CROSS REFERENCE

[0001] This application claims the benefit of U.S. Provisional Application No. 63/392,801, filed July 27, 2022, which is incorporated herein by reference.

BACKGROUND

[0002] Synthetic mRNA provides a template for the synthesis of any given peptide, protein or protein fragment and lends itself to a broad range of pharmaceutical applications. To function in vivo, mRNA requires safe, effective, and stable delivery systems that protect the nucleic acid from degradation and that allow cellular uptake and mRNA release. Lipid nanoparticle (LNP)- mRNA formulations have been developed and are under clinical evaluation for the prevention and treatment of viral infections, cancer, and genetic diseases. Many high throughput methods for screening LNPs in vitro have been developed.

SUMMARY

[0003] The present disclosure provides compositions and methods for high throughput LNP screening in vivo. In some embodiments, provided herein are methods for screening a plurality of different lipid nanoparticles (LNPs), comprising: a) administering to a subject a plurality of nucleic acid molecules, wherein one nucleic acid molecule of said plurality of nucleic acid molecules encodes a reporter protein and a peptidyl barcode; and b) detecting the reporter protein in a sample obtained from the subject; wherein the one nucleic acid molecule is encapsulated by a LNP of the plurality of different LNPs, and wherein the peptidyl barcode is identifiable in an assay, therefore, identifying the LNP encapsulated the one nucleic acid molecule. In some embodiments, the reporter protein is selected from the group consisting of a cytosolic reporter protein, a membrane bound reporter protein, a secreted reporter protein, and any combination thereof. In some embodiments, the reporter protein comprises a fluorescent reporter protein or an enzyme. In some embodiments, the enzyme is selected from the group consisting of a luciferase, a horseradish peroxidase, an alkaline phosphatase, a β- galactosidase, and any combination thereof. In some embodiments, the reporter protein comprises a therapeutic peptide. In some embodiments, the detecting comprises detecting using a plate assay. In some embodiments, the plate assay comprises a luciferase assay, colorimetric assay, chemiluminescence assay, enzyme-linked immunoassay (ELISA), enzyme-linked immunosorbent spot (ELISpot), or a fluorescence-based microplate assay. In some embodiments, the plate assay comprises an aptamer binding assay. In some embodiments, the detecting comprises detecting with flow cytometry. In some embodiments, the detecting comprises detecting with an antibody. In some embodiments, the detecting does not comprise detecting with mass spectrometry. In some embodiments, the peptidyl barcode is configured to bind a capture peptide. In some embodiments, the peptidyl barcode comprises a protein tag. In some embodiments, the protein tag comprises a histidine tag (his tag), a flag tag, or a hemagglutinin tag (HA tag). In some embodiments, the protein tag is selected from a group consisting of: a CBP tag, a flag tag, a GST tag, an HA tag, an HBH tag, an MBP tag, a myc tag, a his tag, an S tag, a SUMO tag, a TAP tab, a TRX tag, and a V5 tag. In some embodiments, the peptidyl barcode comprises a protein aptamer or a ligand of a protein aptamer. In some embodiments, the method further comprising immobilizing the peptidyl barcode by binding to the capture peptide. In some embodiments, the capture peptide is selected from the group consisting of an antibody, a protein aptamer, a ligand of a protein aptamer, and any combination thereof, wherein the capture peptide is immobilized on a solid support. In some embodiments, the assay of the method comprises a plate assay, an aptamer binding assay, or flow cytometry. In some embodiments, the plate assay comprises a luciferase assay, colorimetric assay, chemiluminescence assay, enzyme-linked immunoassay (ELISA), enzyme-linked immunosorbent spot (ELISpot), or a fluorescence-based microplate assay. In some embodiments, the subject comprises a mammal. In some embodiments, the subject comprises a rodent, a non-human primate, or a human. In some embodiments, the administering comprises administering via an injection. In some embodiments, the injection comprises an intravenous injection, an intraperitoneal injection, a subcutaneous injection, an intradermal injection, an intramuscular injection, an intraocular injection, an intravitreal injection, an intracranial injection, or an intrathecal injection. In some embodiments, the administering comprises administering via intratracheal instillation or nebulization. In some embodiments, the administering comprises administering topically. In some embodiments, the one nucleic acid molecule comprises a ribonucleic acid (RNA) sequence. In some embodiments, the one nucleic acid molecule comprises a deoxyribonucleic acid (DNA) sequence. In some embodiments, the one nucleic acid molecule is selected from the group consisting of a RNA, DNA, a DNA/RNA hybrid, a nucleic acid analog, a chemically modified nucleic acid, a chimera composed of two or more nucleic acids or nucleic acid analogs, and any combination thereof. In some embodiments, the sample comprises a cell lysate, a tissue lysate, serum, plasma, saliva, urine, or a cerebral spinal fluid. In some embodiments, the lipid nanoparticle (LNP) comprises a lipid composition; wherein the lipid composition comprises an ionizable lipid or a pharmaceutically acceptable salt thereof; wherein the ionizable lipid comprises an amine head group and at least one hydrophobic tail R _Li ^p _id having a structure of pharmaceutically acceptable salt thereof; wherein

R _k1 is independently a C1-C12 bivalent aliphatic or heteroaliphatic radical;

R _k3 is independently a C1-C12 alkyl, C2-C12 alkenyl, C2-C12 alkynyl, C3-C12 cycloalkyl, C3- C12 heterocycloalkyl, aryl, or heteroaryl;

R _k2 is independently a C1-C20 alkyl, C2-C20 alkenyl, C2-C20 alkynyl, C3-C20 cycloalkyl, C1-C20 heteroalkyl, C3- C20 heterocycloalkyl, aryl, or heteroaryl;

R _k4 and R _k5 are each independently H or C1-C12 bivalent aliphatic radical; and M is O or NR _k6 , wherein R _k6 is H or C1-C12 bivalent aliphatic radical.

[0004] In some embodiments, the amine head group is represented by: wherein Ra, Ra’, Ra”, and Ra’” are each independently, H, C1-20 alkyl, C2-C20 alkenyl, C2-C20 alkynyl, C3-C20 cycloalkyl or heterocycloalkyl, C1-C20 heteroalkyl, C3-C20 aryl or heteroaryl, or a R _Li ^p _id; and Z is a C1-C20 bivalent aliphatic radical, a C1-C20 bivalent heteroaliphatic radical, a bivalent aryl radical, or a bivalent heteroaryl radical.

[0005] In some embodiments, the ionizable lipid is represented by Formula (II): or a pharmaceutically acceptable salt thereof, wherein:

R ^b is a substituted or unsubstituted alkyl; n1 and n2 are each independently 1, 2, 3, 4, 5, or 6; and

R ^b1 , R ^b2 , R ^b3 and R ⁴ are each independently H or R _Li ^p _id , wherein at least one of R ^b1 , R ^b2 , R ^b3 and R ⁴ is not H.

[0006] In some embodiments, the amine head group is selected from the group consisting of

[0007] In some embodiments, the at least one hydrophobic tail comprises,

[0008] In some embodiments, the ionizable lipid comprises a lipid from TABLE 2. In some embodiments, the lipid composition further comprises a steroid. In some embodiments, the steroid comprises cholesterol or a cholesterol derivative. The method of any one of claims 3O- 38, wherein the lipid composition further comprises a helper lipid. In some embodiments, the helper lipid comprises phospholipids or zwitterionic lipids comprising 1,2-dioleoyl-sn-glycero- 3 -phosphoethanolamine (DOPE) or 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC).In some embodiments, the lipid composition further comprises a polymer conjugated lipid. In some embodiments, the polymer conjugated lipid comprises a polyethylene glycol (PEG) conjugated lipid. In some embodiments, the polymer conjugated lipid comprises 1,2- distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polye thylene glycol)-2000 (DSPE-PEG2k) or l,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG- PEG2k).In some embodiments, the lipid composition further comprises a steroid, a helper lipid, and a polymer conjugated lipid. In some embodiments, the lipid composition further comprises a steroid and a helper lipid. In some embodiments, the ionizable lipid is present in the lipid composition at a weight percentage from about 30% to about 90%.In some embodiments, the steroid is present in the lipid composition at a weight percentage from about 10% to about 40%.In some embodiments, the helper lipid is present in the lipid composition at a weight percentage from about 1% to about 20%. In some embodiments, the weight ratio of the ionizable lipid/steroid/helper lipid is about 2/1/1. In some embodiments, the lipid composition further comprises a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutically acceptable carrier comprises a sugar, wherein the sugar comprises mannitol, sucrose, maltose, or trehalose. In some embodiments, the carrier is present in the composition at a weight percentage from about 5% to about 60%. In some embodiments, the ionizable lipid comprises at least two hydrophobic tails, wherein not all hydrophobic tails are identical. In some embodiments, the ionizable lipid comprises at least two hydrophobic tails, wherein two or more hydrophobic tails are identical.

INCORPORATION BY REFERENCE

[0009] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] The novel features of the disclosure are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present disclosure will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the disclosure are utilized, and the accompanying drawings of which:

[0011] FIG. 1A illustrates the design of a representative mRNA sequence encoding an intracellular reporter protein. FIG. 1B shows the design of an mRNA encoding a secreted protein.

[0012] FIG. 2 illustrates a non-limiting exemplary process for mRNA production.

[0013] FIGs. 3A-3I illustrate non-limiting exemplary synthetic lipids that can be used for the mRNA library delivery described herein.

