the present disclosure have a particle diameter or average particle diameter below about 193 nm. In some aspects, the aLNP of the present disclosure have a particle diameter or average particle diameter below about 192 nm. In some aspects, the aLNP of the present disclosure have a particle diameter or average particle diameter below about 191 nm. In some aspects, the aLNP of the present disclosure have a particle diameter or average particle diameter below about 190 nm. In some aspects, the aLNP of the present disclosure have a particle diameter or average particle diameter below about 180 nm. In some aspects, the aLNP of the present disclosure have a particle diameter or average particle diameter below about 170 nm.

[0266] In some aspects, the aLNP of the present disclosure have a particle diameter or average particle diameter of about 300 nm. In some aspects, the aLNP of the present disclosure have a particle diameter or average particle diameter of about 290 nm. In some aspects, the aLNP of the present disclosure have a particle diameter or average particle diameter of 280 nm. In some aspects, the aLNP of the present disclosure have a particle diameter or average particle diameter of about 270 nm. In some aspects, the aLNP of the present disclosure have a particle diameter or average particle diameter of about 260 nm. In some aspects, the aLNP of the present disclosure have a particle diameter or average particle diameter of about 250 nm. In some aspects, the aLNP of the present disclosure have a particle diameter or average particle diameter of about 240 nm. In some aspects, the aLNP of the present disclosure have a particle diameter or average particle diameter of about 230 nm. In some aspects, the aLNP of the present disclosure have a particle diameter or average particle diameter of about 220 nm. In some aspects, the aLNP of the present disclosure have a particle diameter or average particle diameter of about 210 nm. In some aspects, the aLNP of the present disclosure have a particle diameter or average particle diameter of about 200 nm. In some aspects, the aLNP of the present disclosure have a particle diameter or average particle diameter of about 199 nm. In some aspects, the aLNP of the present disclosure have a particle diameter or average particle diameter of about 198 nm. In some aspects, the aLNP of the presentdisclosure have a particle diameter or average particle diameter of about 197 nm. In some aspects, the aLNP of the present disclosure have a particle diameter or average particle diameter of about 196 nm. In some aspects, the aLNP of the present disclosure have a particle diameter or average particle diameter of about 195 nm. In some aspects, the aLNP of the present disclosure have a particle diameter or average particle diameter of about 194 nm. In some aspects, the aLNP of the present disclosure have a particle diameter or average particle diameter of about 193 nm. In some aspects, the aLNP of the present disclosure have a particle diameter or average particle diameter of about 192 nm. In some aspects, the aLNP of the present disclosure have a particle diameter or average particle diameter of about 191 nm. In some aspects, the aLNP of the present disclosure have a particle diameter or average particle diameter of about 190 nm. In some aspects, the aLNP of the present disclosure have a particle diameter or average particle diameter of about 180 nm. In some aspects, the aLNP of the present disclosure have a particle diameter or average particle diameter of about 170 nm.

[0267] In some aspects, the aLNP of the present disclosure have a particle diameter or average particle diameter between about 20 nm and about 300 nm. In some aspects, the aLNP of the present disclosure have a particle diameter or average particle diameter between about 30 nm and about 300 nm. In some aspects, the aLNP of the present disclosure have a particle diameter or average particle diameter between about 40 nm and about 300 nm. In some aspects, the aLNP of the present disclosure have a particle diameter or average particle diameter between about 50 nm and about 300 nm. In some aspects, the aLNP of the present disclosure have a particle diameter or average particle diameter between about 60 nm and about 300 nm. In some aspects, the aLNP of the present disclosure have a particle diameter or average particle diameter between about 70 nm and about 300 nm. In some aspects, the aLNP of the present disclosure have a particle diameter or average particle diameter between about 80 nm and about 300 nm. In some aspects, the aLNP of the present disclosure have a particle diameter or average particle diameter between about 90 nm and about 300 nm. In some aspects, the aLNP of the present disclosure have a particle diameter or average particle diameter between about 100 nm and about 300 nm. In some aspects, the aLNP of the present disclosure have a particle diameter or average particle diameter between about 110 nm and about 300 nm. In some aspects, the aLNP of the present disclosure have a particle diameter or average particle diameter between about 120 nm and about 300 nm. In some aspects, the aLNP of the present disclosure have a particle diameter or average particle diameter between about 130 nm and about 300 nm. In some aspects, the aLNP of the present disclosure have a particle diameter or average particle diameter between about 140 nm and about 300 nm. In some aspects, the aLNP ofthe present disclosure have a particle diameter or average particle diameter between about 150 nm and about 300 nm. In some aspects, the aLNP of the present disclosure have a particle diameter or average particle diameter between about 160 nm and about 300 nm. In some aspects, the aLNP of the present disclosure have a particle diameter or average particle diameter between about 170 nm and about 300 nm. In some aspects, the aLNP of the present disclosure have a particle diameter or average particle diameter between about 180 nm and about 300 nm. In some aspects, the aLNP of the present disclosure have a particle diameter or average particle diameter between about 190 nm and about 300 nm. In some aspects, the aLNP of the present disclosure have a particle diameter or average particle diameter between about 200 nm and about 300 nm. In some aspects, the aLNP of the present disclosure have a particle diameter or average particle diameter between about 210 nm and about 300 nm. In some aspects, the aLNP of the present disclosure have a particle diameter or average particle diameter between about 220 nm and about 300 nm. In some aspects, the aLNP of the present disclosure have a particle diameter or average particle diameter between about 230 nm and about 300 nm. In some aspects, the aLNP of the present disclosure have a particle diameter or average particle diameter between about 240 nm and about 300 nm. In some aspects, the aLNP of the present disclosure have a particle diameter or average particle diameter between about 250 nm and about 300 nm. In some aspects, the aLNP of the present disclosure have a particle diameter or average particle diameter between about 260 nm and about 300 nm. In some aspects, the aLNP of the present disclosure have a particle diameter or average particle diameter between about 270 nm and about 300 nm. In some aspects, the aLNP of the present disclosure have a particle diameter or average particle diameter between about 280 nm and about 300 nm. In some aspects, the aLNP of the present disclosure have a particle diameter or average particle diameter between about 290 nm and about 300 nm.

[0268] In some aspects of the present disclosure, the term “particle diameter” refers to the hydrodynamic diameter, i.e. the diameter of a perfect solid sphere that would exhibit the same hydrodynamic friction as the particle. The (average) particle diameter can be measured by dynamic light scattering (DLS), e.g. using a Malvern Zetasizer Nano S (Malvern Instruments, Malvern, UK), such as equipped with a He-Ne 633 nm laser configured with a scattering angle of 173°. In some aspects, the term “(average) hydrodynamic particle diameter” refers to the diameter of a single particle or the average particle diameter as determined for a collection of particles as present, e.g., in a population of aLNP of the present disclosure.l.C.ii Concentration

[0269] In some aspects, a preparation comprising aLNP of the present disclosure comprises at least about 2, at least about 10, at least about 50, at least about 100, at least about 200, at least about 500, at least about 1000, at least about 10,000, at least about 25,000, at least about 50,000, at least about 100,000, at least about 250,000, at least about 500,000, at least about 1,000,000, at least about 10,000,000, or at least about 100,000,000 or more aLNP per ml (or per pl).

[0270] In some aspects, a preparation comprising aLNP of the present disclosure comprises at about 2, about 10, about 50, about 100, about 200, about 500, about 1000, about 5,000, about 10,000, about 25,000, about 50,000, about 100,000, about 250,000, about 500,000, about 1,000,000, about 2,000,000, about 3,000,000, about 4,000,000, about 5,000,000, about 6,000,000, about 7,000,000, about 8,000,000, about 9,000,000, about 10,000,000, about 20,000,000, about 30,000,000,, about 40,000,000, about 50,000,000, about 60,000,000, about 70,000,000, about 80,000,000, about 90,000,000, or about 100,000,000 or more aLNP per ml (or per pl). l.C.iii Homogeneity / Polydispersity

[0271] The “poly dispersity” (or polydispersity index, PDI) as used herein is a measure of the heterogeneity of a sample based on particle diameter. In some aspects, the method of determining the hydrodynamic diameter and the poly dispersity (PDI) of an aLNP preparation of the present disclosure is by measuring DLS, e.g. using a Malvern Zetasizer Nano S (Malvern Instruments, Malvern, UK), such as equipped with a He-Ne 633 nm laser configured with a scattering angle of 173°.

[0272] In a specific aspect, a DLS protocol to determine the PDI of an aLNP preparation of the present disclosure comprises the following steps:(i) diluting the aLNP samples are diluted in a buffer, e.g., PBS;(ii) performing DLS, e.g., using a Malvern Zetasizer Nano S (Malvern Instruments, Malvern, UK) equipped with a He-Ne 633 nm laser configured with a scattering angle of 173°;(iii) analyzing the DLS results, e.g., using Malvern’s Zetasizer v7.13 software (Malvern Instruments, Malvern, UK).

[0273] In some aspects of the method disclosed above, a minimum of 3 measurements are performed per sample. In some aspects, the measurement duration is automatically set by the software depending on the samples’ characteristics. In some aspects, a viscosity of 0.8872 centipoises (cP) and refractive index (RI) of 1.330 for the dispersant and an RI of 1.590 and absorption of 0.010 for the material in suspension are set in the software of the instrument. In some aspects, the samples are diluted in PBS, and measured at 25 °C. A PDI below 0.3 is consideredoptimal. Thus, in some aspects, the PDI of an aLNP preparation of the present disclosure has a PDI below about 0.3.

[0274] In some aspects, an aLNP preparation of the present disclosure has a polydispersity index (i.e. PDI) in particle diameter of less than about 0.6, less than about 0.5, less than about 0.4, less than about 0.35, less than about 0.30, less than about 0.25, less than about 0.2, or less than about 0.15. In some aspects, an aLNP preparation of the present disclosure has a PDI between about 0.01 and about 0.5. In some aspects, an aLNP preparation of the present disclosure has a PDI between about 0.01 and about 0.4. In some aspects, an aLNP preparation of the present disclosure has a PDI between about 0.01, about 0.02, about 0.03, about 0.04, about 0.05, about 0.06, about 0.07, about 0.08, about 0.09, about 0.1, about 0.15, about 0.20, about 0.25, about 0.25, about 0.30, about 0.35, about 0.40, about 0.45, or about 0.50. In some aspects, an aLNP preparation of the present disclosure has a PDI between about 0.01 and about 0.05, about 0.05 and about 0.10, about 0.10 and about 0.15, about 0.15 and about 0.20, about 0.20 and about 0.25, about 0.25 and about 0.30, about 0.30 and about 0.35, about 0.35 and 0.40, about 0.40 and about 0.45, about 0.45 and about 0.50, about 0.10 and about 0.20, about 0.20 and about 0.30, about 0.30 and about 0.40, about 0.40 and about 0.50, about 0.1 and about 0.3, or about 0.2 and about 0.4. l.C.iv Charge

[0275] In some aspects, the aLNP of the present disclosure have a zeta potential of between about -100 mV and about -10 mV. The term “zeta potential” as used herein is a measure of the overall charge that a particle or plurality of particles acquire(s) in a particular medium. The “zeta potential” can refer to the charge of a single particle or the (average) overall charge of a collection of particles as determined for a collection of particles. Generally, larger zeta potentials predict a more stable dispersion, which means that all the particles in suspension will tend to repel each other thus preventing aggregation. The zeta potential is expressed in mV and can be determined, e.g., by electrophoretic light scattering (ELS). Exemplary measurement conditions are described below.

[0276] In some aspects, zeta potential can be measured by ELS as follows:(i) samples are diluted in buffer, e.g., O. lx PBS (1 volume of PBS and 9 volumes of purified water);(ii) the zeta potential is determined using a Zetasizer Nano Z instrument (Malvern Instruments, Malvern, UK);(iii) the ELS data is analyzed using Malvern’s Zetasizer v7.13 software.

[0277] In some aspects, a minimum of 3 measurements are performed per sample, and the measurement duration is automatically set by the software depending on the samples’characteristics. In some aspects, the samples are measured at 25 °C. In some aspects, a viscosity of 0.8872 cP, RI of 1.330 and dielectric constant of 78.5 for the dispersant and an RI of 1.590 and absorption of 0.010 for the material in suspension are set in the software of the instrument.

[0278] In some aspects, the aLNP of the present disclosure have a zeta potential of between about -200 mV and about -5 mV. In some aspects, the aLNP of the present disclosure have a zeta potential of between about -150 mV and about -10 mV. In some aspects, the aLNP of the present disclosure have a zeta potential of between about -100 mV and about -15 mV. In some aspects, the aLNP of the present disclosure have a zeta potential of between about -50 mV and about -20 mV.

[0279] In some aspects, the aLNP of the present disclosure have a zeta potential of about -5 mV, about -10 mV, about 015 mV, about -20 mV, about -25 mV, about -30 mV, about -35 mV, about - 40 mV, about -45 mV, about -50 mV, about -55 mV, about -60 mV, about -65 mV, about -70 mV, about -75 mV, about -80 mV, about -85 mV, about -90 mV, about -95 mV, about -100 mV, about-105 mV, about -110 mV, about -115 mV, about -120 mV, about -125 mV, about -130 mV, about-135 mV, about -140 mV, about -145 mV, about -150 mV, about -155 mV, about -160 mV, about-165 mV, about -170 mV, about - 175 mV, about -180 mV, about -185 mV, about -190 mV, about-195 mV, or about -200 mV. l.D Payloads

[0280] The aLNP of the present disclosure can comprise one or more payloads. In some aspects, the aLNP can carry a payload (i) in the aqueous core of the aLNP (e.g., an anticancer agent or a therapeutic peptide), (ii) attached to the inner surface of the lipid bilayer, (iii) inserted in the bilayer, (iv) attached to the surface of the aLNP, or (v) any combination thereof. In some aspects, the payload is an immunologic adjuvant.

[0281] As used herein, the term "payload" refers to a biologically active molecule (e.g., a therapeutic agent) that acts on a target (e.g., a target cell) that is contacted with an aLNP of the present disclosure. Non-limiting examples of payloads that can be introduced into an aLNP of the present disclosure, include therapeutic agents such as nucleotides (e.g., therapeutic nucleotides or nucleotides comprising a detectable moiety), nucleic acids (e.g., DNA or mRNA molecules that encode a polypeptide such as an enzyme, or RNA molecules that have regulatory function such as miRNA, dsDNA, IncRNA, and siRNA), amino acids (e.g., amino acids comprising a detectable moiety), polypeptides (e.g., enzymes), lipids, carbohydrates, or small molecules (e.g., small molecule drugs and toxins).

[0282] In certain aspects, a payload comprises an antigen or a nucleic acid (e.g., an mRNA) encoding an antigen. As used herein, the term "antigen" refers to any agent that when introducedinto a subject elicits an immune response (cellular or humoral) to itself. In some aspects, the antigen is used to elicit an immune response, i.e., as a vaccine, e.g., in a cancer vaccine. In other aspects, a payload comprises an adjuvant. In other aspects, the payload comprises a nucleic acid, e.g., an mRNA encoding a therapeutic protein, e.g., an antibody or a component of a gene editing system.

[0283] In some aspects, the payload molecule is in the aqueous core of the aLNP. In some aspects, the payload molecule is inserted or partially inserted in the hydrophobic core of the lipid bilayer of the aLNP. In some aspects, the payload molecule is covalently attached to the internal surface of the lipid bilayer of the aLNP, facing the aqueous core. In some aspects, the payload molecule is covalently attached to the external surface of the lipid, facing the external medium. In some aspects, the payload molecule is covalently linked to the aLNP via a maleimide moiety. In some aspects, the payload molecule is covalently linked to the aLNP via a linker, e.g., a cleavable linker.

[0284] The aLNP of the present disclosure can be used to deliver a variety of payloads, e.g., immunological adjuvants, therapeutic agent proteins and polynucleotides, detectable labels, and cell penetrating payloads. In some aspects, the payloads are encapsulated in the aLNP. In some aspects of the present disclosure, the payload comprises a polypeptide, a peptide, a polynucleotide, a chemical compound, or any combination thereof. In some aspects, an aLNP of the present disclosure can comprise a single payload. In other aspects, an aLNP of the present disclosure can comprises multiple payloads.

[0285] In some aspects, the payload comprises, consists or consists essentially of a polynucleotide. In some aspects, the polynucleotide is an mRNA, an antisense oligonucleotide (ASO), a phosphorodiamidate morpholino oligonucleotide (PMO), a siRNA, a miRNA, a shRNA, a plasmid, or a vector. In some aspects, the payload comprises a therapeutic small molecule. In some aspects, the small molecule is a proteolysis-targeting chimera (PROTAC). In some aspects, the small molecule is a nucleotide. In some aspects, the nucleotide is a stimulator of interferon genes protein (STING) agonist. l.D.i Immunologic adjuvants

[0286] In some aspects, the payload of an aLNP of the present disclosure comprises an immunologic adjuvant. As used herein, the term “immunologic adjuvant” refers to a substance or compound that modulates, preferably increases, the (innate or adaptive) immune response to another immunological agent, such as a vaccine and / or a cancer immunological agent. This typically means that the potency of the immunological agent is increased and / or lessimmunological agent is needed to achieve a similar efficacy of immune response, in comparison to when the immunologic adjuvant is not used.

[0287] In some aspects, the immunologic adjuvant of the present disclosure can mimic the activity of pathogen-associated molecular patterns (PAMPs), which include lipopolysaccharides, molecular cages for antigens, components of bacterial cell walls, and nucleic acids such as singlestranded RNA, double-stranded RNA, single-stranded DNA, unmethylated CpG dinucleotide- containing DNA, or DNA-RNA hybrids. In some aspects, the immunologic adjuvant comprises a substance or compound that further enhances the immune response nonspecifically or specifically through binding to pattern recognition receptors (PRRs) including but not limited to C-type lectin receptors (CLRs), RIG-like receptors (RLRs), nucleotide-binding oligomerization domain (NOD) proteins, stimulator of interferon genes (STING) and toll-like receptors (TLRs).

[0288] In some aspects, the immunologic adjuvant is a TLR agonist. In some aspects, the TLR agonist is an agonist for one or more of an endosomal TLR. In some aspects, the TLR agonist targets TLR3, TLR7, TLR8, TLR9, or a combination thereof. In the context of the present disclosure, the term “Toll-Like Receptor” refers to TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, TLR11, TLR12, TLR13, or a combination thereof. In some aspects, the toll-like receptor is TLR3.

[0289] In some aspects, the immunological adjuvant comprises a compounds selected from the group consisting of CpG (Cytosine followed by Guanine residue), CpG motif, methylated CpG, CpG oligodeoxynucleotide (ODN, e.g. 5-25 bp) (e.g., CpG 1018, which is a TLR9 agonist), Poly(LC) (i.e., polyinosinic:polycytidylic acid, which is a TLR3 agonist), poly(I:Ci2U) (which is a TLR3 agonist), poly(G:C) (which is a TLR3 agonist), poly(A:U) (which is a TLR3 agonist), poly(U) (which is a TLR7 / 8 agonist), and a combination thereof. In some aspects, the CpG ODN can be a class A, class B or class C CpG ODN, or a combination thereof.

[0290] The term “CpG” as used herein means a nucleic acid (analogue) having a cytosine followed by a guanine linked by a phosphate bond in which the pyrimidine ring of the cytosine is unmethylated. The term “methylated CpG” as used herein means methylation of the cytosine on the pyrimidine ring, preferably occurring at the 5-position of the pyrimidine ring. The term “CpG motif’ as used herein means a pattern of bases that includes an unmethylated central CpG surrounded by at least one base flanking (on the 3' and the 5' side of) the central CpG. The term “CpG ODN” as used herein means an oligodeoxynucleotide comprises one or more unmethylated CpG and preferably is least about ten nucleotides in length. The “CpG ODN” is preferably singlestranded. The entire CpG ODN can be unmethylated or portions can be unmethylated. In oneaspect, at least the C of the 5' CG 3' is unmethylated. The “CpG ODN” can be a class A, class B or class C CpG ODN. The skilled person is familiar with the difference classes of CpG ODNs and the different types of CpG ODN that can be used in context of the current disclosure (e.g. Zhang et al. Pharmaceutics. 2021 Dec 28; 14(1):73.),

[0291] In some aspects, the immunologic adjuvant is a TLR3 agonist. TLR3 interacts with the ribose-phosphate backbone of double stranded RNA and has no specific sequence requirement. In some aspects, the immunologic adjuvant comprises a TLR1 agonist, a TLR2 agonist, a TLR3 agonist, a TLR4 agonist, a TLR5 agonist, a TLR6 agonist, a TLR7 agonist, a TLR8 agonist, a TLR9 agonist, a TLR10 agonist, a TLR11 agonist, a TLR12 agonist, a TLR13 agonist, or a combination thereof.

[0292] In some aspects, the immunologic adjuvant comprises poly(I:C) (i.e. polyinosinic:poly cytidylic acid). In some aspects, the poly(I:C) according to the present disclosure comprises single-stranded polyinosinic acid (Poly I) and single-stranded polycytidylic acid (Poly C), that are not associated by hydrogen bonding or covalent bonding at the time of administration as well as double-stranded or complexed Poly I / Poly C. In some aspects, the Poly(I:C) is a mismatched double-stranded RNA with one strand being a polymer of inosinic acid, the other a polymer of cytidylic acid.

[0293] In some aspects, the immunologic adjuvant comprises poly(I:Ci2U). In some aspects, the poly(I:Ci2U) is a poly(I:C) with a U mismatch at every 12th base of the C strand. In some aspects, Poly (A:U) (Polyadenylic-polyuridylic acid) or poly (C:G) (Polycytidylic-guanylic acid could be used as an alternative analogue to poly(I:C). l.D.ii Gene editing system components

[0294] In some aspects, the payload of an aLNP of the present disclosure comprises a polynucleotide comprising one or more components of a gene editing system. In some aspects, the payload comprises a gRNA. In some aspects, the payload comprises an mRNA encoding a nuclease. In some aspects, the payload comprises a gRNA and an mRNA encoding a nuclease.

[0295] CRISPR / Cas: In some aspects, the gene editing system used with an aLNP of the present disclosure can comprise a CRISPR system. In some aspects, the payload of an aLNP of the present disclosure comprises an mRNA encoding a CRISPR Cas nuclease, e.g., a mRNA encoding a Cas9 nuclease.

[0296] In some aspects, the CRISPR / Cas nuclease is codon-optimized for the desired cell type in which it is to be expressed. In some aspects, the CRIPS / Cas gene editing system can also employ a guide RNA (gRNA) that comprises two separate molecules. An exemplary two-molecule gRNAcomprises a crRNA-like ("CRISPR RNA" or "targeter-RNA" or "crRNA" or "crRNA repeat") molecule and a corresponding tracrRNA-like ("trans-acting CRISPR RNA" or "activator-RNA" or "tracrRNA" or "scaffold") molecule.