[0014] FIG. 4 depicts a schematic illustration of exemplary multiplex screening of LNP formulations in vivo.

[0015] FIG. 5 depicts a schematic illustration of an exemplary sandwich ELISA assay for high throughput screening.

[0016] FIGs. 6A-6F illustrate results of an LNP screening. FIG. 6A shows that the mRNA used were of the expected size, indicating good quality of mRNA. FIG. 6B shows the reporter protein-peptidyl barcode protein constructs were of the expected size via Western blot. FIG. 6C shows total luciferase protein expression patterns for each individual LNP-FLuc-tag and for the mixed LNP formulations. FIG. 6D depicts luciferase protein expression in anti-Flug antibody coated plates. FIG. 6E depicts luciferase protein expression in anti-his antibody coated plates. FIG. 6F depicts Luciferase protein expression in anti- HA antibody coated plates.

DETAILED DESCRIPTION

[0017] Messenger RNA (mRNA) as a platform for efficient protein expression in vivo has many advantages over other therapeutic approaches. A large size and negative charges of mRNA are obstacles to it efficiently reaching the cytosol. Naked mRNA can be spontaneously taken up by different cell types, but this usually results in degradation in acidic endolysosomal compartments. Lipid nanoparticles (LNPs) are an efficient way to protect mRNA from ubiquitous RNAses, shield it from immune cells and deliver it to cells while enabling escape from the endosome. LNPs generally consist of four major components: an ionizable lipid, a sterol, a helper lipid, e.g., a phospholipid, and apolymer conjugated lipid, e.g., a lipid-anchored polyethylene glycol (PEG). The phospholipid and sterol work together to stabilize the LNP, the lipid-anchored PEG provides vial and storage stability and the ionizable lipid is critical for cellular uptake and endosomal escape, allowing for release of the mRNA into the cytosol. By changing the ratio and identities of the lipid components, the efficacy and tolerability of the formulation can be altered. Importantly, mRNA/LNP formulation requires careful process controls to ensure reproducibility of manufacturing and stability.

[0018] Currently, a major limitation in the field of mRNA therapeutics is a lack of safe, specific, and efficacious delivery vehicles, partly due to the poor capability of in vitro studies, which can be carried out in a relatively high-throughput fashion, to predict in vivo biodistribution and efficacy, as well as the low-throughput nature of traditional in vivo studies. While these issues have been partly addressed by the application of methods such as batch analysis and nucleic acid barcoding, these methods are not without shortcomings that could limit their utility as screening approaches in preclinical studies. For example, the extent to which batch analysis can reduce the number of animals required for library screening depends on the composition of the library itself; in cases where a large fraction of a library consists of similarly performing LNPs, batch analysis can require a comparable number of animals when compared to single LNP analysis. Furthermore, while nucleic acid barcoding provides a wealth of information regarding the separate biodistributions of dozens of nanoparticles within the same animal, previous studies have shown that trafficking of LNPs to a specific tissue does not necessarily lead to expression within that tissue and that. Tissues that have very low accumulation of a certain LNP can have functional delivery at a much higher rate than tissues with significantly higher accumulation. In summary, there remains the need for developing high throughput in vivo LNP screening methods that can more accurately predict in vivo function and/or efficacy of LNP-mRNA therapeutics.

[0019] Provided herein are compositions and methods directed to an in vivo LNP screening system, which utilizes peptidyl barcodes to simultaneously evaluate multiple LNPs, e.g., a plurality of LNPs, within the same subject, e.g., an animal. The plurality of LNPs provided herein can be different. The nucleic acid molecules provided herein can be different. Each type of LNPs can comprise a corresponding type of nucleic acid molecules. The nucleic acid molecules can comprise nucleic acid sequences encoding a peptidyl barcode. The nucleic acid sequences encoding a peptidyl barcode can be different. The nucleic acid sequences can encode different peptidyl barcodes. Each type of nucleic acid molecules can comprise a corresponding type of nucleic acid sequences encoding a peptidyl barcode that is unique to the type of nucleic acid molecule. Therefore, each type of LNPs can comprise a corresponding type of nucleic acid sequences encoding a peptidyl barcode that is unique to the type of LNPs. Each type of peptidyl barcode encoded by the nucleic acid sequences of the nucleic acid molecules encapsulated within the LNP can be distinct to the LNP. The LNPs can be referred to as “barcoded LNPs”. Barcoded LNPs can then be pooled and administered in a single dose to the same subject (e.g., an animal). After administering to a subject, the nucleic acid molecules comprising the nucleic acid sequences encoding the peptidyl barcodes are expressed in vivo. Proteins comprising the peptidyl barcodes can be produced in vivo in a plurality of cells, tissues, or organs of the subject. The peptidyl barcodes can be configured to identify the type of LNPs encapsulating the nucleic acid molecules that comprise the nucleic acid sequences encoding the peptidyl barcode.

[0020] The nucleic acid sequences provided herein can encode a reporter protein and a peptidyl barcode. The nucleic acid sequences encoding a reporter protein and a peptidyl barcode can be different. The nucleic acid sequences can encode different peptidyl barcodes. The nucleic acid sequences can encode different reporter proteins. The nucleic acid sequences can encode the same reporter proteins. The LNPs encapsulating nucleic acid molecules comprising nucleic acid sequences encoding a reporter protein and a peptidyl barcode can be referred to as “barcoded LNPs”. Barcoded LNPs can then be pooled and administered in a single dose to the same subject (e.g., an animal). After administering to a subject, the nucleic acid molecules comprising the nucleic acid sequences encoding the reporter protein and the peptidyl barcodes are expressed in vivo. Chimeric proteins comprising a reporter protein and a peptidyl barcode can be produced in vivo in a plurality of cells, tissues, or organs of the subject. The peptidyl barcodes can be configured to identify the type of LNPs encapsulating the nucleic acid molecules that comprise the nucleic acid sequences encoding the reporter protein and the peptidyl barcode. The reporter protein can be configured to identify the type of LNPs encapsulating the nucleic acid molecules that comprise the nucleic acid sequences encoding the reporter protein and the peptidyl barcode. Therefore, each unique LNP formulation may yield its own distinct measurement that is a direct quantification of protein production by the nucleic acid molecules encapsulated within the LNP at the LNP target cells, tissues, or organs. [0021] In some aspects, the methods provided herein comprises using an immunoassay (e.g., ELISA). In some embodiments, the peptidyl barcode can be identifiable in an ELISA. In some embodiments, the reporter protein can be detected in an ELISA. In some embodiments, the reporter protein is not detected by mass spectrometry or liquid chromatography-mass spectrometry (LC-MS). Detecting proteins by ELISA can have many advantages over LC-MS. ELISA and its many variations are straightforward to perform and data analysis for ELISA does not require highly specialized personnel. ELISA may be performed on microplates without the use of expensive equipment or software. Even multiplexing ELISA methods, which require automation of liquid handling robots, are a lot less costly than LC-MS. Therefore, ELISA is more efficient due to its low training requirements for operators. In many cases, it is easy to develop specialized ELISA kits with commercial vendors, which can further reduce the complexity and the cost. In contrast, LC-MS can be costly due to high cost in equipment, software, and personnel. LC-MS can be complicated to operate and the resulting spectral data can be difficult to interpret.

[0022] Additionally, sample preparation for LC-MS can be time and resource consuming. ELISA on the other hand, requires zero to minimal sample preparation. Tissue lysates or cell lysates can be further processed however suitable for downstream applications. For example, cell lysates may have to be treated with enzymes (e.g., trypsin, TEV protease) and purified (e.g., C18 column) before they are applied to LC-MS equipment. In some embodiments, the methods provided herein requires zero to minimal sample preparation. In some embodiments, samples obtained from the subject can comprise tissue lysates or cell lysates. In some embodiments, the tissue lysates or cell lysates can be directly applied to ELISA without further processing.

[0023] Due to the microplate format, many ELISA samples can be processed in parallel to give high throughput. Multiplexed ELISA offers additional ability to assess multiple analytes in each sample. ELISA has been widely practiced and validated in the regulatory science and clinical science environment. Immunoassays are approved for use across a broad range of applications and both new and existing laboratories can easily conform to the status quo. Often it is much easier for manufacturers to optimize and troubleshoot ELISA protocols. Last but not least, ELISA coupled with the advances in signal amplification, typically offer a high level of sensitivity. Due to the immuno-selection of the analyte of interest other non-binding analytes can be removed, which improves signal to background. Therefore, ELISA can be a cost effective, easily adapted, and efficient method to detect the peptidyl barcodes and reporter proteins provided herein.

Barcoded lipid nanoparticles

[0024] In some embodiments, the compositions and methods provided herein are directed to a barcoded lipid nanoparticle (LNP). Barcoded LNPs can comprise nucleic acid molecules comprising one or more nucleic acid sequences. A plurality of barcoded LNPs may comprise a plurality of different nucleic acid molecules. A plurality of barcoded LNPs may comprise a plurality of different nucleic acid sequences. The nucleic acid sequences can encode a peptidyl barcode. The plurality of nucleic acid sequences can encode different peptidyl barcodes. The nucleic acid sequences can encode a peptidyl barcode and a reporter protein. The plurality of nucleic acid sequences can encode different peptidyl barcodes and different reporter proteins. The plurality of nucleic acid sequences can encode different peptidyl barcodes and the same reporter proteins. Barcoded LNPs can then be pooled and administered in a single dose to the same subject (e.g., animal). Successful expression of the peptidyl barcodes and/or the reporter proteins can be detected in the assays provided herein. The peptidyl barcode can be configured to identify the barcoded LNPs encapsulating the nucleic acid molecules that comprise the nucleic acid sequences encoding the peptidyl barcode and the reporter protein. Identifying and/or distinguishing the barcoded LNPs can be done using the assays provided herein. For example, the barcoded LNPs can be identified and/or distinguished via enzyme-linked immunoassay (ELISA).