[0297] A crRNA comprises both the DNA-targeting segment (single stranded) of the gRNA and a stretch of nucleotides that forms one-half of a double stranded RNA (dsRNA) duplex of the protein-binding segment of the gRNA. A corresponding tracrRNA (activator-RNA) comprises a stretch of nucleotides that forms the other half of the dsRNA duplex of the protein-binding segment of the gRNA. Thus, a stretch of nucleotides of a crRNA are complementary to and hybridize with a stretch of nucleotides of a tracrRNA to form the dsRNA duplex of the protein-binding domain of the gRNA. As such, each crRNA can be said to have a corresponding tracrRNA. The crRNA additionally provides the single stranded DNA-targeting segment. Accordingly, a gRNA comprises a sequence that hybridizes to a target sequence, and a tracrRNA. Thus, a crRNA and a tracrRNA (as a corresponding pair) hybridize to form a gRNA. If used for modification within a cell, the exact sequence and / or length of a given crRNA or tracrRNA molecule can be designed to be specific to the species in which the RNA molecules will be used.

[0298] In some aspects, the CRISPR / Cas gene editing system can employ a fused crRNA- tracrRNA construct (i.e., a single transcript) that functions with the codon-optimized Cas9. This single RNA is often referred to as a guide RNA or gRNA. Within a gRNA, the crRNA portion is identified as the "target sequence" for the given recognition site and the tracrRNA is often referred to as the "scaffold." To generate a gRNA, a short DNA fragment containing the target sequence is inserted into a guide RNA expression nucleic acid. The gRNA expression nucleic acid comprises the target sequence (in some aspects around 20 nucleotides), a form of the tracrRNA sequence (the scaffold) as well as a suitable promoter that is active in the cell and necessary elements for proper processing in eukaryotic cells. In some aspects, the payload of the aLNP comprises the gRNA expression nucleic acid comprising the target sequence (in some aspects around 20 nucleotides), a form of the tracrRNA sequence (the scaffold) as well as a suitable promoter. In some aspects, custom, complementary oligonucleotides are annealed to form a double stranded DNA and are then cloned into the gRNA expression nucleic acid, which is included as payload in the aLNP. In some aspects, the payload comprises a two-molecule gRNA or a fused crRNA-tracrRNA construct.

[0299] In some aspects, the payload comprises a Cas9 nuclease provided in the form of a protein. In some aspects, the Cas9 protein can be provided in the form of a complex with the gRNA. In other aspects, the Cas9 nuclease can be provided in the form of a nucleic acid encoding the protein. The nucleic acid encoding the Cas9 nuclease can be RNA (e.g., messenger RNA (mRNA)) orDNA. In some aspects, the gRNA can be provided in the form of RNA. In other aspects, the gRNA can be provided in the form of DNA encoding the RNA. In some aspects, the gRNA can be provided in the form of separate crRNA and tracrRNA molecules, or separate DNA molecules encoding the crRNA and tracrRNA, respectively.

[0300] In some aspects, the gRNA comprises a third nucleic acid sequence encoding a Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) RNA (crRNA) and a trans-activating CRISPR RNA (tracrRNA). In some aspects, the Cas protein to be used with the aLNP is a type I Cas protein. In some aspects, the Cas protein is a type II Cas protein. In some aspects, the type II Cas protein is Cas9. In some aspects, the type II Cas, e.g., Cas9 protein, is a human codon- optimized Cas.

[0301] In certain aspects, the Cas protein is a "nickase" that can create single strand breaks (i.e., "nicks") at the target site without cutting both strands of double stranded DNA (dsDNA). Cas9, for example, comprises two nuclease domains — a RuvC-like nuclease domain and an HNH-like nuclease domain — which are responsible for cleavage of opposite DNA strands. Mutation in either of these domains can create a nickase. Examples of mutations creating nickases can be found, for example, WO / 2013 / 176772A1 and WO / 2013 / 142578A1, each of which is herein incorporated by reference.

[0302] In certain aspects, two separate Cas proteins (e.g., nickases) specific for a target site on each strand of dsDNA can create overhanging sequences complementary to overhanging sequences on another nucleic acid, or a separate region on the same nucleic acid. The overhanging ends created by contacting a nucleic acid with two nickases specific for target sites on both strands of dsDNA can be either 5' or 3' overhanging ends. For example, a first nickase can create a single strand break on the first strand of dsDNA, while a second nickase can create a single strand break on the second strand of dsDNA such that overhanging sequences are created. The target sites of each nickase creating the single strand break can be selected such that the overhanging end sequences created are complementary to overhanging end sequences on a different nucleic acid molecule. The complementary overhanging ends of the two different nucleic acid molecules can be annealed by the methods disclosed herein. In some aspects, the target site of the nickase on the first strand is different from the target site of the nickase on the second strand.

[0303] In some aspects, the first nucleic acid comprises a mutation that disrupts at least one amino acid residue of nuclease active sites in the Cas protein, wherein the mutant Cas protein generates a break in only one strand of the target DNA region, and wherein the mutation diminishes non-homologous recombination in the target DNA region. In some aspects, the first nucleic acidthat encodes the Cas protein further comprises a nuclear localization signal (NLS). In some aspects, the nuclear localization signal is a SV40 nuclear localization signal.

[0304] Talen: In some aspects, the gene editing system used as payload in an aLNP of the present disclosure can comprise a TALEN system. TAL effector nucleases are a class of sequence-specific nucleases that can be used to make double-strand breaks at specific target sequences in the genome of a prokaryotic or eukaryotic organism. TAL effector nucleases are created by fusing a native or engineered transcription activator-like (TAL) effector, or functional part thereof, to the catalytic domain of an endonuclease, such as, for example, Fokl.

[0305] The unique, modular TAL effector DNA binding domain allows for the design of proteins with potentially any given DNA recognition specificity. Thus, the DNA binding domains of the TAL effector nucleases can be engineered to recognize specific DNA target sites and thus, used to make double-strand breaks at desired target sequences. See, WO 2010 / 079430; Morbitzer et al. (2010) PNAS 10.1073 / pnas.1013133107; Scholze & Boch (2010) Virulence 1 :428-432; Christian et al. Genetics (2010) 186:757-761; Li et al. (2010) Nuc. Acids Res. (2010) doi: 10.1093 / nar / gkq704; and Miller et al. (2011) Nature Biotechnology 29: 143-148; all of which are herein incorporated by reference.

[0306] Examples of suitable TAL nucleases, and methods for preparing suitable TAL nucleases, are disclosed, e.g., in US Patent Application No. 2011 / 0239315 Al, 2011 / 0269234 Al, 2011 / 0145940 Al, 2003 / 0232410 Al, 2005 / 0208489 Al, 2005 / 0026157 Al, 2005 / 0064474 Al, 2006 / 0188987 Al, and 2006 / 0063231 Al (each hereby incorporated by reference).

[0307] In various aspects, TAL effector nucleases are engineered that cut in or near a target nucleic acid sequence in, e.g., a genomic locus of interest, wherein the target nucleic acid sequence is at or near a sequence to be modified by a targeting vector. The TAL nucleases suitable for use with the various methods and compositions provided herein include those that are specifically designed to bind at or near target nucleic acid sequences to be modified in a target cell using the aLNP system of the present disclosure.

[0308] In some aspects, each monomer of the TALEN comprises 12-25 TAL repeats, wherein each TAL repeat binds a 1 bp subsite. In some aspects, the nuclease agent is a chimeric protein comprising a TAL repeat-based DNA binding domain operably linked to an independent nuclease. In some aspects, the independent nuclease is a Fokl endonuclease. In some aspects, the nuclease agent comprises a first TAL-repeat-based DNA binding domain and a second TAL-repeat-based DNA binding domain, wherein each of the first and the second TAL-repeat-based DNA binding domain is operably linked to a Fokl nuclease, wherein the first and the second TAL-repeat-basedDNA binding domain recognize two contiguous target DNA sequences in each strand of the target DNA sequence separated by about 6 bp to about 40 bp cleavage site, and wherein the FokI nucleases dimerize and make a double strand break at a target sequence.

[0309] In some aspects, the nuclease agent of the aLNP payload comprises a first TAL-repeat- based DNA binding domain and a second TAL-repeat-based DNA binding domain, wherein each of the first and the second TAL-repeat-based DNA binding domain is operably linked to a FokI nuclease, wherein the first and the second TAL-repeat-based DNA binding domain recognize two contiguous target DNA sequences in each strand of the target DNA sequence separated by a 5 bp or 6 bp cleavage site, and wherein the FokI nucleases dimerize and make a double strand break.

[0310] Zinc-finger nucleases: In some aspects, the gene editing system of the aLNP payload can comprise a zinc-finger nuclease (ZFN) system. In some aspects, each monomer of the ZFN comprises 3 or more zinc finger-based DNA binding domains, wherein each zinc finger-based DNA binding domain binds to a 3 bp subsite. In other aspects, the ZFN is a chimeric protein comprising a zinc finger-based DNA binding domain operably linked to an independent nuclease. In some aspects, the independent endonuclease is a FokI endonuclease. In some aspects, the nuclease agent comprises a first ZFN and a second ZFN, wherein each of the first ZFN and the second ZFN is operably linked to a FokI nuclease, wherein the first and the second ZFN recognize two contiguous target DNA sequences in each strand of the target DNA sequence separated by about 6 bp to about 40 bp cleavage site or about a 5 bp to about 6 bp cleavage site, and wherein the FokI nucleases dimerize and make a double strand break. See, for example, US20060246567; US20080182332; US20020081614; US20030021776; WO / 2002 / 057308A2; US20130123484; US20100291048; and, WO / 2011 / 017293 A2, each of which is herein incorporated by reference.

[0311] Meganucleases: In some aspects, the gene editing system of the aLNP payload can comprise a meganuclease system. Meganucleases (or homing endonucleases or HEases) have been classified into four families based on conserved sequence motifs, the families are the "LAGLIDADG," "GIY-YIG," "H-N-H," and "His-Cys box" families. These motifs participate in the coordination of metal ions and hydrolysis of phosphodiester bonds.

[0312] HEases are notable for their long recognition sites, and for tolerating some sequence polymorphisms in their DNA substrates. Meganuclease domains, structure and function are known, see for example, Guhan and Muniyappa (2003) Crit Rev Biochem Mol Biol 38: 199-248; Lucas et al., (2001) Nucleic Acids Res 29:960-9; Jurica and Stoddard, (1999) Cell Mol Life Sci 55: 1304- 26; Stoddard, (2006) Q Rev Biophys 38:49-95; and Moure et al., (2002) Nat Struct Biol 9:764.

[0313] In some aspects, a payload of the aLNP comprises a naturally occurring variant, and / or engineered derivative meganuclease. Methods for modifying the kinetics, cofactor interactions, expression, optimal conditions, and / or recognition site specificity, and screening for activity are known, see for example, Epinat et al., (2003) Nucleic Acids Res 31 :2952-62; Chevalier et al., (2002) Mol Cell 10:895-905; Gimble et al., (2003) Mol Biol 334:993-1008; Seligman et al., (2002) Nucleic Acids Res 30:3870-9; Sussman et al., (2004) J Mol Biol 342:31-41; Rosen et al., (2006) Nucleic Acids Res 34:4791-800; Chames et al., (2005) Nucleic Acids Res 33:el78; Smith et al., (2006) Nucleic Acids Res 34:el49; Gruen et al., (2002) Nucleic Acids Res 30:e29; Chen and Zhao, (2005) Nucleic Acids Res 33:el54; W02005105989; W02003078619; W02006097854; W02006097853; W02006097784; and W02004031346.