[0025] In some aspects, the methods provided herein are directed to screening a plurality of different lipid nanoparticles (LNPs). In some embodiments, the LNPs are barcoded LNPs. In some embodiments, the method comprises a) administering to a subject a plurality of nucleic acid molecules, wherein one nucleic acid molecule of said plurality of nucleic acid molecules encodes a reporter protein and a peptidyl barcode; and b) detecting the reporter protein in a sample obtained from the subject; wherein the one nucleic acid molecule is encapsulated by a LNP of the plurality of different LNPs, and wherein the peptidyl barcode is identifiable in an assay, therefore, identifying the LNP encapsulated the one nucleic acid molecule. In some embodiments, the LNPs provided herein are referred to as “barcoded LNPs”. Barcoded LNPs can then be pooled and administered in a single dose to the same subject (e.g., animal).

[0026] In some embodiments, the barcoded LNP comprises a nucleic acid molecule that comprises a nucleic acid sequence encoding a reporter protein. A reporter protein is a protein that can be easily detected by biochemical methods. In some embodiments, the reporter protein comprises a membrane bound reporter protein, a secreted reporter protein, or a cytosolic protein, or any combination thereof. In some embodiments, reporter protein is selected from the group consisting of a cytosolic reporter protein, a membrane bound reporter protein, a secreted reporter protein, and any combination thereof. In some embodiments, nucleic acid sequences provided herein encode a fluorescent reporter protein. In some embodiments, the reporter protein comprises a fluorescent reporter protein or an enzyme. In some embodiments, the reporter protein comprises a fluorescent reporter protein. In some embodiments, the fluorescent reporter protein comprises a green fluorescent protein (GFP), a yellow fluorescent protein (YFP), or a red fluorescent protein (RFP), or any fluorescent protein derived from any one of these, or any combinations thereof. In some embodiments, the fluorescent reporter protein comprises a tdTomato. In some embodiments, the florescent reporter protein comprises any florescent protein listed in the florescent protein data base FPbase, accessible at: https://www.fpbase.org/.

[0027] In some embodiments, nucleic acid sequences provided herein encode an enzyme. In some embodiments, the reporter protein comprises an enzyme. In some embodiments, the enzyme is selected from the group consisting of a luciferase, a horseradish peroxidase, an alkaline phosphatase, a β-galactosidase, and any combination thereof. In some embodiments, the luciferase comprises a firefly luciferase, a renilla luciferase, or a nanoLuc luciferase. In some embodiments, the firefly luciferase can be derived from the genes comprising luc, luc+, luc2, luc2P, luc2CP. In some embodiments, the renilla luciferase can be derived from the genes comprising Rluc, hRluc, hRlucP, hRlucCP. In some embodiments, the NanoLuc luciferase can be derived from the genes comprising Nluc, NlucP, secNluc.

[0028] In some embodiments, nucleic acid sequences provided herein encode a therapeutic peptide. In some embodiments, the reporter protein comprises a therapeutic peptide.

[0029] In some embodiments, the nucleic acid sequences encode any reporter protein that can be detected by an assay disclosed herein. In some embodiments, the reporter protein can be detected using a plate assay. In some embodiments, the plate assay comprises a luciferase assay, colorimetric assay, chemiluminescence assay, enzyme-linked immunoassay (ELISA), enzyme-linked immunosorbent spot (ELISpot), or a fluorescence-based microplate assay. In some embodiments, the plate assay comprises an aptamer binding assay. In some embodiments, the reporter protein can be detected using flow cytometry. In some embodiments, the reporter protein can be detected with an antibody. For example, the reporter protein can be detected by a primary antibody against the reporter protein, wherein the primary antibody against the reporter protein is conjugated with a fluorophore. In some embodiments, the reporter protein can be detected by a primary antibody against the reporter protein and a secondary antibody, wherein the secondary antibody comprises a reporter molecule, such as a fluorophore.

[0030] In some embodiments, the reporter protein provided herein is not detected by mass spectrometry.

[0031] In some embodiments, the barcoded LNP comprises a nucleic acid molecule that comprises a nucleic acid sequence encoding a peptidyl barcode. A peptidyl barcode is a peptide that can be used to distinguish or identify the barcoded LNP encapsulating the nucleic acid sequences encoding the peptidyl barcode. The peptidyl barcode can be used to enrich the proteins encoded by the nucleic acid molecules provided herein, in some embodiments the peptidyl barcode is configured to bind a capture molecule. [0032] In some embodiments, the nucleic acid sequences encode peptidyl barcodes, wherein the peptidyl barcodes comprise a protein tag. In some embodiments, the protein tag comprises a histidine tag (his tag), a flag tag, or a hemagglutinin tag (HA tag). In some embodiments, the protein tag is selected from a group consisting of: a CBP tag, a flag tag, a GST tag, an HA tag, an HBH tag, an MBP tag, a myc tag, a his tag, an S tag, a SUMO tag, a TAP tab, a TRX tag, an E tag, an E2 tag, a KT3, a T7, a VSVG, an OLLAS, a Protein C, an NE tag, an Xpress tag, an Avi, and a V5 tag, and any combinations thereof. In some embodiments, the protein tag comprises a CBP tag, a flag tag, a GST tag, an HA tag, an HBH tag, an MBP tag, a myc tag, a his tag, an S tag, a SUMO tag, a TAP tab, a TRX tag, an E tag, an E2 tag, a KT3, a T7, a VSVG, an OLLAS, a Protein C, an NE tag, an Xpress tag, an Avi, or a V5 tag, or a portion of any one of these, or any combinations thereof. In some embodiments, the tag can comprise any peptide or protein that is configured to bind a capture molecule. In some embodiments, any peptide or protein can be a tag, which can be operably linked to the reporter protein to form a fusion protein. In some embodiments, the peptide or protein that can be the protein tag is a soluble protein. In some embodiments, the fusion protein comprises a linker region. The fusion protein can be identified on a plate based assay via the protein tag (e.g., the peptide or protein).

[0033] In some embodiments, the peptidyl barcode comprises a protein aptamer or a ligand of a protein aptamer.

[0034] In some embodiments, the peptidyl barcode comprises any peptide or protein that is configured to bind a capture molecule. In some embodiments, any peptide or protein can be operably linked to the reporter protein to form a fusion protein. In some embodiments, the peptide or protein that can be the peptidyl barcode is a soluble protein. In some embodiments, the fusion protein comprises a linker region. The fusion protein can be identified on a plate based assay via the peptidyl barcode (e.g., the peptide or protein). In some embodiments, the peptidyl barcode is configured to be immobilized by binding to the capture molecule (e.g., capture peptide). The capture molecule can be any molecule that can bind the peptidyl barcode with high affinity. The capture molecule can be any molecule that can preferentially binds the peptidyl barcode. In some embodiments, the capture molecule can be a peptide (e.g., a capture peptide). In some embodiments, the capture molecule is not a peptide. For example, a capture molecule can be a non-peptidyl ligand of a peptide, such as a polysaccharide or nucleic acid. [0035] In some embodiments, the capture peptide is selected from the group consisting of an antibody, a protein aptamer, a ligand of a protein aptamer, and any combination thereof, wherein the capture peptide is immobilized on a solid support. In some embodiments, the solid support can be a plate, for example, a 96-well microplate. In some embodiments, the solid support can be a bead, for example, agarose beads, Sepharose beads, or magnetic beads.

[0036] In some embodiments, the peptidyl barcode can be identifiable in an assay. In some embodiments, the assay comprises a plate assay, an aptamer binding assay, or flow cytometry. In some embodiments, the plate assay comprises a luciferase assay, colorimetric assay, chemiluminescence assay, enzyme-linked immunoassay (ELISA), enzyme-linked immunosorbent spot (ELISpot), or a fluorescence-based microplate assay.

[0037] In some embodiments, the peptidyl barcode can be identifiable in an ELISA. In some embodiments, the reporter protein can be detected in an ELISA. In some embodiments, the reporter protein is not detected by mass spectrometry or liquid chromatography-mass spectrometry (LC-MS). In some embodiments, the ELISA comprises an indirect ELISA (or “sandwich” ELISA). In some embodiments, the ELISA can be configured to be high throughput (e.g., multiplexing ELISA). In multiplexing ELISA, multiple analytes may be measured in the same sample by using a variety of specific antibodies bound to a surface that is applicable to automation, such as a magnetic sphere or a protein microarray.

[0038] Detecting the reporter protein with immunoassays (e.g., ELISA) can have many advantages over LC-MS. ELISA and its many variations are straightforward to perform and data analysis for ELISA does not require highly specialized personnel. ELISA may be performed on microplates without the use of expensive equipment or software. Even multiplexing ELISA methods, which require automation of liquid handling robots, are a lot less costly than LC-MS. Therefore, ELISA is more efficient due to its low training requirements for operators. In many cases, it is easy to develop specialized ELISA kits with commercial vendors, which can further reduce the complexity and the cost. In contrast, LC- MS can be costly due to high cost in equipment, software, and personnel. LC-MS can be complicated to operate and the resulting spectral data can be difficult to interpret.

[0039] Additionally, sample preparation for LC-MS can be time and resource consuming. ELISA on the other hand, requires zero to minimal sample preparation. Tissue lysates or cell lysates can be further processed however suitable for downstream applications. For example, cell lysates may have to be treated with enzymes (e.g., trypsin, TEV protease) and purified (e.g., C18 column) before they are applied to LC-MS equipment. In some embodiments, the methods provided herein requires zero to minimal sample preparation. In some embodiments, samples obtained from the subject can comprise tissue lysates or cell lysates. In some embodiments, the tissue lysates or cell lysates can be directly applied to ELISA without further processing. In some embodiments, the methods provided herein does not comprise using LC- MS. [0040] Due to the microplate format, many ELISA samples can be processed in parallel to give high throughput. Multiplexed ELISA offers additional ability to assess multiple analytes in each sample. ELISA has been widely practiced and validated in the regulatory science and clinical science environment. Immunoassays are approved for use across a broad range of applications and both new and existing laboratories can easily conform to the status quo. Often it is much easier for manufacturers to optimize and troubleshoot ELISA protocols. Last, but not least, ELISA coupled with the advances in signal amplification, typically offer a high level of sensitivity. Due to the immuno-selection of the analyte of interest other non-binding analytes can be removed, which improves signal to background. Therefore, ELISA can be a cost effective, easily adapted, and efficient method to detect the peptidyl barcodes and reporter proteins provided herein.

[0041] In some embodiments, the reporter protein and the peptidyl barcode are operatively linked by a linker. In some embodiments, the linker comprises a short oligo- or polypeptide linker, for example, between 2 and 10 amino acids (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids) in length. In one embodiment, a glycine-serine doublet can be used as a suitable linker. In one embodiment, a single amino acid, e.g., an alanine, a glycine, can be used as a suitable linker. In some embodiments, the peptidyl barcode can be at the N-terminus of the reporter protein. In some embodiments, the peptidyl barcode can be at the C-terminus of the reporter protein. In some embodiments, the reporter protein can be produced from a protein comprising a signal peptide. The signal peptide can be processed and cleaved by a cell, leaving the reporter protein without a signal peptide.

[0042] As depicted in FIG. 1A, the nucleic acid sequence encoding the peptidyl barcode (e.g., a tag sequence or a protein sequence) can be at the 5’ or 3’ end of the nucleic acid sequence encoding the reporter protein. As depicted in FIG. 1B, the nucleic acid sequence encoding the peptidyl barcode (e.g., a tag sequence or a protein sequence) can be at the 3’ end of the nucleic acid sequence encoding the reporter protein. In this case, the 5’ end of the nucleic acid encoding the reporter protein can comprise a nucleic acid sequence encoding a signal peptide.

[0043] The LNPs of the present disclosure (e.g., barcoded LNPs) can be administered to a subject. In some embodiments, a plurality of different LNPs are mixed together and administered to a subject in a single dose. In some embodiments, more than 5, more than 10, more than 15, more than 20, more than 25, more than 30, more than 35, more than 40, more than 45, more than 50, more than 55, more than 60, more than 65, more than 70, more than 75, more than 80, more than 85, more than 90, more than 95, more than 100, more than 120, more than 150, more than 170, more than 200, more than 220, more than 250, more than 270, more than 300 different LNPs are mixed together and administered to a subject in a single dose.

[0044] In some embodiments, the subject comprises a mammal. In some embodiments, the subject comprises a rodent, a non-human primate, or a human.

[0045] In some embodiments, the LNPs are administered via injections. In some embodiments, the LNPs are administered directly to the systemic circulation. In some embodiments, the LNPs are administered to a local tissue or organ of the subject. In some embodiments, injections can comprise an intravenous injection, an intraperitoneal injection, a subcutaneous injection, an intramuscular injection, an intraocular injection, an intravitreal injection, an intracranial injection, or an intrathecal injection. In some embodiments, the LNPs are administered to the trachea, airways, or the lungs. In some embodiments, the LNPs are administered via intratracheal instillation or nebulization. In some embodiments, the LNPs are administered topically.

Lipid Compositions

[0046] In one aspect, provided herein relates to a plurality of different lipid nanoparticles (LNPs) comprising a lipid composition provided herein. The lipid composition comprises an ionizable lipid. The LNPs encapsulate the nucleic acid molecules provided herein, and the nucleic acid molecules comprises the nucleic acid sequences encoding the peptidyl barcodes and the reporter proteins. In some embodiments, the plurality of LNPs comprise different LNPs. In some embodiments, the different LNPs comprise different ionizable lipids. In some embodiments, the different LNPs comprise the same ionizable lipids. In some embodiments, the different LNPs comprise different lipid formulations. In some embodiments, the different LNPs comprise the same lipid formulations. Lipid formulations can refer to the type and/or the amount of ingredients within a lipid composition. In some embodiments, the different LNPs comprise different ionizable lipids with the same formulation. In this case, the goal of the screening can be to test the different ionizable lipids. In some embodiments, the different LNPs comprise the same ionizable lipids with different lipid formulations. In this case, the goal of the screening can be to test the different lipid formulations.

[0047] In some embodiments, the lipid composition comprises an ionizable lipid, wherein the ionizable lipid comprises an amine head group and at least one hydrophobic tail R _Li ^p _id having a structure of

or a pharmaceutically acceptable salt thereof; wherein

R _k1 is independently a C1-C12 bivalent aliphatic or heteroaliphatic radical;

R _k3 is independently a C1-C12 alkyl, C2-C12 alkenyl, C2-C12 alkynyl, C3-C12 cycloalkyl, C3-C12 heterocycloalkyl, aryl, or heteroaryl;

R _k2 is independently a C1-C20 alkyl, C2-C20 alkenyl, C2-C20 alkynyl, C3-C20 cycloalkyl, C1-C20 heteroalkyl, C3- C20 heterocycloalkyl, aryl, or heteroaryl;

R _k4 and R _k s are each independently H or C1-C12 bivalent aliphatic radical;

M is O or NR _k6 , wherein R _k6 is H or C1-C12 bivalent aliphatic radical.

[0048] In some embodiments, the ionizable lipid comprises an amine head group and at least one hydrophobic tail R _Li ^p _id having a structure of Formula (I): or a pharmaceutically acceptable salt thereof; wherein: indicates a point of attachment to a nitrogen in the amine head group; R ₁ and R ₂ are each independently a C1-C12 bivalent aliphatic or heteroaliphatic radical; which each of L ₁ , L ₂ , L ₃ , and L ₄ is, independently, a bond, O, S, or NR ^c ; G is O, S, or NR ^d ; Q is OR ^e , SR ^f , or NR ^g R ^h ; and each of r and t is independently 1-6; each of R ^c , R ^d , R ^e , R ^f , R ^g , and R ^h is independently H, C1-C10 alkyl, C1-C10 heteroalkyl, aryl, or heteroaryl;

Y and U are each independently a bond, O, S, NR ¹⁰ , or Se; n is 0 or 1 ; R ₃ and R ₄ , are each independently H, C1-C10 alkyl, C1-C10 heteroalkyl, aryl, or heteroaryl; or R ₃ and R ₄ together with the atom to which they are attached, form C=O; R ₅ is a C1-C20 alkyl, C2-C20 alkenyl, C2-C20 alkynyl, C3-C20 cycloalkyl, C1-C20 heteroalkyl, C3-C20 heterocycloalkyl, aryl, or heteroaryl.

[0049] In some embodiments, n is 0. In some embodiments, n is 1.

[0050] In some embodiments, n is 1 and R ₃ and R ₄ together with the atom to which they are attached, form C=O. In some embodiments, Y is CH ₂ and U is O. In some embodiments, Y and U are both O. In some embodiments, U is CH ₂ and Y is O. In some embodiments, Y and U are NR ¹⁰ . In some embodiments, Y is O and U is NR ¹⁰ . In some embodiments, Y is S and U is NR ¹⁰ .

[0051] In some embodiments, n is 0, and Y and U are both S.

[0052] In some embodiments, n is 0, and one of Y and U is O.

[0053] In some embodiments, n is 0, and one of Y and U is Se.

[0054] In some embodiments, R ₁ is a C1-C12 alkyl, linear or branched. In some embodiments, R ₁ is a C1-C10 alkyl, linear or branched. In some embodiments, R ₁ is a C1-C8 alkyl, linear or branched. In some embodiments, R ₁ is a C1-C6 alkyl, linear or branched. In some embodiments, R ₁ is a C1-C4 alkyl, linear or branched. In some embodiments, R ₁ is a C2 alkyl, e.g., In some embodiments, R ₁ is a C3 alkyl, e.g., In some embodiments, R ₁ is a C4 alkyl. In some embodiments, R ₁ is a C1-C12 heteroaliphatic radical.

[0055] In some embodiments, R ₂ is a C1-C12 alkyl, linear or branched. In some embodiments, R ₂ is a C1-C10 alkyl, linear or branched. In some embodiments, R ₂ is a C1-C8 alkyl, linear or branched. In some embodiments, R ₂ is a C1-C6 alkyl, linear or branched. In some embodiments, R ₂ is a C1-C4 alkyl, linear or branched. In some embodiments, R ₂ is a C2 alkyl, e.g., In some embodiments, R ₂ is a C3 alkyl, e.g., In some embodiments, R ₂ is a C4 alkyl. In some embodiments,

R ₂ is a C1-C12 heteroaliphatic radical. [0056] In some embodiments, the amine head group is represented by wherein Ra, Ra’, Ra”, and Ra’” are each independently, H, C1-20 alkyl, C2-C20 alkenyl, C2- C20 alkynyl, C3-C20 cycloalkyl or heterocycloalkyl, C1-C20 heteroalkyl, C3-C20 aryl or heteroaryl, or a R ^Lipid , ; and Z is a C1-C20 bivalent aliphatic radical, a C1-C20 bivalent heteroaliphatic radical, a bivalent aryl radical, or a bivalent heteroaryl radical.