[0314] Any meganuclease can be used as payload with the aLNP system described herein, including, but not limited to, I-Scel, I-Scell, 1-SceIII, LScelV, I-SceV, I-SecVI, LSceVII, I-Ceul, I-CeuAIIP, LCrel, LCrepsblP, LCrepsbllP, 1-CrepsbIIIP, LCrepsblVP, LTlil, I-Ppol, PLPspI, F- Scel, F-Scell, F-Suvl, F-TevI, F-TevII, LAmal, LAnil, LChuI, LCmoel, LCpal, LCpall, LCsmI, LCvuI, LCvuAIP, LDdil, LDdill, LDirl, LDmoI, I-Hmul, LHmuII, LHsNIP, LLlal, I-Msol, I- Naal, LNanI, I-NcIIP, LNgrlP, LNitl, LNjal, I-Nsp236IP, I-PakI, I-PboIP, LPcuIP, LPcuAI, I- PcuVI, LPgrIP, LPoblP, LPorllP, I-PbpIP, LSpBetalP, I-Scal, I-SexIP, 1-SneIP, LSpomI, I- SpomCP, LSpomlP, LSpomllP, LSquIP, LSsp6803I, LSthPhiJP, I-SthPhiST3P, I-SthPhiSTe3bP, LTdelP, LTevI, I-TevII, I-TevIII, LUarAP, LUarHGPAIP, I-UarHGPA13P, I-VinIP, LZbilP, PL Mtul, PLMtuHIP, PLMtuHIIP, Pl-Pful, Pl-PfuII, Pl-Pkol, PLPkoII, PI-Rma43812IP, PL SpBetalP, Pl-Scel, PI-Tful, PI-TfuII, PI-Thyl, PI-Tlil, PI-Tlill, or any active variants or fragments thereof.

[0315] In some aspects, the meganuclease recognizes double-stranded DNA sequences of 12 to 40 base pairs. In some aspects, the meganuclease recognizes one perfectly matched target sequence in one of the heterologous plasmids described herein. In some aspects, the meganuclease is a homing nuclease. In some aspects, the homing nuclease is a "LAGLID ADG" family of homing nuclease. In some aspects, the "LAGLID ADG" family of homing nuclease is selected from I-Scel, LCrel, and LDmol.

[0316] Restriction endonucleases: In some aspects, the gene editing system of aLNP payload can comprise a restriction endonuclease, which includes Type I, Type II, Type III, and Type IV endonucleases. Type I and Type III restriction endonucleases recognize specific recognition sites, but typically cleave at a variable position from the nuclease-binding site, which can be hundreds of base pairs away from the cleavage site (recognition site). In Type II systems the restrictionactivity is independent of any methylase activity, and cleavage typically occurs at specific sites within or near to the binding site. Most Type II enzymes cut palindromic sequences, however Type Ila enzymes recognize non-palindromic recognition sites and cleave outside of the recognition site, Type lib enzymes cut sequences twice with both sites outside of the recognition site, and Type Ils enzymes recognize an asymmetric recognition site and cleave on one side and at a defined distance of about 1-20 nucleotides from the recognition site. Type IV restriction enzymes target methylated DNA. Restriction enzymes are further described and classified, for example in the REBASE database (webpage at rebase.neb.com; Roberts et al., (2003) Nucleic Acids Res 31 :418-20), Roberts et al., (2003) Nucleic Acids Res 31 : 1805-12, and Belfort et al., (2002) in Mobile DNA II, pp. 761-783, Eds. Craigie et al., (ASM Press, Washington, D.C.). l.D.iii Therapeutics mRNAs

[0317] In some aspects, the payload of the aLNP comprises an mRNA molecule, wherein the mRNA molecule is encapsulated within the LNP. The mRNA can be completely or partially encapsulated within the LNP. In some aspects, the payload comprises a single species of mRNA. In other aspects, the payload comprises one or more (e.g., a cocktail) mRNAs. mRNA can comprise at least one, two, three, four, five, six, seven, eight, nine, ten, or more modified nucleotides such as 2'0Me nucleotides. Preferably, uridine and / or guanosine nucleotides in the mRNA are modified with 2'0Me nucleotides. In some aspects, the mRNA can further comprise modified (e.g., 2'0Me- modified) adenosine and / or modified (e.g., 2'OMe-modified) cytosine nucleotides. In some aspects, the mRNA can further comprise, linkage modifications, e.g., phosphorothioate linkages.

[0318] In some aspects, the mRNA(s) are fully encapsulated in the aLNP. With respect to formulations comprising an mRNA cocktail, the different types of mRNA species present in the cocktail (e.g., mRNA having different sequences) can be co-encapsulated in the same aLNP, or each type of mRNA species present in the cocktail can be encapsulated in a separate aLNP. The mRNA cocktail can be formulated in the aLNP described herein using a mixture of two or more individual mRNAs (each having a unique sequence) at identical, similar, or different concentrations or molar ratios. In one aspect, a cocktail of mRNAs (corresponding to a plurality of mRNAs with different sequences) is formulated using identical, similar, or different concentrations or molar ratios of each mRNA species, and the different types of mRNAs are co-encapsulated in the same aLNP. In another aspect, each type of mRNA species present in the cocktail is encapsulated in different aLNP at identical, similar, or different mRNA concentrations or molar ratios, and the aLNP thus formed (each containing a different mRNA payload) are administeredseparately (e.g., at different times in accordance with a therapeutic regimen), or are combined and administered together as a single unit dose (e.g., with a pharmaceutically acceptable carrier).

[0319] In some aspects, the mRNA comprises an mRNA vaccine, e.g., an mRNA vaccine to treat, e.g., COVID-19 (SARS-CoV2 infection), influenza, RSV infection, rabies, HPV infection, malaria, EBV infection, tuberculosis, CMV infection, Herpes zoster, Zika virus infection, HBV infection, yellow fever, PIV infection, hMPV infection, rotavirus infection, Nipah or virus infection. In some aspects, the mRNA comprises an mRNA encoding an antibody to treat, e.g., COVID-19, HIV infection, or Chikungunya virus infection. In some aspects, the mRNA encodes one or more components of gene editing system. In some aspects, the mRNA encodes a vaccine for the treatment of cancer, e.g., melanoma, NSCLC, cervical cancer, breast cancer, ovarian cancer, liver cancer, gastric cancer, pancreatic cancer, colorectal cancer, bladder cancer, prostate cancer, head and neck cancer, adenoidcystic carcinoma, cSCC, basal cell cancer, renal cell cancer, or AML, In some aspects, the vaccine for the treatment of cancer is a personal vaccine. In some aspects, the mRNA encodes a CAR (see below). In some aspects, the mRNA encodes an antibody or antigen-binding portion thereof. In some aspects, the mRNA encodes an antibody disclosed below or an antigen-binding portion thereof (e.g., the antigen-binding portion of a CAR). In some aspects, the mRNA encodes a protein for protein replacement therapy. In some aspects, the mRNA encodes a component of the CRISPR / Cas nuclease system.

[0320] In some aspects, the mRNA encodes a protein for protein replacement therapy in genetic diseases such as cystic fibrosis, propionic academia, methylmalonic academia, CSDla, phenylketonuria, CN-1, OTC, or hemophilia. In some aspects, the mRNA encodes a protein for protein replacement therapy in autoimmune disorders. In some aspects, the mRNA encodes a protein for protein replacement therapy in metabolic disorders, e.g., type 2 diabetes. In some aspects, the mRNA encodes a protein for protein replacement therapy in cardiovascular disease, e.g., hypercholesterolemia or myocardial ischemia. In some aspects, the mRNA encodes a protein for protein replacement therapy in fibrosis, e.g., hypertropic scarring, liver fibrosis, lung fibrosis, anemia, or primary sclerosing cholangitis. See Qin et al (2022) “mRNA-based therapeutics: powerful and versatile tools to combat diseases.” Signal Transduction and Targeted Therapy 7: 166; Huang et al. (2022) “The landscape of mRNA nanomedicine” Nature Medicine 28:2273-2287; and Liu et al. (2022) “mRNA-based cancer therapeutics” Nature Reviews Cancer 23:526-543, which are herein incorporated by reference in their entireties.l.D.iv Chimeric Antigen Receptors (CARs)

[0321] In some aspects the payload of the aLNP comprises a CAR or polynucleotide encoding a CAR. As used herein, the term “Chimeric Antigen Receptor” or alternatively a “CAR” refers to a recombinant polypeptide construct comprising at least (i) an extracellular antigen binding domain, (ii) a transmembrane domain, and (iii) a cytoplasmic signaling domain comprising a functional signaling domain derived from a stimulatory molecule and / or costimulatory molecule. In its simplest form, a CAR comprises a set of polypeptides, typically two, which when in an immune effector cell, provides the cell with specificity for a target cell, typically a cancer cell, and with intracellular signal generation. In some aspects, the set of polypeptides are in the same polypeptide chain, e.g., comprise a chimeric fusion protein. In some aspects, the set of polypeptides are not contiguous with each other, e.g., are in different polypeptide chains. In some aspects, the set of polypeptides include a dimerization switch that, upon the presence of a dimerization molecule, can couple the polypeptides to one another, e.g., can couple an antigen-binding domain to an intracellular signaling domain. In some aspects, the stimulatory molecule of the CAR is the zeta chain associated with the T cell receptor complex (CD3 zeta). In some aspects, the cytoplasmic signaling domain comprises a primary signaling domain (e.g., a primary signaling domain of CD3 zeta). In some aspects, the cytoplasmic signaling domain further comprises one or more functional signaling domains derived from at least one costimulatory molecule defined below. In some aspects, the costimulatory molecule is chosen from the costimulatory molecules described herein, e.g., 4- IBB, CD27, and / or CD28.

[0322] In some aspects, the CAR comprises a chimeric fusion protein comprising an antigenbinding domain (extracellular antigen binding domain), a transmembrane domain and an intracellular signaling domain comprising a functional signaling domain derived from a stimulatory molecule, wherein the antigen-binding domain and the transmembrane domain are linked by a CAR spacer. In some aspects, the CAR comprises a chimeric fusion protein comprising an antigen-binding domain (extracellular antigen binding domain ) linked to a transmembrane domain via a CAR spacer and an intracellular signaling domain comprising a functional signaling domain derived from a costimulatory molecule and a functional signaling domain derived from a stimulatory molecule. In some aspects, the CAR comprises a chimeric fusion protein comprising an extracellular antigen binding domain linked to a transmembrane domain via a CAR spacer and an intracellular signaling domain comprising two functional signaling domains derived from one or more costimulatory molecule(s) and a functional signaling domain derived from a stimulatory molecule. In some aspects, the CAR comprises an extracellular antigen binding domain linked toa transmembrane domain via a CAR spacer and an intracellular signaling domain comprising at least two functional signaling domains derived from one or more costimulatory molecule(s) and a functional signaling domain derived from a stimulatory molecule. In some aspects, the CAR comprises an optional leader sequence at the amino-terminus (N-terminus) of the CAR. In some aspects, the CAR further comprises a leader sequence at the N-terminus of the antigen-binding domain, wherein the leader sequence is optionally cleaved from the antigen-binding domain (e.g., a scFv) during cellular processing and localization of the CAR to the cellular membrane.

[0323] In some aspects, the present disclosure provide polynucleotides encoding a CAR comprising, e.g., (i) an extracellular antigen binding domain, (ii) a transmembrane domain, (iii) an intracellular domain, and (iv) a CAR spacer comprising an amino acid sequence derived from a human immunoglobulin (Ig) hinge region and / or loop region (z.e., a CAR spacer), and optionally a linker (e.g, a Gly-Ser rich linker) wherein the spacer is located between the extracellular antigen binding domain and the transmembrane domain. In some aspects, the present disclosure provides a recombinant nucleic acid construct comprising a transgene encoding a CAR of the present disclosure. The present disclosure also provides a CAR encoded by one or more of the polynucleotide sequences or the vectors disclosed herein. In some aspects, the CAR of present disclosure is designed as a standard CAR, a split CAR, an off-switch CAR, an on-switch CAR, a first-generation CAR, a second-generation CAR, a third-generation CAR, a fourth-generation CAR, or a fifth generation CAR.