[0057] In some embodiments, the ionizable lipid is represented by Formula (II): or a pharmaceutically acceptable salt thereof, wherein: i) R ^b is a substituted or unsubstituted alkyl, hydroxyalkyl, alkoxyalkyl, or aryl; ii) n1 and n2 are each independently 1, 2, 3, 4, 5, or 6; and iii) R ^b1 , R ^b2 , R ^b3 and R ^b4 are each independently H, or R _Li ^p _id wherein at least one of R ^b1 , R ^b2 , R ^b3 and R ^b4 is not H.

[0058] In some embodiments, R ^b is a C1-C6 alkyl, linear or branched. In some embodiments, R ^b is a substituted C1-C6 alkyl, linear or branched. In some embodiments, a substituent comprises hydroxyl, carbonyl, thiocarbonyl, alkoxyl, phosphoryl, phosphate, phosphonate, phosphinate, amino, amide, cyclic amine, amidine, imine, cyano, nitro, azido, sulfhydryl, alkylthio, sulfate, sulfonate, sulfamoyl, sulfonamido, sulfonyl, heterocyclyl, aralkyl, or aromatic or heteroaromatic moiety. In some embodiments, the substituent comprises hydroxyl, NH-Boc, or any suitable substituent thereof.

[0059] In some embodiments, n1 is 1, 2, 3, 4, 5, or 6. In some embodiments, n1 is 2 or 3.

[0060] In some embodiments, n2 is 1, 2, 3, 4, 5, or 6. In some embodiments, n2 is 2 or 3.

[0061] In some embodiments, n1 and n2 are identical. In some embodiments, n1 and n2 are different. In some embodiments, both n1 and n2 are 2. In some embodiments, both n1 and n2 are 3. In some embodiments, both n1 and n2 are 4.

[0062] In some embodiments, R ^b1 is not H. In some embodiments, R ^b2 is not H. In some embodiments, R ^b3 is not H. In some embodiments, R ^b4 is not H.

[0063] In some embodiments, at least two of R ^b1 , R ^b2 , R ^b3 and R ^b4 are not H. In some embodiments, at least three of R ^b1 , R ^b2 , R ^b3 and R ^b4 are not H. In some embodiments, none of R ^b1 , R ^b2 , R ^b3 and R ^b4 is H.

[0064] In some embodiments, the amine head group is selected from the group consisting of

[0065] In some embodiments, the at least one hydrophobic tail has a structure of wherein R _k1 and R _k3 are each independently a C1-C10 alkyl;

R _k2 is a C1-C20 alkyl, C2-C20 alkenyl, C2-C20 alkynyl, C3-C20 cycloalkyl, C1-C20 heteroalkyl, C3-C20 heterocycloalkyl, aryl, or heteroaryl;

R _k4 and R _k5 are each independently H, or C1-C10 alkyl.

[0066] In some embodiments, R _k1 is a C1-C10 alkyl, linear or branched. In some embodiments,

R _k1 is a C1-C4 alkyl, linear or branched. In some embodiments, R _k1 is a C2 alkyl, e.g.,

. In some embodiments, R _k1 is a C3 alkyl, e.g., , or In some embodiments, R _k1 is a C4 alkyl.

[0067] In some embodiments, R _k3 is a C1-C10 alkyl, linear or branched. In some embodiments,

R _k3 is a C1-C4 alkyl, linear or branched. In some embodiments, R _k3 is a C2 alkyl, e.g., . In some embodiments, R _k3 is a C3 alkyl, e.g., . In some embodiments, R _k3 is a C4 alkyl.

[0068] In some embodiments, R _k4 is H. In some embodiments, R _k4 is C1-C10 alkyl, In some embodiments, R _k4 is C1-C4 alkyl. In some embodiments, R _k4 is C4-C10 alkyl.

[0069] In some embodiments, R _k5 is H. In some embodiments, R _k5 is C1-C10 alkyl, In some embodiments, R _k5 is C1-C4 alkyl. In some embodiments, R _k5 is C4-C10 alkyl.

[0070] In some embodiments, R _k2 is a C1-C20 alkyl. In some embodiments, R _k2 is a C2-C20 alkenyl. In some embodiments, R _k2 is a C2-C20 alkynyl. In some embodiments, R _k2 is a C3- C20 cycloalkyl. In some embodiments, R _k2 is a C1-C20 heteroalkyl. In some embodiments, R _k2 is a C3-C20 heterocycloalkyl, aryl, or heteroaryl.

[0071] In some embodiments, the at least one hydrophobic tail comprises

[0072] In some embodiments, the at least one hydrophobic tail is selected from the TABLE 1.

TABLE 1. Exemplary Hydrophobic Tail

[0073] In some embodiments, the ionizable lipid comprises:

[0074] In some embodiments, the ionizable lipid comprises at least two hydrophobic tails. In some embodiments, the at least two hydrophobic tails are independently of structure In some embodiments, the at least two hydrophobic tails are identical. In some embodiments, the at least two hydrophobic tails are not identical. In some embodiments, one of the at least two hydrophobic tails is different from the rest.

[0075] In some embodiments, the lipid composition comprises at least three hydrophobic tails. In some embodiments, the at least three hydrophobic tails are independently of structure In some embodiments, the at least three hydrophobic tails are identical. In some embodiments, the at least three hydrophobic tails are not identical. In some embodiments, one of the at least three hydrophobic tails is different from the rest. [0076] In some embodiments, the lipid composition comprises two hydrophobic tails. In some embodiments, the two hydrophobic tails are independently of structure . In some embodiments, the two hydrophobic tails are identical. In some embodiments, the two hydrophobic tails are not identical.

[0077] In some embodiments, the lipid composition comprises three hydrophobic tails. In some embodiments, the three hydrophobic tails are independently of structure i _n some embodiments, the three hydrophobic tails are identical. In some embodiments, two of the three hydrophobic tails are identical and the third hydrophobic tail is different. In some embodiments, all three hydrophobic tails are different. [0078] In some embodiments, the lipid composition comprises four hydrophobic tails. In some embodiments, the four hydrophobic tails are independently of structure In some embodiments, the four hydrophobic tails are identical. In some embodiments, three of the four hydrophobic tails are identical and the fourth hydrophobic tail is different. In some embodiments, two of the four hydrophobic tails are identical, the other two hydrophobic tails are identical, and the two are different from other two. In some embodiments, two of the four hydrophobic tails are identical while the other two are different from each other and are different from the two. In some embodiments, all four hydrophobic tails are different.

[0079] In some embodiments, the ionizable lipid is selected from TABLE 2.

TABLE 2. Exemplary Ionizable Lipid

[0080] In some embodiments, the composition provided herein further comprises a steroid. In some embodiments, the steroid comprises a cholesterol or a cholesterol derivative. In some embodiments, the composition provided herein further comprises a helper lipid. In some embodiments, the helper lipid comprises a phospholipid, such as l,2-dioleoyl-sn-glycero-3- phosphoethanolamine (DOPE) or l,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC). In some embodiments, the composition provided herein further comprises a polymer conjugated lipid. In some embodiments, the polymer conjugated lipid comprises l,2-distearoyl-sn-glycero-3- phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000 (DSPE-PEG2k) or 1,2- dimyristoyl-rac-glycero-3 -methoxypolyethylene gly col-2000 (DMG-PEG2k) .

[0081] In some embodiments, the lipid composition comprises an ionizable lipid disclosed in this application, a steroid, a helper lipid, and a polymer conjugated lipid.

[0082] In some embodiments, the ionizable lipid is present in the lipid composition at a weight percentage from about 10% to about 20%, about 10% to about 30%, about 10% to about 40%, about 10% to about 50%, about 10% to about 60%, about 10% to about 80%, about 10% to about 90%, from about 20% to about 30%, from about 20% to about 30%, from about 20% to about 40%, from about 20% to about 50%, from about 20% to about 60%, from about 20% to about 70%, from about 20% to about 80%, from about 20% to about 90%, from about 30% to about 40%, from about 30% to about 50%, from about 30% to about 60%, from about 30% to about 70%, from about 30% to about 80%, from about 30% to about 90%, from about 40% to about 50%, from about 40% to about 60%, from about 40% to about 70%, from about 40% to about 80%, from about 40% to about 90%, from about 50% to about 60%, from about 50% to about 70%, from about 50% to about 80%, from about 50% to about 90%, from about 60% to about 70%, from about 60% to about 80%, from about 60% to about 90%, from about 70% to about 80%, from about 70% to about 90%, or from about 80% to about 90%.

[0083] In some embodiments, the helper lipid is present in the lipid composition at a weight percentage from about 1% to about 5%, from about 1% to about 10%, from about 1% to about 20%, from about 5% to about 10%, from about 5% to about 20%, or from about 10% to about 20%.

[0084] In some embodiments, the steroid is present in the lipid composition at a weight percentage from about 10% to about 20%, from about 10% to about 30%, from about 10% to about 40%, from about 20% to about 30%, from about 20% to about 40%, or from about 30% to about 40%.

[0085] In some embodiments, the polymer conjugated lipid is present in the lipid composition at a weight percentage from about 1% to about 5%, from about 1% to about 10%, from about 1% to about 20%, from about 5% to about 10%, from about 5% to about 20%, or from about 10% to about 20%.

[0086] In some embodiments, the weight ratio of the ionizable lipid/steroid/helper lipid/polymer conjugated lipid is about 14/4/1/1, about 15/4/1/1, about 16/4/1/1, about 17/4/1/1, about 18/4/1/1, about 19/4/1/1, about 20/4/1/1, about 14/4/2/1, about 15/4/2/1, about 16/4/2/1, about 16.8/4/2/1, about 17/4/2/1, about 18/4/2/1, about 19/4/2/1, or about 20/4/2/1.

[0087] In some embodiments, the lipid composition comprises an ionizable lipid disclosed in this application, a steroid and a helper lipid. In some embodiments, the ionizable lipid is present in the lipid composition at a weight percentage from about 30% to about 90%. In some embodiments, the helper lipid is present in the lipid composition at a weight percentage from about 5% to about 40%. In some embodiments, the steroid is present in the lipid composition at a weight percentage from about 5% to about 40%. In some embodiments, the weight ratio of the ionizable lipid/steroid/helper lipid is about 1/1/1, 2/1/1, about 3/1/1, about 4/1/1, about 5/1/1, about 6/1/1, about 2/2/1, about 3/2/1, about 4/2/1, about 5/2/1, or about 6/2/1.

[0088] In some embodiments, the lipid composition comprises a nucleic acid molecule. In some embodiments, the lipid composition comprises a nucleic acid sequence. In some embodiments, the nucleic acid sequence encodes a peptidyl barcode. In some embodiments, the nucleic acid sequence encodes a peptidyl barcode and a reporter protein.

[0089] In some embodiments, the lipid composition further comprises a pharmaceutically acceptable carrier. In some embodiments, a carrier comprises a pharmaceutically acceptable excipient. The carrier can comprise a sugar, wherein the sugar comprises mannitol, sucrose, maltose, or trehalose. The carrier can comprise EC-16, (2-hydroxypropyl)-β-cyclodextrin ((HP-β-CD), stearic acid, Perfluoroundecanoic, Saponin, Mannitol, Borneol, Amikacin-EC16, Kanamycin-EC16, Neomycin-EC16, or Bile salts. In some embodiments, the carrier is present in the composition at a weight percentage from about 5% to about 60%. In some embodiments, the excipient is present in the composition at a weight percentage from about 1% to about 70%, from about 5% to about 60%, from about 5% to about 50%, from about 5% to about 40%, from about 5% to about 30%, from about 10% to about 50%, from about 10% to about 40%, from about 10% to about 30%, or from about 10% to about 20%.

[0090] In some embodiments, the lipid composition further comprises a pharmaceutical agent, wherein the pharmaceutical agent comprises a polynucleotide, an oligonucleotide, a polypeptide, an oligopeptide, a small molecule compound, or any combination thereof. In some embodiments, the polynucleotide is a messenger ribonucleic acid (mRNA).

[0091] In some embodiments, the pharmaceutical agent comprises a polynucleotide that encodes a gene product thereof.

[0092] In some embodiments, nucleic acid molecules are assembled in the lipid composition at a weight ratio of the nucleic acid/lipid composition of from about 1:200 to about 1:100, from about 1:200 to about 1:50, from about 1:200 to about 1:40, from about 1:200 to about 1:30, from about 1:200 to about 1:20, from about 1:200 to about 1:10, from about 1:200 to about 1:5, from about 1:200 to about 1:1, from about 1:100 to about 1:50, from about 1:100 to about 1:40, from about 1:100 to about 1:25, from about 1:100 to about 1:20, from about 1:100 to about 1:15, from about 1: 100 to about 1:10, from about 1:100 to about 1:5 or from about 1:100 to about 1:1.

[0093] In some embodiments, the composition is formulated for systemic or local administration. In some embodiments, the composition is formulated for intravenous administration. In some embodiments, the composition is formulated for intramuscular administration. In some embodiments, the composition is formulated for injections. In some embodiments, the composition is formulated for an intravenous injection, an intraperitoneal injection, a subcutaneous injection, an intramuscular injection, an intraocular injection, an intravitreal injection, an intracranial injection, or an intrathecal injection. In some embodiments, the compositions are formulated for delivery to the trachea, airways, or the lungs. In some embodiments, the compositions are formulated for intratracheal instillation or nebulization. In some embodiments, the compositions are formulated for topical administrations.

Additional Lipids

[0094] In some embodiments, the lipid composition further comprises an additional lipid comprising a steroid or a steroid derivative, a PEG lipid, and a helper lipid (e.g., phospholipids or other zwitterionic lipids).

[0095] In some embodiments, the lipid composition further comprises a helper lipid. In some embodiments, the helper lipid comprises a lipid that contributes to the stability or delivery efficiency of the lipid compositions. In some embodiments, the helper lipid comprises a zwitterionic lipid. In some embodiments, the helper lipid comprises a phospholipid. In some embodiments, the phospholipid may contain one or two long chain (e.g., C ₆ -C ₂₄ ) alkyl or alkenyl groups, a glycerol or a sphingosine, one or two phosphate groups, and, optionally, a small organic molecule. The small organic molecule may be an amino acid, a sugar, or an amino substituted alkoxy group, such as choline or ethanolamine. In some embodiments, the phospholipid is a phosphatidylcholine. In some embodiments, the phospholipid is distearoylphosphatidylcholine or dioleoylphosphatidylethanolamine. In some embodiments, other zwitterionic lipids are used, where zwitterionic lipid defines lipid and lipid-like molecules with both a positive charge and a negative charge. In some embodiments of the lipid compositions, the phospholipid is not an ethylphosphocholine. In some embodiments, the helper lipid can comprise 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) or 1,2- dioleoyl-sn-glycero-3-phosphocholine (DOPC).

[0096] In some embodiments, the compositions may further comprise a molar percentage of the phospholipid to the total lipid composition from about 5 to about 30.

[0097] In some embodiments, the helper lipid is present in the lipid composition at a weight percentage from about 1% to about 5%, from about 1% to about 10%, from about 1% to about 20%, from about 5% to about 10%, from about 5% to about 20%, or from about 10% to about 20%.

[0098] In some embodiments, the lipid composition comprises the phospholipid at a molar percentage from about 8% to about 23%. In some embodiments, the lipid composition comprises the phospholipid at a molar percentage from about 10% to about 20%. In some embodiments, the lipid composition comprises the phospholipid at a molar percentage from about 15% to about 20%. In some embodiments, the lipid composition comprises the phospholipid at a molar percentage from about 8% to about 15%. In some embodiments, the lipid composition comprises the phospholipid at a molar percentage from about 10% to about 15%. In some embodiments, the lipid composition comprises the phospholipid at a molar percentage from about 12% to about 18%. In some embodiments, the lipid composition comprises the phospholipid at a molar percentage of at least about 8%, at least about 10%, at least about 12%, at least about 15%, at least about 18%, at least about 20%, or at least about 23%. In some embodiments, the lipid composition comprises the phospholipid at a molar percentage of at most about 8%, at most about 10%, at most about 12%, at most about 15%, at most about 18%, at most about 20%, or at most about 23%.

[0099] In some embodiments, the lipid composition further comprises a steroid or steroid derivative. In some embodiments, the steroid or steroid derivative comprises any steroid or steroid derivative. As used herein, in some embodiments, the term “steroid” is a class of compounds with a four ring 17 carbon cyclic structure which can further comprises one or more substitutions including alkyl groups, alkoxy groups, hydroxy groups, oxo groups, acyl groups, or a double bond between two or more carbon atoms. In one aspect, the ring structure of a steroid comprises three fused cyclohexyl rings and a fused cyclopentyl ring as shown in the formula: . In some embodiments, a steroid derivative comprises the ring structure above with one or more non-alkyl substitutions. In some embodiments, the steroid or steroid derivative is a sterol wherein the formula is further defined as: . In some embodiments, the steroid or steroid derivative is a cholestane or cholestane derivative. In a cholestane, the ring structure is further defined by the formula: . As described above, a cholestane derivative includes one or more non-alkyl substitution of the above ring system. In some embodiments, the cholestane or cholestane derivative is a cholestene or cholestene derivative or a sterol or a sterol derivative. In other embodiments, the cholestane or cholestane derivative is both a cholesterol and a sterol or a derivative thereof.

[00100] In some embodiments, the compositions may further comprise a molar percentage of the steroid to the total lipid composition from about 20 to about 60. In some embodiments, the steroid is present in the lipid composition at a weight percentage from about 10% to about 20%, from about 10% to about 30%, from about 10% to about 40%, from about 20% to about 30%, from about 20% to about 40%, or from about 30% to about 40%.

[00101] In some embodiments, the lipid composition comprises the steroid or steroid derivative at a molar percentage from about 15% to about 46%. In some embodiments, the lipid composition comprises the steroid or steroid derivative at a molar percentage from about 20% to about 40%. In some embodiments, the lipid composition comprises the steroid or steroid derivative at a molar percentage from about 25% to about 35%. In some embodiments, the lipid composition comprises the steroid or steroid derivative at a molar percentage from about 30% to about 40%. In some embodiments, the lipid composition comprises the steroid or steroid derivative at a molar percentage from about 20% to about 30%. In some embodiments, the lipid composition comprises the steroid or steroid derivative at a molar percentage of 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%, or at least about 46%. In some embodiments, the lipid composition comprises the steroid or steroid derivative at a molar percentage of at most about 15%, at most about 20%, at most about 25%, at most about 30%, at most about 35%, at most about 40%, at most about 45%, or at most about 46%.