[0324] In some specific aspects, the payload of the aLNP comprises a CAR or polynucleotide encoding a CAR wherein the CAR’s antigen binding portion is an anti-CD19, anti-BCMA, anti- HER2, anti-CD20, anti-CD22, anti-IL13Ra2, anti-GPC3, or combination thereof. In some aspects the payload comprises a CAR or polynucleotide encoding a CAR wherein the CAR’s antigen binding portion comprises an scFv derived from a therapeutic antibody disclosed herein, e.g., a therapeutic antibody disclosed herein targeting an antigen expressed on the surface of T cells. In some aspects, the CAR is a monospecific CAR. In some aspects, the CAR is a bispecific CAR. l.D.v Therapeutic antibodies

[0325] In some aspects, the aLNP payload comprises a therapeutic antibody or an antigenbinding portion thereof, e.g., a therapeutic antibody disclosed below or an antigen-binding portion thereof. In some aspects the payload can comprise a fusion protein comprising a therapeutic antibody disclosed below or an antigen-binding portion thereof.

[0326] It is to be noted that the used of the therapeutic antibodies and antigen-binding portions thereof disclose below is not limited to serve as payloads in the delivery systems of the presentdisclosure. Thus, in some aspects, a therapeutic antibody disclosed below or an antigen-binding portion thereof can be or can be part of a surface anchored targeting molecule in a aLNP delivery system of the present disclosure, and direct the aLNP to a cell or tissue expressing the molecule to which an antibody disclosed below binds specifically.

[0327] In some aspects, VH and / or VL domains of antibodies disclosed below can be used as part of the targeting portion of a CAR. In some aspects, a CAR used as payload in a LNP delivery system of the present disclosure can comprise a scFv comprising VH and VL domains from a therapeutic antibody.

[0328] In some aspects, the payload comprises an antibody or antigen-binding portion thereof targets an apoptosis regulator. In some aspects, the apoptosis regulator is pro-apoptotic gene product, e.g, FasL (Fas ligand), BAX, BID, BAK or BAD. In some aspects, the apoptosis regulator is an anti-apoptotic gene product, e.g, Bcl-XI, an IAP (e.g., XIAP), or Bcl-2. In some aspects, the apoptosis regulator is a prosurvival factor such as cFLIP, BNIP3, FADD, Akt, or NF-KB. l.D.vi Therapeutic Oligonucleotides.

[0329] In some aspects, the payload of the aLNP comprises a nucleic acid, e.g., an RNA or a DNA. Nucleic acid active agents suitable for delivery using the aLNP of the present disclosure include all types of RNA and all types of DNA, including also oligonucleotides such as probes and primers used in the polymerase chain reaction (PCR), hybridizations, or DNA sequencing. In some aspects, the nucleic acid comprises mRNA, miRNA, miRNA sponge, tough decoy miRNA (TD), antimir (antagomir), small RNA, rRNA, siRNA, shRNA, gDNA, cDNA, pDNA, PNA, BNA, antisense oligonucleotide (ASO), aptamer, cyclic dinucleotide, or any combination thereof.

[0330] In some aspects, the payload comprises a short interfering RNA (siRNA), which is a double-stranded RNA that can induce sequence-specific post-transcriptional gene silencing, thereby decreasing or even inhibiting gene expression. For example, siRNAs can trigger the specific degradation of homologous RNA molecules, such as mRNAs, within the region of sequence identity between both the siRNA and the target RNA. Non-limiting exemplary siRNAs are disclosed in WO 02 / 44321, which is incorporated by reference in its entirety.

[0331] In some aspects, the payload comprises a short hairpin RNA (shRNA). In some aspects, the payload comprises a miRNA or a miRNA inhibitor (antimiR). In some aspects, the payload can be between about 10 and about 30 nucleotides in length, for example from about 14 to about 25 nucleotides in length. In some aspects, the payload has a length of 16 to 30 nucleotides, 18 to 25 nucleotides, particularly 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length.

[0332] Sequences for miRNAs are available publicly, for example, through the miRBase registry (Griffiths-Jones, et al., Nucleic Acids Res., 36(Database Issue):D154-D158 (2008); Griffiths- Jones, et al., Nucleic Acids Res., 36(Database Issue):D140-D144 (2008); Griffiths-Jones, et al., Nucleic Acids Res., 36(Database Issue) :D1O9-D111 (2008)) and other publically accessible databases.

[0333] In some aspects, the miRNA inhibitors are oligomers or polymers of ribonucleic acid (RNA) or deoxyribonucleic acid (DNA) or modifications thereof. In some aspects, the miRNA antagonists are antimir. Antimirs are a specific class of miRNA inhibitors that are described, for example, in US2007 / 0213292 to Stoffel et al. Antimirs are RNA-like oligonucleotides that contain various modifications for RNase protection and pharmacologic properties such as enhanced tissue and cellular uptake. Antimirs differ from normal RNA by having complete 2'-O-methylation of sugar, phosphorothioate backbone and a cholesterol-moiety at 3 '-end.

[0334] Non-limiting examples of antimirs and other miRNA inhibitors are described in W02009 / 020771, W02008 / 091703, W02008 / 046911, W02008 / 074328, W02007 / 090073, W02007 / 027775, W02007 / 027894, W02007 / 021896, W02006 / 093526, W02006 / 112872, W02007 / 112753, W02007 / 112754, W02005 / 023986, or W02005 / 013901, all of which are hereby incorporated by reference.

[0335] In some aspects, the nucleic acids are phosphodiester antisense oligonucleotides, and any oligonucleotides where the sugar-phosphate "backbone" has been derivatized or replaced with "backbone analogues" such as with phosphorothioate, phosphorodithioate, phosphoroamidate, alkyl phosphotriester, or methylphosphonate linkages. In some aspects, the nucleic acids active agents are antisense oligonucleotides, and any oligonucleotides or oligodeoxynucleotides with non-phosphorous backbone analogues such as sulfamate, 3'-thioformacetal, methylene(methylimino) (MMI), 3'-N-carbamate, or morpholino carbamate.

[0336] In some aspects, the biologically active molecule (payload) is an antimir. As used herein, the terms "antimir," "anti microRNA," "anti miRNA," and variants thereof refer to molecules (e.g., synthetically generated molecules) that are used to neutralize microRNA (miRNA) function in cells for desired responses. MiRNA are complementary sequences (approx. 20-22bp) to mRNA that are involved in the cleavage of RNA or the suppression of the translation. By controlling the miRNA that regulate mRNAs in cells, antimirs (also called anti-miRNA oligonucleotides, AMOs, or antagomirs) can be used as further regulation as well as for therapeutic for certain cellular disorders. This regulation can occur through a steric blocking mechanism as well as hybridization to miRNA.

[0337] These interactions within the body between antimirs and a miRNA can be for therapeutics in disorders in which over / under expression occurs or aberrations in miRNA lead to coding issues. Some of the miRNA-linked disorders that are encountered in the humans include cancers, muscular diseases, autoimmune disorders, and viruses.

[0338] Various components of antimirs can be manipulated to affect the binding affinity and potency of the antimir. The 2’-sugar of the antimirs can be modified by introducing fluorine or various methyl groups, almost all with an increase in binding affinity. However, some of these modified 2’-sugar antimirs lead to negative effects on cell growth. Modifying the 5'-3' phosphodiester backbone linkage to a phosphorothioate (P-S) backbone linkage is also known to have an effect on target affinity. Using the P-S mutation was shown to decrease the Tm of the oligonucleotide, which leads to a lower target affinity. A final requirement for antimirs is mismatch specificity and length restrictions. Due to miRNAs in the same families sharing "seed" (shared) sequences and differ by only a couple of additional nucleotides; one antimir can potentially target multiple miRNA sequences.

[0339] In some aspects, the payload of the aLNP comprises an antisense oligonucleotide (AON) for gene editing via ADAR, wherein the antisense oligonucleotide recruits endogenous ADAR (adenosine deaminase acting on RNA). See U.S. Patent Nos. 10,941,402; 11,274,300; 10,988,763; and 10,676,737; or U.S. Publ. Nos. US 2021-0230590; US 2022-0127609; US 2022-0177894; US 2022-0340900; US 2023-0235322; US 2022-0307023; and US 2023-0323346, which are herein incorporated by reference in their entireties. In some aspects, the aLNP comprises an AON for gene editing via ADAR. In some aspects, the ADAR is an endogenous ADAR enzyme. In some aspects, the ADAR is an exogenous ADAR enzyme.

[0340] In some aspects, the aLNP comprises an AON for gene editing via ADAR and a mRNA encoding an ADAR enzyme. In some aspects, the present disclosure provides a first population of aLNP comprising an AON for gene editing via ADAR, and a second population of aLNP comprising an mRNA encoding an ADAR enzyme. In some aspects, both populations of aLNP are administered concurrently. In some aspects, the aLNP comprising the AON and the aLNP comprising the mRNA encoding the ADAR enzyme are administered sequentially, e.g., the aLNP comprising the mRNA encoding the ADAR enzyme can be administered first, followed by the aLNP comprising the AON, e.g., when exogenous ADAR enzyme expression reaches a detectable level, or a threshold level, for example, in a specific tissue or organ.

[0341] In some aspects, the therapeutic oligonucleotide is selected from the group consisting of is selected from the group consisting of 1018 ISS, AB-729, abetimus, AEG35156, afovirsen,aganirsen, agatolimod, alicaforsen, ALNAAT-02, amlivirsen, anivamersen, apatorsen, aprinocarsen, APTA-16, AR- 177, ARC 19499, archexin, AROANG-3, AR0AP0C-3, ARO-HSD, AS1411, ASM-8, asvasiran, atesidorsen, ATL-1102, ATU-027, avacincaptad pegol, AVI-4126, AVI-7288, AVI-7537, AVT-02, AZD-8233, AZD-8701, baliforsen, bamosiran, bazlitoran, BC007, beclanorsen, belcesiran, bepirovirsen, bevasiranib, BUB-080, BMN 044, BMN 053, brivoligide, casimersen, cavrotolimod, cemdisiran, cenersen, CIVI 008, cimdelirsen, cobitolimod, cobomarsen, CODA-001, cofirasersen, cosdosiran, CpG 7909, CPG-8954, cupabimod, custirsen, danvatirsen, daplusiran, defibrotide, dematirsen, donidalorsen, drisapersen, DYN-101, edifoligide, egaptivon pegol, EIF-4E, eluforsen, emapticap pegol, eplontersen, eteplirsen, fazisiran, fesomersen, fitusiran, fomivirsen, frenlosirsen, gataparsen, givosiran, GNKG-168, golodirsen, GPI-2A, GTI-2040, GTI-2501, GTX-102, HBVAXPRO, imetelstat, IMT-504, inclisiran, inotersen (TEGSEDI™), ION-224, ION-253, ION-363, ION-464, ION-541, ION-859, IONIS-AGTLRX, IONIS-APO(a)-Rx, IONISAR-2.5Rx, IONIS-C9Rx, IONIS-DNM2-2.5Rx, IONISENAC-2.5Rx, IONIS-FB-LRX, IONIS-FXILRX, IONIS-FXIRX, IONIS-GCGRRX, IONIS-HBVLRX, IONIS- MAPTRx, IONIS-PKKRX, IONISTMPRSS-6LRx, IONIS-TTRRX, ISIS EIF4E Rx, ISIS- 104838, ISIS-1082, ISIS-113715, ISIS-2503, ISIS-333611, ISIS-426115, ISIS-449884, ISIS-463588, ISIS- 5132, ISIS-702843, ISIS-757456, ISIS-863633, ISTH-0036, JNJ-3989, lademirsen, lexanersen, lexaptepid pegol, litenimod, LSP-GR3, lumasiran, mipomersen, miravirsen, monarsen, mongersen, MT-5745, MTL-CEBPA, ND-L02-s0201, nedosiran, NS-089, nusinersen, oblimersen, olaptesed pegol, olezarsen, olpasiran, OLX-101, patisiran, pegaptanib, PEGnivacogin, pegpleranib, pelacarsen, prexigebersen, PUL-042, QPI-1007, QR-1123, QRX-421a, radavirsen, remlarsen, renadirsen, revusiran, RG-012, RG-101, RG-6346, RGLS-4326, rimigorsen, rosomidnar, rovanersen, sapablursen, SB010, sepofarsen, siG-12D-LODER, SLN124, SR-063, SRP-5051, STK-001, STP-705, suvodirsen, tadnersen, temavirsen, teprasiran, tilsotolimod, tivanisiran, tofersen, tominersen, tomligisiran, TOP-1731, trabedersen, trecovirsen, varodarsen, VEGLIN 3, vidutolimod, viltolarsen, VIR-2218, volanesorsen, vupanorsen, vutrisiran, WVE-003, WVE-004, WVEN-531, zilebesiran, and zilganersen. l.D.vii Chemotherapy Agents