[00102] In some embodiments, the lipid composition further comprises a polymer conjugated lipid. In some embodiments, the polymer conjugated lipid is a PEG lipid. In some embodiments, the PEG lipid is a diglyceride which also comprises a PEG chain attached to the glycerol group. In other embodiments, the PEG lipid is a compound which contains one or more C ₆ -C ₂₄ long chain alkyl or alkenyl group or a C ₆ -C ₂₄ fatty acid group attached to a linker group with a PEG chain. Some non-limiting examples of a PEG lipid includes a PEG modified phosphatidylethanolamine and phosphatidic acid, a PEG ceramide conjugated, PEG modified dialkylamines and PEG modified 1,2-diacyloxypropan-3-amines, PEG modified diacylglycerols and dialkylglycerols. In some embodiments, PEG modified diastearoylphosphatidylethanolamine or PEG modified dimyristoyl-sn-glycerol. In some embodiments, the PEG modification is measured by the molecular weight of PEG component of the lipid. In some embodiments, the PEG modification has a molecular weight from about 100 to about 15,000. In some embodiments, the molecular weight is from about 200 to about 500, from about 400 to about 5,000, from about 500 to about 3,000, or from about 1,200 to about 3,000. The molecular weight of the PEG modification is from about 100, 200, 400, 500, 600, 800, 1,000, 1,250, 1,500, 1,750, 2,000, 2,250, 2,500, 2,750, 3,000, 3,500, 4,000, 4,500, 5,000, 6,000, 7,000, 8,000, 9,000, 10,000, 12,500, to about 15,000. Some non-limiting examples of lipids that may be used in the present application are taught by U.S. Patent 5,820,873, WO 2010/141069, or U.S. Patent 8,450,298, which is incorporated herein by reference.

[00103] In some embodiments, the PEG lipid has a structural formula: , wherein: R ₁₂ and R ₁₃ are each independently alkyl(c≤24), alkenyl(c≤24), or a substituted version of either of these groups; R _e is hydrogen, alkyl(c≤8), or substituted alkyl(c≤8); and x is 1-250. In some embodiments, R _e is alkyl(c≤8) such as methyl. R ₁₂ and R ₁₃ are each independently alkyl(c≤4-20). In some embodiments, x is 5-250. In one embodiment, x is 5-125 or x is 100-250. In some embodiments, the PEG lipid is 1,2- dimyristoyl-sn-glycerol, methoxypolyethylene glycol.

[00104] In some embodiments, the PEG lipid has a structural formula: , wherein: m is an integer between 1 and 100 and n ₂ and n ₃ are each independently selected from an integer between 1 and 29. In some embodiments, m is 5, 10, 15, 20, 25, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100, or any range derivable therein. In some embodiments, n ₁ is from about 30 to about 50. In some embodiments, m is from 5 to 23. In some embodiments, m is 11 to about 17. In some embodiments, n ₃ is from 5 to 23. In some embodiments, n ₃ is 11 to about 17.

[00105] In some embodiments, the polymer conjugated lipid comprises 1 ,2-distearoyl- sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000 (DSPE-PEG2k) or l,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG2k). [00106] In some embodiments, the lipid composition comprises the polymer-conjugated lipid at a molar percentage from about 0.5% to about 20%. In some embodiments, the lipid composition comprises the polymer-conjugated lipid at a molar percentage from about 1 % to about 8%. In some embodiments, the lipid composition comprises the polymer-conjugated lipid at a molar percentage from about 2% to about 7%. In some embodiments, the lipid composition comprises the polymer-conjugated lipid at a molar percentage from about 3% to about 5%. In some embodiments, the lipid composition comprises the polymer-conjugated lipid at a molar percentage from about 5% to about 10%. In some embodiments, the lipid composition comprises the polymer-conjugated lipid at a molar percentage of at least about 0.5%, at least about 1%, at least about 1.5%, at least about 2%, at least about 2.5%, at least about 3%, at least about 3.5%, at least about 4%, at least about 4.5%, at least about 5%, at least about 5.5%, at least about 6%, at least about 6.5%, at least about 7%, at least about 7.5%, at least about 8%, at least about 8.5%, at least about 9%, at least about 9.5%, or at least about 10%. In some embodiments, the lipid composition comprises the polymer-conjugated lipid at a molar percentage of at most about 0.5%, at most about 1%, at most about 1.5%, at most about 2%, at most about 2.5%, at most about 3%, at most about 3.5%, at most about 4%, at most about 4.5%, at most about 5%, at most about 5.5%, at most about 6%, at most about 6.5%, at most about 7%, at most about 7.5%, at most about 8%, at most about 8.5%, at most about 9%, at most about 9.5%, at most about 10%, at most about 15%, or at most 20%.

[00107] In some embodiments, the polymer conjugated lipid is present in the lipid composition at a weight percentage from about 1% to about 5%, from about 1% to about 10%, from about 1% to about 20%, from about 5% to about 10%, from about 5% to about 20%, or from about 10% to about 20%.

Nucleic acids

[00108] The nucleic acid molecules and nucleic acid sequences provided herein can comprise ribonucleic acid (RNA) molecules and RNA sequences. The nucleic acid molecules and nucleic acid sequences provided herein can comprise deoxyribonucleic acid (DNA) molecules and DNA sequences. In some embodiments, the nucleic acids provided herein comprises a RNA, a DNA, a DNA/RNA hybrid, a nucleic acid analog, a chemically modified nucleic acid, a chimera composed of two or more nucleic acids or nucleic acid analogs, or any combination thereof. In some embodiments, the nucleic acid molecule and/or nucleic acid sequence is selected from the group consisting of a RNA, a DNA, a DNA/RNA hybrid, a nucleic acid analog, a chemically modified nucleic acid, a chimera composed of two or more nucleic acids or nucleic acid analogs, and any combination thereof. [00109] In some embodiments, the LNPs provided herein (e.g., barcoded LNPs) comprise nucleic acid molecules (e.g., mRNA) encoding a peptidyl barcode and a reporter protein. In some cases, the mRNA encapsulated by the lipid composition provided herein further comprises regulatory sequences that can facilitate and/or promote expression of the peptidyl barcode and the reporter protein. The regulatory sequences can comprise a 5’ cap, a 5’ untranslated region, a promoter, a signal peptide sequence, a 3’ untranslated region, and a poly-A tail. In some cases, the regulatory sequences comprise an enhancer (e.g., CMV enhancer) to further enhance expression of the peptidyl barcode and the reporter protein.

[00110] In some cases, the nucleic acid sequences encoding a peptidyl barcode and a reporter protein further encode a signal peptide, wherein the signal peptide can be cleaved during post-translational processing.

[00111] In some cases, the nucleic acid sequences encapsulated by the LNPs of the present disclosure comprises a naturally occurring or an artificial promoter.

[00112] some cases, the nucleic acid sequences encapsulated by the LNPs comprises natural, synthetic, and/or artificial nucleotide analogues or bases. In some cases, the synthetic or artificial nucleotide analogues or bases comprise modifications at one or more of deoxyribose moieties, ribose moiety, phosphate moiety, nucleoside moiety, or a combination thereof.

[00113] In some cases, a nucleotide analogue or artificial nucleotide base comprises a nucleic acid with a modification at a 2' hydroxyl group of the ribose moiety. In some instances, the modification includes an H, OR, R, halo, SH, SR, NH2, NHR, NR2, or CN, wherein R is an alkyl moiety. Illustrative alkyl moiety includes, but are not limited to, halogens, sulfurs, thiols, thioethers, thioesters, amines (primary, secondary, or tertiary), amides, ethers, esters, alcohols and oxygen. In some instances, the alkyl moiety further comprises a modification. In some instances, the modification comprises an azo group, a keto group, an aldehyde group, a carboxyl group, a nitro group, a nitroso, group, a nitrile group, a heterocycle (e.g., imidazole, hydrazino or hydroxylamino) group, an isocyanate or cyanate group, or a sulfur containing group (e.g., sulfoxide, sulfone, sulfide, or disulfide). In some instances, the alkyl moiety further comprises a hetero substitution. In some instances, the carbon of the heterocyclic group is substituted by a nitrogen, oxygen or sulfur. In some instances, the heterocyclic substitution includes but is not limited to, morpholino, imidazole, and pyrrolidino.

[00114] In some cases, the modification at the 2' hydroxyl group is a 2'-O-methyl modification or a 2'-O-methoxyethyl (2’-O-MOE) modification. In some cases, the 2'-O- methyl modification adds a methyl group to the 2' hydroxyl group of the ribose moiety whereas the 2'O-methoxyethyl modification adds a methoxyethyl group to the 2' hydroxyl group of the ribose moiety.

[00115] In some cases, the modification at the 2' hydroxyl group is a 2'-O-aminopropyl modification in which an extended amine group comprising a propyl linker binds the amine group to the 2' oxygen. In some instances, this modification neutralizes the phosphate-derived overall negative charge of the oligonucleotide molecule by introducing one positive charge from the amine group per sugar and thereby improves cellular uptake properties due to its zwitterionic properties.