[0342] In some aspects, the aLNP payload comprises a chemotherapy agent. In some aspects, the chemotherapy agent comprises an alkylating agent, antimetabolite, anti -microtubule, topoisomerase inhibitor, cytotoxic antibiotic, or a combination thereof. In some aspects, the chemotherapy agent comprises, e.g., cyclophosphamide, methotrexate, 5-fluorouracil, doxorubicin, docetaxel, vinblastine, vincristine, prednisolone, bleomycin, etoposide, cisplatin,epirubicin, capecitabine, ifosfamide, folinic acid, oxaliplatin, vinorelbine, procarbazine, mistune, dacarbazine, or a combination thereof. In some aspects, the payload comprises a nucleoside analog selected from the group consisting of azacitidine, capecitabine, carmofur, cladribine, clofarabine, cytarabine, decitabine, floxuridine, fludarabine, fluorouracil, gemcitabine, mercaptopurine, nelarabine, pentostatin, tegafur, and tioguanine. In some aspects, the payload comprises an antifolate selected from the group consisting of methotrexate, pemetrexed, and raltitrexed. In some aspects, the payload comprises a topisomerase I inhibitor such as irinotecan or topotecan. In some aspects, the payload comprises an anthracycline such as daunorubicin, doxorubicin, epirubicin, idarubicin, mitoxantrone or valrubicine. In some aspects, the payload comprises a podophyllotoxin such as etoposide or teniposide. In some aspects, the payload comprises a taxane such a cabazitaxel, docetaxel, or paclitaxel. In some aspects, the payload comprises a Vinca alkaloid selected from the group consisting of vinblastine, vincristine, vindesine, vinflunine, and vinrelbine. In some aspects, the payload comprises an alkylating agent selected from the group consisting of bendamustine, busulfan, carmustine, chlorambucil, chormethine, cyclophosphamide, dacarbazine, folemustine, ifosfamide, lomustine, melphalan, streptozotocin, and temozolomide. In some aspects, the payload comprises a platinum compound selected from the group consisting of carboplatin, cisplatin, nedaplatin, and oxaliplatin.

[0343] In some aspects, the payload comprises a targeted antineoplastic therapeutic agent. In some aspects, the targeted antineoplastic therapeutic agent comprises an antibody, e.g., a monoclonal antibody, or an antigen-binding portion thereof. In some aspects, the monoclonal antibodsy comprises alemtuzumab (anti-CD52), bevacizumab (anti-VEGF), cetuximab (anti- EGFR), denosumab (antri-RNAKL), gemtuzumab ozogamicin (anti-CD33), ibritumomab tiuxetan (anti-CD20), ipilimumab (anti-CTLA4), nivolumab (anti-PDl), ofatumumab (anti-CD20), panitumumab (anti-EGFR), pembrolizumab (anti-PDl), pentuzimab (anti-HER2), rituximab (anti- CD20), tositumomab (anti-CD20), trastuzumab (anti-HER2) or an antigen binding portion thereof.

[0344] In some aspects, the aLNP payload comprises a small molecules tyrosine kinase inhibitor. In some aspects, the tyrosine kinase inhibitor is selected from the group consisting of afatinib (EGFR, HER2 and HER4 inhibitor), aflibercept (VEGF and PGF inhibitor), axitinib (multikinase inhibitor), bosutinib (Bcr-Abl and SRc kinase inhibitor), crizotinib (ALK, HGFR, and RON inhibitor), dasatinib (BCR-ABL, SRC family, c-Kit, EPHA2 and PDGFR-P kinase inhibitor), erlotinib (EGFR inhibitor), gefitinib (EGFR inhibitor), imatinib (Bcr-Abl kinase inhibitor), lapatinib (HER2 inhibitor), nilotinib (Bcr-Abl kinase inhibitor), pazopanib (Multikinase inhibitor, including c-KIT, FGFR, PDGFR and VEGFR), ponatinib (Multikinase inhibitor (BEGFR,PDGFR, FGFR, EPH receptors and SRC families of kinases, and KIT, RET, TIE2 and FLT3), that also inhibits T135I Bcr-Abl kinase), regorafenib (Multikinase inhibitor for RET, VEGFR1, VEGFR2, VEGFR3, KIT, PDGFR-alpha, PDGFR-beta, FGFR1, FGFR2, TIE2, DDR2, Trk2A, Eph2A, RAF-1, BRAF, BRAFV600E, SAPK2, PTK5, and Bcr-Abl.), ruxolitinib (JAK1 and JAK2 inhibitor), sorafenib (Multikinase inhibitor, including VEGF and PDGF receptor kinases), sunitinib (Multikinase inhibitor, including VEGF & PDGF receptor tyrosine kinases), and vandelanic (Tyrosine kinase inhibitor (TKI) with selective activity against RET, VEGFR-2 and EGFR).

[0345] In some aspects, the aLNP payload comprises an mTOR inhibitor such as everolimus or temsirolimus. In some aspects, the aLNP payload comprises a retinoid selected from the group consisting of bexarotene (RXR agonist), isotretinoin (RXR and RAR agonist), tamibarotene (RAR agonist), and tretinoin (RXR and RAR agonist). In some aspects, the aLNP payload comprises an immunomodulatory agent (IMID) such as lenalidomide, thalidomide, or pomalidomide. In some aspects, the aLNP payload comprises a histone deacetylase inhibitor such as romidepsin, valproate, or vorinostat. l.D.viii Detectable substances

[0346] In some aspects, the aLNP payload is a detectable substance. Detectable substances include, but are not limited to, various organic small molecules, inorganic compounds, nanoparticles, enzymes or enzyme substrates, fluorescent materials, luminescent materials, bioluminescent materials, chemiluminescent materials, radioactive materials, and contrast agents. Labels are contemplated by the present disclosure, including, but not limited to, optically detectable labels. Labels can be attached to another payload of the present disclosure, e.g., an mRNA, and / or to a component of the aLNP using standard chemistries such that the label can be removed upon cleavage of a cleavable linker. A detectable label can useful in therapeutic, diagnostic, imaging (e.g., radioimaging), or basic research applications.

[0347] In some aspects, the detectable label is a radioactive label. Examples of a radioactive label include, but are not limited to, the isotopes3H,14C,32P,35S,36C1,51Cr,57Co,58Co,59Fe,90Y,121I,124I,125I,131I,mIn,117LU,211At,198Au,67Cu,225Ac,213Bi, "Tc,186Re and89Zr.

[0348] In some aspects, the detectable label is a chemiluminescent label, fluorescent label, enzyme, biotin, or a combination thereof. In some aspects, the detectable label is a peptide tag. In some aspects, the detectable label is a polyhistidine tag, polyarginine tag, glutathione-S-transferase (GST), maltose binding protein (MBP), chitin binding protein (CBP), Strep-tag, thioredoxin (TRX), poly(NANP), FLAG tag, ALFA-tag, V5-tag, Myc-tag, hemagglutinin (HA) tag, Spot tag,T7 tag, NE tag, or green fluorescence protein (GFP), or a combination thereof. In some aspects, the polyhistidine tag consists of from about 4 to about 10 histidine residues. In some aspects, the polyhistidine tag consists of about 4, about 5, about 6, about 7, about 8, about 9, or about 10 histidine residues. Additional examples of detectable labels and methods for introducing detectable labels into a polypeptide or polynucleotide are known and include routine chemical, molecular biology and recombinant DNA techniques. See, e.g., Hnatowich et al., Science, 220(4597):613- 615, 1983; Yao et al., Int. J. Mol. Sci., 17(2): 194, 2016; Kimple et al., Curr. Protoc. Protein Sci., 73 :Unit 9.9, 2013; Sambrook J, Fritsch EF. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press; Cold Spring Harbor, N.Y.: 1989; Molecular Cell Biology, 4thedition, Section 3.5, Purifying, Detecting and Characterizing Proteins; and Mahmoodi et al., Cogent Biology, 5(1):DOL 10 / 1080 / 23312025.2019.1665406, which are herein incorporated by reference in their entireties.2. Methods of Manufacture2.A aLNP manufacture

[0349] The present disclosure also provides methods for preparing compositions comprising aLNP. In some aspects the method comprises the following steps:(a) providing one or more organic solution comprising one or more lipid, preferably at least one anionic lipid and / or at least one neutral lipid;(b) providing one or more aqueous solution comprising one or more nucleic acid; and,(c) combining the one or more organic solution and the one or more aqueous solution, thereby producing the composition comprising aLNP as disclosed herein.

[0350] In some aspects, the combining in step (c) refers to mixing the one or more organic solutions, and the one or more aqueous solutions (substantially) in laminar flow, e.g., in a microfluidics device. The combining step can be performed using methods such as solvent injection, pulsed jet flow, depressurization of an expanded liquid organic solution into aqueous suspension (DELOS), supercritical anti-solvent (SAS), or film-hydration.

[0351] In some aspects, one or more of steps (a)-(c) of the method are performed by a microfluidic device. The term “microfluidic device” as used herein means a device comprising a combination of two or more (micro-)channels, preferably etched and / or molded into a material (e.g. glass, silicon or polymer), wherein at least two (micro-)channels are connected together in order to achieve a desired feature (e.g. mixing, pumping, sorting), preferably to achieve a laminar flow and mixing of fluids. In some aspects, the method for preparing a composition comprising nucleic acid-lipid particles according to the disclosure does not apply purification using dialysis.

[0352] In some aspects, the combining or mixing in step (c) is in a (substantially) laminar flow and / or the mixing of the aqueous and organic solutions in step (c) of the method is laminar mixing. The mixing is preferably effected by passive diffusion of molecules from a high concentration to a lower concentration domain in laminar flow. As used herein, the term “laminar flow” means that the fluids follow smooth paths in layers, with each layer moving smoothly past the adjacent layers. The “laminar flow” is preferably defined by a Reynolds number of about 2,000 or less (e.g., about 1,900, about 1,800, about 1,700, about 1,600, about 1,500, about 1,400, about 1,300, about 1,200, about 1,100, about 1,000 or less), more preferably in conjunction with a smooth flow (e.g. not inducing chaotic advection, chaotic mixing, and / or lateral mixing). The Reynolds is preferably determined by considering the flow as a flow through a pipe, wherein the Reynolds number is defined as: puDHUDHQDHRe = - = - = - p v vA

[0353] DH is the hydraulic diameter of the channel (m), Q is the volumetric flow rate (m3 / s), A is the pipe's cross-sectional area (m2), u is the mean speed of the fluid (SI units: m / s), is the dynamic viscosity of the fluid (Pa s = N s / m2= kg / (m s)), v is the kinematic viscosity of the fluid (v = p / p (m2 / s)), p is the density of the fluid (kg / m3).

[0354] Better encapsulation efficiency and better PDI are be achieved with laminar mixing, as compared to turbulent of chaotic mixing. Higher encapsulation efficiency and lower PDI is particularly achieved in laminar mixing and having the combination of at least one organic solution and two sequential aqueous solutions.

[0355] In some aspects, the present disclosure does not exclude that step (c) employs chaotic flow mixing and / or the mixing of the aqueous and organic solutions in step (c) of the method under chaotic flow. The term “chaotic flow” as used herein means a flow that is not a laminar flow, and is neither constant in time nor presenting any regular periodicity. A turbulent flow can be an example of a chaotic flow. “Chaotic mixing” as used herein means that mixing occurs under chaotic flow.

[0356] In an aspect, the present disclosure does not exclude that the flow in step (c) is a turbulent flow and / or the mixing of the aqueous and organic solutions in step (c) of the method is turbulent mixing. “Turbulent mixing” as used herein means that mixing occurs under a turbulent flow. The term “turbulent flow” as used herein means a fluid motion characterized by fluid flow not in parallel layers and / or with disruption between those layers. A “turbulent flow” is preferably dominated by inertial forces, which typically produce chaotic eddies, vortices and other flowinstabilities. A “turbulent flow” preferably means that the flow is characterized by chaotic changes in pressure and flow velocity. In an aspect, the term “turbulent flow” encompasses any type of flow that is not a laminar flow. In an aspect, the turbulent flow is defined by a Reynolds number larger than about 4,000, preferably larger than about 5,000, more preferably larger than about 6,000. A turbulent flow can be an example of a chaotic flow.

[0357] Typically, a laminar flow is interspersed with turbulent flow until at a Reynolds number of about 2,000 to about 4,000, or about 2,500 to about 3,500. A flow of between about 2,000 and about 4,000, or between about 2,500 to about 3,500 can herein be defined as a laminar and / or turbulent flow.

[0358] In an aspect, chaotic and / or turbulent mixing (or chaotic and / or turbulent flow) according to the disclosure is achieved by providing a mixer device, preferably a micromixer chip on a microfluidic device. The mixer device can achieve a chaotic and / or turbulent flow by mixing of a laminar flow.

[0359] In an aspect, the solvent selected for the dissolution of the lipids to produce the aLNP of the present disclosure is one or more selected preferentially but not limited from the group of aqueous miscible solvents, such as methanol, ethanol, isopropanol, butanol, acetonitrile, acetone, dimethyl sulfoxide. More preferably, the organic solvent is an aqueous miscible solvent classified as a class 3 solvent. Class 3 includes no solvent known as a human health hazard at levels normally accepted in pharmaceuticals (permissible dose accepted of 50 mg or more per day) (Q3C (R6): Impurities: guideline for residual solvents EMA / CHMP / ICH / 82260 / 2006). Examples of class 3 solvents are ethanol, acetone, dimethyl sulfoxide, isopropanol, ethyl ether, methyl acetate, 1- pentanol, 1-propanol. Most preferably, the organic solvent(s) is / are ethanol, isopropanol, or a mixture of them. The ethanol solution can comprise at least about 10, at least about 20, 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 99% (v / v) ethanol.

[0360] The ethanol solution can be pure ethanol (100% ethanol). In addition or alternatively, the ethanol solution can comprise no more than at least about 99, at least about 95, at least about 90, at least about 80, at least about 70, at least about 60, or at least about 50% (v / v) ethanol.

[0361] The isopropanol solution can comprise at least at least about 10, at least about 20, 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 99% (v / v) isopropanol. The isopropanol solution can be pure isopropanol (100% isopropanol). In addition or alternatively, the isopropanol solutioncan comprise no more than at least about 99, at least about 95, at least about 90, at least about 80, at least about 70, at least about 60, or at least about 50% (v / v) isopropanol.

[0362] In some aspects, ethanol is used as organic solution. An increase in ethanol concentration can led to larger and more monodisperse particles. Hydrophobic interactions can determine the mechanism for the encapsulation of nucleic acids in the aLNP. In the presence of ethanol, nucleic acids change its conformation, exposing hydrophobic nucleotide bases that can interact with lipid tails, allowing the formation of aLNP containing the nucleic acids in the hydrophobic core of the bilayer(s).

[0363] In some aspects, the aqueous solution is water. In an aspect, the aqueous solution in the context of the current disclosure contains an osmolar agent such as NaCl or sucrose and / or a pH buffer group, such as one or more selected but no limited from the group consisting of phosphate, histidine, HEPES, Tris, acetate, carbonate, and citrate. In addition or alternatively, the aqueous solution as disclosed herein is RNase and DNase free.

[0364] In some aspects, the aqueous solution as disclosed herein has a pH of between about 5.0 and about 9.5. In some aspects, the aqueous solution as disclosed herein has a pH of between about 5.5 and about 9.0. In some aspects, the aqueous solution as disclosed herein has a pH of between about 6.0 and about 8.5. In some aspects, the aqueous solution as disclosed herein has a pH of between about 6.0 and about 8.0.

[0365] In some aspects, the flow rate ratio (A:B) between the one or more aqueous solutions (A) and the one or more organic solutions (B) is between 48: 1 and 1 : 10, preferably 24: 1 and 1 :8, more preferably 18: 1 and 1 :3, even more preferably 10: 1 and 1 : 1.

[0366] In a preferred aspect, the at least one anionic and / or at least one neutral lipid is provided in the organic solution disclosed herein in a concentration of 0.01 - 500 mM, preferably 0.1- 50 mM, more 1-20 mM.

[0367] In a preferred aspect, the one or more nucleic acid is provided in the aqueous solution in a concentration of 5 - 50000 pg / ml, preferably 10-50000 pg / ml, more preferably 50 - 5000 pg / ml, more preferably 100 - 2000 pg / ml, even more preferably 200 - 1000 pg / ml.

[0368] In a preferred aspect, the organic solution in step a) comprises at least 10 mol%, preferably at least 20 mol%, more preferably at least 30 mol%, even more preferably at least 40 mol%, most preferably at least 50 mol% (e.g. at least 60, 70, 80, 90, 95, or 99 mol%) of at least one anionic and / or at least one neutral lipid, relative to total mol lipid in the organic solution.

[0369] In an aspect, the organic solution comprises 5-50 mol%, preferably 10-40 mol%, more preferably 20-30 mol% anionic lipid and / or neutral lipid, relative to total mol lipid in the organic solution.

[0370] In a preferred aspect, the organic solution in step a) comprises at most 40 mol%, preferably at most 30 mol%, more preferably at most 20 mol%, even more preferably at most 10 mol%, most preferably at most 5 mol% (e.g. at most 4, 3, 2,1, 0.5, or 0.1 mol%) of (at least one) cationic lipid, relative to total mol lipid in the organic solution.

[0371] In a preferred aspect, the organic solution in step a) comprises at least 1 mol%, preferably at least 5 mol%, more preferably at least 10 mol%, even more preferably at least 20 mol%, most preferably at least 30 mol% of at least one sterol, relative to the total mol lipid (including sterol) in the organic solution. In an aspect, the organic solution comprises 5-66 mol%, preferably 10-40 mol%, more preferably 20-35 mol% sterol, relative to total mol lipid (including sterol) in the organic solution.

[0372] In a preferred aspect, the organic solution in step a) is free of cationic lipids and / or comprises at most 1 mol% of (at least one) cationic lipid relative to the total mol lipid in the organic solution.

[0373] In a preferred aspect, the organic solution in step a) is free of cationic polymer, and / or comprises at most 1 mol.%, preferably at most 0.1 mol.%, more preferably at most 0.01 mol.% of at least one (cationic) polymer, calculated on the total mol lipid of the organic solution.

[0374] In a preferred aspect, the method and / or particles of the disclosure do not involve or comprise multivalent cation (such as Ca2+, Mn2+, Mg2+) cationic lipid and / or a (cationic) polymer or at most 10, 5, 1 mol.% relative to total mol lipid of the aLNPs.

[0375] In an aspect, the method of the disclosure provides (or achieves) a nucleic acid encapsulation efficiency in the nucleic acid-aLNP of at least 40%, preferably at least 60%, more preferably at least 80%, even more preferably at least 90%, most preferably at least 95%. In addition or alternatively, the method of the disclosure preferably provides (or achieves) a nucleic acid encapsulation efficiency in the nucleic acid-aLNP of 60 - 95%, preferably 70-90%, more preferably 75-85%. The “encapsulation efficiency (of nucleic acid)” as used herein means the percentage of nucleic acid that is incorporated into the nucleic acid-aLNP, when compared with the total amount of nucleic acid present in the formulation, and is calculated as follows:

[0376] In some aspect, the method of manufacture disclosed herein comprises one or more steps or methodologies disclosed in PCT Publ. No. W02024084089, e.g., the use of impingement jet mixing, which is herein incorporated by reference in its entirety.2.B Small, medium, and large scale aLNP manufacture

[0377] The present disclosure also provides methods for the manufacture of aLNP at small-scale, medium-scale, and large-scale.2.C Conjugation methods

[0378] Different molecules, e.g., endosomal escape peptides, targeting moieties, payloads, etc., described generically in this section as “heterologous moieties” can be conjugated to the surface of an aLNP of the present disclosure using any of a number of methods known in the art. Thus, the incorporation of an endosomal escape peptides, a targeting moiety, e.g., a molecule that specifically targets an aLNP disclosed herein to a specific cell type or tissue, or any heterologous moiety in general can take place using any conjugation approach know in the art, e.g., those disclosed in Adhikari P et al. “Antibody-Drug Conjugates, Methods and Protocols.” Methods Mol. Biol. 2019;2078:51-69; or McPherson & Hobson “Antibody-Drug Conjugates, Methods and Protocols.” Methods Mol. Biol. 2019;2078:23-36, which are incorporated herein by reference in their entireties. In a particular aspect of the present disclosure, the molecules are conjugated to the surface of the aLNP using click chemistry, e.g., maleimide chemistry.

[0379] In some aspects, the methods disclosed herein can be applied to conjugate a heterologous moiety (e.g., a targeting antibody or endosomal escape peptide) to the surface of an aLNP. In other aspects, the methods disclosed herein can also be used to attach, e.g., a lipid to an endosomal escape peptide to generate an endosomal escape lipopeptide.

[0380] In some aspects, the conjugation comprises reacting a maleimide group covalently attached to the lipid with a sulfhydryl group of the heterologous molecule, e.g., an endosomal escape peptide. In some aspects, the lipid is DSPE-PEG2000-maleimide, DMG-PEG2000- maleimide, cholesterol-PEG2000-maleimide, DSPE-PEG5000-maleimide, DMG-PEG5000- maleimide, cholesterol-PEG5000-maleimide or a combination thereof.

[0381] In some aspects, the conjugation comprises reacting a maleimide group attached to...

Claims

WHAT IS CLAIMED IS:

1. An endosomal escape peptide between 15 and 50 amino acids in length comprising at least two amino acid subsequences selected from the group consisting of EALAHH (SEQ ID NO: 1336), DALAHH (SEQ ID NO: 1337), EALAHW (SEQ ID NO: 1338), DALAHY (SEQ ID NO: 1339), DALAHG (SEQ ID NO: 1340), and DALAHW (SEQ ID NO: 1341), wherein one optional non-polar amino acid can be intercalated between the subsequences, and wherein one tryptophan is located at the N-terminus and / or C-terminus of the endosomal escape peptide.

2. The endosomal escape peptide of claim 1, wherein the endosomal escape peptide comprises the EALAHH (SEQ ID NO: 1336) subsequence and is selected from the group consisting of SEQ ID NOS: 1038, 1039, 1040, 1041, 1310, 1311, 1312, 1313, 1314, 1319, 1328, 1329, 1330, 1331, 1332, 1333, and 1334.

3. The endosomal escape peptide of claim 1, wherein the endosomal escape peptide comprises the DALAHH (SEQ ID NO: 1337) subsequence and is selected from the group consisting of SEQ ID NOS: 1311, 1328, 91329, 1330, and 1331.

4. The endosomal escape peptide of claim 1, wherein the endosomal escape peptide comprises the EALAHW (SEQ ID NO: 1338) subsequence and is selected from the group consisting of SEQ ID NOS: 1038, 1039, 1040, 1041, 1310, 1311, 1312, 1313, 1314, 1319, 1328, 1329, 1330, 1331, 1332, 1333, and 1334.

5. The endosomal escape peptide of claim 1, wherein the endosomal escape peptide comprises the DALAHY (SEQ ID NO: 1339) subsequence and has the amino acid sequence set forth in SEQ ID NO: 1329.

6. The endosomal escape peptide of claim 1, wherein the endosomal escape peptide comprises the DALAHG (SEQ ID NO: 1340) subsequence and has the amino acid sequence set forth in SEQ ID NO: 1328.

7. The endosomal escape peptide of claim 1, wherein the endosomal escape peptide comprising the DALAHW (SEQ ID NO: 1341) subsequence and has the amino acid sequence set forth in of SEQ ID NO: 1330.

8. The endosomal escape peptide of claim 1, wherein the one optional non-polar amino acid intercalated between the subsequences is selected from the group consisting of alanine, valine, leucine, isoleucine, methionine, and cysteine.

9. The endosomal escape peptide of any one of claims 1 to 8, further comprising a N- terminal or C-terminal cysteine.

10. The endosomal escape peptide of any onoe of claims 1 to 9, wherein the endosomal escape peptide is conjugated to a lipid, thereby yielding a lipopeptide.

11. The endosomal escape peptide of claim 10, wherein the endosomal escape peptide is conjugated to the lipid via a click chemistry reaction.

12. The endosomal escape peptide of claim 10, wherein the endosomal escape peptide is conjugated to the lipid via an amide linkage, via an ester linkage, via an hemisuccinate linker, through an amide-amide bond, or via a keto linker such as a disulfide or hydrazone.

13. The endosomal escape peptide of claim 10, wherein the lipid is selected from the group consisting of stearic acid, palmitic acid, oleic acid, cholesterol, sitosterol, DSPE, DSPE-PEG, DPPE, DOPE, myristoyl diglyceride, palmitoyl diglyceride and stearyl diglyceride.

14. An anionic lipid nanoparticle (aLNP) comprising a fusogenic lipid and endosomal escape peptide of any one of claims 1 to 13, wherein the aLNP has net negative charge at neutral pH.

15. The aLNP of claim 14, wherein the fusogenic lipid is DOPE.16 The aLNP of claim 15, wherein the molar ratio of fusogenic lipid is between 5 mol% and60 mol%.

17. The aLNP of any one of claims 14 to 16, further comprising at least one bilayer-forming lipid.

18. The aLNP of claim 17, wherein the at least one bilayer forming lipid comprises a neutral lipid, an anionic lipid, or a combination thereof.

19. The aLNP of claim 18, wherein the neutral lipid is selected from the group consisting of DSPC, DPPC, SM, DMPC, DPPE, DSPE, DMPE, and POPC.

20. The aLNP of claim 19, wherein the anionic lipid is selected from the group consisting of DSPG, DPPG, DOPG, POPG, DMPG, DSP A, DPP A, DOPA, POP A, DMPA, DSPS, DPPS, DOPS, POPS, a ganglioside, and DMPS.

21. The aLNP of claim 17, wherein the molar ratio the at least one bilayer forming lipid is between 5 mol% and 55 mol%.

22. The aLNP of any one of claims 14 to 21, further comprising a sterol.

23. The aLNP of claim 22, wherein the sterol is sitosterol or cholesterol.

24. The aLNP of claim 14, where in the molar ratio of sterol is between 20 mol% and 50 mol%.

25. The aLNP of any one of claims 14 to 24, wherein the aLNP has a formulation shown in FIG. 16, 17, 18, 20, 21, 23, 24 or 25.

26. The aLNP of any one of claims 14 to 25, wherein the aLNP has(i) an average hydrodynamic diameter of about 50 nm to about 250 nm as determined by dynamic light scattering (DLS);(ii) a poly dispersity index (PDI) of about 0.01 to about 0.3 as determined by dynamic light scattering (DLS);(iii) a zeta potential of between about -100 and about -10 mV as determined by electrophoretic light scattering (ELS); or,(iv) a combination thereof.

27. An aLNP comprising:(i) at least one fusogenic lipid;(ii) a sterol;(iii) at least one non-fusogenic neutral or anionic lipid; and,(iv) an endosomal escape peptide between 15 and 50 amino acids in length comprising at least two amino acid subsequences selected from the group consisting of EALAHH (SEQ IDNO: 1336), DALAHH (SEQ ID NO: 1337), EALAHW (SEQ ID NO: 1338), D AL AH Y (SEQ ID NO: 1339), DALAHG (SEQ ID NO: 1340), and DALAHW (SEQ ID NO: 1341), wherein the aLNP has net negative charge at neutral pH.

28. The aLNP of claim 27, wherein:(i) the fusogenic lipid is selected from the group consisting of phosphatidylethanolamines, phosphatidic acids, phosphatidylserines, monoacylglycerols, glycolipids, such as monogalactosyldiacylglycerol (MGDG); lysophospholipids, and unsaturated fatty acids;(ii) the sterol is selected from the group consisting of cholesterol, stigmasterol or sitosterol;(iii) the non-fusogenic neutral or anionic lipid is selected from the group consisting of EPC, DSPC, DPPC, POPC, DMPC, EPG, DSPG, DPPG, POPG, DMPG, DSP A, DPP A, POP A, DMPA, DSPS, DPPS, POPS, DMPS, sphingomyelin or a ganglioside.

29. The aLNP of claim 28, wherein the aLNP formulation is selected from the group consisting of DOPE:DSPC:DSPG:Sitosterol:EPP, DOPE:DSPC:DSPG:Cholesterol:EPP, DOPE: SM:DSPG: Sitosterol :EPP, DOPE:SM:DSPG:Cholesterol:EPP, DOPE:DSPC:DPPG:Sitosterol:EPP, DOPE:DSPC:DPPG:Cholesterol:EPP,DOPE: SM:DPPG: Sitosterol :EPP, DOPE:SM:DPPG:Cholesterol:EPP, DOPE:SM:EEP, DOPE:DPPC:DPPG:Sitosterol:EPP, DOPE:DPPC:DPPG: Cholesterol :EPP, DOPE:DPPC:DSPG:Sitosterol:EPP, DOPE:DPPC:DSPG:Cholesterol:EPP, DOPE:SM:GM3:Sitosterol:EPP, DOPE:SM:GM3:Cholesterol:EPP, DOPE:SM, DOPE:DSPC:GM3:Sitosterol:EPP, DOPE:DSPC:GM3:Cholesterol:EPP, DOPE:DPPC : GM3 : Sitosterol :EPP, DOPE:DPPC : GM3 : Cholesterol :EPP, DOPE:DSPC:DSPG:GM3:Sitosterol:EPP, DOPE:DSPC:DSPG:GM3:Cholesterol:EPP, DOPE: SM:DSPG:GM3: Sitosterol :EPP, DOPE:SM:DSPG:GM3:Cholesterol:EPP,DOPE:DSPC:DPPG:GM3:Sitosterol:EPP, DOPE:DSPC:DPPG:GM3:Cholesterol:EPP,DOPE: SM:DPPG:GM3: Sitosterol :EPP, DOPE:SM:DPPG:GM3:Cholesterol:EPP, DOPE:POPC:POPG: Sitosterol :EPP, DOPE:POPC:POPG: Cholesterol :EPP, DOPE: SM:DSPG: Sitosterol :EPP, and DOPE:SM:DSPG:Cholesterol:EPP, wherein EEP is an endosomal escape peptide.

30. The aLNP of claim 29, wherein the aLNP formulation is selected from the group consisting of the lipid formulations of the aLNP disclosed in FIGS. 16, 17, 18, 20, 21,23, 24, 25, and 26.

31. The aLNP of any one of claims 14 to 30, wherein the aLNP comprises a payload.

32. The aLNP of claim 31, wherein the payload is selected from the group consisting of a nucleic acid, a protein, a small molecule, a diagnostic reagent, or a combination thereof.

33. The aLNP of claim 32, wherein the nucleic acid payload comprises a DNA, RNA, DNA analogue, RNA analogue, or a combination thereof.

34. The aLNP of claim 33, wherein the RNA comprises an mRNA, an ASO, a shRNA, a siRNA, a miRNA, or a combination thereof.

35. The aLNP of claim 32, wherein the payload is an anti -neoplastic agent selected from the group consisting of(i) a cell-based anti-neoplastic agent;(ii) a lymphocyte-based anti-neoplastic agent selected from the group consisting of B cells, aPT cells, yST cells, NK cells, NKT cells, autologous tumor-infiltrating lymphocytes (TILs), autologous NK cells, CAR-T cells, CAR-B cells, CAR-NK cells, CAR-NKT cells, and any combination thereo;(iii) a myeloid-based anti -neoplastic agent selected from the group consisting of a dendritic cell-based anti -neoplastic agent, a macrophage-based anti -neoplastic agent, a neutrophil based anti -neoplastic agent, or any combination thereof;(iv) an antibody elected from the group consisting of an anti-CTLA4 antibody, an anti-PDl antibody, an anti-PD-Ll antibody, and any combination thereof;(v) an immune checkpoint inhibitor;(vi) a small molecule drug selected from the group consisting of an alkylating agent, an antibiotic, an anti-metabolite, a hormonal antagonist, a photosensitizer, a protein kinase inhibitor, a poly (ADP-ribose) polymerase inhibitor, a taxane, a topoisomerase inhibitor, and any combination thereof;(vii) a radiation therapy;(viii) a cytokine selected from the group consisting of IL-2, , IL-12, IL-15, IL-21, and any combination thereof;(ix) growth factor selected from the CSF family, Flt3L, and any combination thereof;(x) a steroidal or a non-steroidal anti-inflammatory drug; and,(xi) any combination thereof.

36. A pharmaceutical composition comprising the aLNP of any one of claims composition of any one of claims 14 to 35 and a pharmaceutically acceptable excipient.

37. A method to treat a disease or condition in a subject in need thereof comprising administering the aLNP of any of claims 14 to 35, or the pharmaceutical composition of claim 36 to the subj ect.

38. The method according to claim 36, wherein the disease or condition is selected from the group consisting of cancer, infection, chronic inflammation, genetic disease, and autoimmune disease.

39. A diagnostic method comprising contacting a tissue sample of a subject with an aLNP of any one of claims 14 to 35, or the pharmaceutical composition of claim 36, wherein the aLNP comprises a detectable moiety.

40. A diagnostic system comprising an aLNP of any one of claims 14 to 35, or the pharmaceutical composition of claim 36, wherein the aLNP comprises a detectable moiety.

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