[00116] In some cases, the modification at the 2' hydroxyl group is a locked or bridged ribose modification (e.g., locked nucleic acid or LN A) in which the oxygen molecule bound at the 2' carbon is linked to the 4' carbon by a methylene group, thus forming a 2'-C,4'-C-oxy- methylene-linked bicyclic ribonucleotide monomer.

[00117] In some cases, additional modifications at the 2' hydroxyl group include 2'- deoxy, T-deoxy-2'-fluoro, 2'-O-aminopropyl (2'-O-AP), 2'-O-dimethylaminoethyl (2'-O- DMAOE), 2'-O-dimethylaminopropyl (2'-O-DMAP), T-O- dimethylaminoethyloxyethyl (2'- O-DMAEOE), or 2'-O-N-methylacetamido (2'-0-NMA).

[00118] In some cases, a nucleotide analogue comprises a modified base, for example, N1 -methylpseudouridine, 5-propynyluridine, 5-propynylcytidine, 6- methyladenine, 6- methylguanine, N, N, -dimethyladenine, 2-propyladenine, 2propylguanine, 2-aminoadenine, 1- methylinosine, 3-methyluridine, 5 -methylcytidine, 5 -methyluridine and other nucleotides having a modification at the 5 position, 5- (2- amino) propyl uridine, 5-halocytidine, 5- halouridine, 4-acetylcytidine, 1- methyladenosine, 2-methyladenosine, 3-methylcytidine, 6- methyluridine, 2- methylguanosine, 7-methylguanosine, 2, 2-dimethylguanosine, 5- methylaminoethyluridine, 5 -methyloxyuridine, deazanucleotides (such as 7-deaza- adenosine, 6-azouridine, 6-azocytidine, or 6-azothymidine), 5-methyl-2-thiouridine, other thio bases (such as 2-thiouridine, 4-thiouridine, and 2-thiocytidine), dihydrouridine, pseudouridine, queuosine, archaeosine, naphthyl and substituted naphthyl groups, any O-and N-alkylated purines and pyrimidines (such as N6-methyladenosine, 5-methylcarbonylmethyluridine, uridine 5- oxyacetic acid, pyridine -4-one, or pyridine-2-one), phenyl and modified phenyl groups such as aminophenol or 2,4, 6-trimethoxy benzene, modified cytosines that act as G-clamp nucleotides, 8-substituted adenines and guanines, 5-substituted uracils and thymines, azapyrimidines, carboxyhydroxyalkyl nucleotides, carboxyalkylaminoalkyi nucleotides, and alkylcarbonylalkylated nucleotides. Modified nucleotides also include those nucleotides that are modified with respect to the sugar moiety, as well as nucleotides having sugars or analogs thereof that are not ribosyl. For example, the sugar moieties, in some cases are or are based on, mannoses, arabinoses, glucopyranoses, galactopyranoses, 4'-thioribose, and other sugars, heterocycles, or carbocycles. The term nucleotide also includes universal bases. By way of example, universal bases include but are not limited to 3 -nitropyrrole, 5-nitroindole, or nebularine.

[00119] In some cases, one or more modifications optionally occur at the internucleotide linkage. In some instances, a modified internucleotide linkages can include, but is not limited to, phosphorothioates; phosphorodithioates; methylphosphonates; 5'- alkylenephosphonates; 5'-methylphosphonate; 3 '-alkylene phosphonates; borontrifluoridates; borano phosphate esters and selenophosphates of 3’-5’linkage or 2’-5’linkage; phosphotriesters; thionoalkylphosphotriesters; hydrogen phosphonate linkages; alkyl phosphonates; alkylphosphonothioates; arylphosphonothioates; phosphoroselenoates; phosphorodiselenoates; phosphinates; phosphoramidates; 3’- alkylphosphoramidates; aminoalkylphosphoramidates; thionophosphoramidates; phosphoropiperazidates; phosphoroanilothioates; phosphoroanilidates; ketones; sulfones; sulfonamides; carbonates; carbamates; methylenehydrazos; methylenedimethylhydrazos; formacetals; thioformacetals; oximes; methyleneiminos; methylenemethyliminos; thioamidates; linkages with riboacetyl groups; aminoethyl glycine; silyl or siloxane linkages; alkyl or cycloalkyl linkages with or without heteroatoms of, for example, 1 to 10 carbons that are saturated or unsaturated and/or substituted and/or contain heteroatoms; linkages with morpholino structures, amides, or polyamides wherein the bases are attached to the aza nitrogens of the backbone directly or indirectly; and combinations thereof.

[00120] In some cases, one or more modifications comprise a modified phosphate backbone in which the modification generates a neutral or uncharged backbone. In some instances, the phosphate backbone is modified by alkylation to generate an uncharged or neutral phosphate backbone. As used herein, alkylation includes methylation, ethylation, and propylation. In some cases, an alkyl group, as used herein in the context of alkylation, refers to a linear or branched saturated hydrocarbon group containing from 1 to 6 carbon atoms. In some instances, exemplary alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n- pentyl, isopentyl, neopentyl, hexyl, isohexyl, 1, 1 -dimethylbutyl, 2,2-dimethylbutyl, 3.3- dimethylbutyl, and 2-ethylbutyl groups. In some cases, a modified phosphate is a phosphate group as described in U.S. Patent No. 9481905.

[00121] In some embodiments, additional modified phosphate backbones comprise methylphosphonate, ethylphosphonate, methylthiophosphonate, or methoxyphosphonate. In some cases, the modified phosphate is methylphosphonate. In some cases, the modified phosphate is ethylphosphonate. In some cases, the modified phosphate is methylthiophosphonate. In some cases, the modified phosphate is methoxyphosphonate.

[00122] In some cases, one or more modifications further optionally include modifications of the ribose moiety, phosphate backbone and the nucleoside, or modifications of the nucleotide analogues at the 3' or the 5' terminus. For example, the 3' terminus optionally include a 3' cationic group, or by inverting the nucleoside at the 3 '-terminus with a 3 '-3' linkage. In another alternative, the 3'-terminus is optionally conjugated with an aminoalkyl group, e.g., a 3' C5-aminoalkyl dT. In an additional alternative, the 3'-terminus is optionally conjugated with an abasic site, e.g., with an apurinic or apyrimidinic site. In some instances, the 5'-terminus is conjugated with an aminoalkyl group, e.g., a 5'-O-alkylamino substituent. In some cases, the 5'-terminus is conjugated with an abasic site, e.g., with an apurinic or apyrimidinic site.

[00123] In some embodiments, the nucleic acid sequences encapsulated by the LNPs (e.g., barcoded LNPs) comprises a pseudouridine.

Methods

[00124] The present disclosure provides methods for screening a plurality of different lipid nanoparticles (LNPs), comprising: a) administering to a subject a plurality of nucleic acid molecules, wherein one nucleic acid molecule of said plurality of nucleic acid molecules encodes a reporter protein and a peptidyl barcode; and b) detecting the reporter protein in a sample obtained from the subject; wherein the one nucleic acid molecule is encapsulated by a LNP of the plurality of different LNPs, and wherein the peptidyl barcode is identifiable in an assay, therefore, identifying the LNP encapsulated the one nucleic acid molecule.

[00125] In some embodiments, the method for screening a plurality of different LNPs can identify or distinguish the LNP via the peptidyl barcode encoded by the nucleic acid sequence encapsulated by the LNP. In some embodiments, the method for screening a plurality of different LNPs can identify or distinguish the LNP via the peptidyl barcode and the reporter protein encoded by the nucleic acid sequence encapsulated by the LNP. In some embodiments, the method can be used for high throughput screening of the LNPs.

[00126] In some embodiments, the method comprises administering the plurality of different LNPs through any suitable routes comprising parenteral delivery (e.g., injections), such as intravenous, intramuscular, subcutaneous, intramedullary injections, as well as intrathecal, direct intraventricular, intraperitoneal, intranasal, or intraocular injections.

[00127] In some embodiments, the method provides potent delivery of the LNPs to a plurality of different cell types, tissues, or organs of a subject. In some embodiments, the method comprises delivering the LNPs to more than 5, more than 10, more than 15, more than 20, more than 25, more than 30 cell types, tissues, or organs of the subject.

[00128] In some embodiments, the delivery of the plurality of different LNPs to a cell result in expression of a plurality of different proteins. In some embodiments, a plurality of different proteins comprises a plurality of different peptidyl barcodes. In some embodiments, the plurality of different proteins comprises reporter proteins.

[00129] In some embodiments, provided is a high throughput method for screening LNPs in vivo. A mixture of different LNPs can be mixed and administered into one animal in a single dose. In some embodiments, more than 3, more than 5, more than 10, more than 13, more than 15, more than 17, more than 20, more than 23, more than 25, more than 30, more than 33, more than 35, more than 37, more than 40, more than 43, more than 45, more than 50, more than 53, more than 55, more than 57, more than 60, more than 63, more than 65, more than 70, more than 73, more than 75, more than 77, more than 80, more than 83, more than 85, more than 90, more than 93, more than 95, more than 97, more than 100 different LNPs can be mixed and administered into one animal in a single dose.

[00130] In some embodiments, the nucleic acid molecules are present in the LNP formulation at a dose of about 10, 9, 8, 7, 6, 5, 4, 3, 2, 1.5, 1.0, 0.5, 0.2, 0.1, 0.05, 0.02, 0.01, .005, 0.002, or 0.001 milligram per kilogram (mg/kg, or mpk) body weight, or of a range between (inclusive) any two of the foregoing values. In some embodiments, the nucleic acid molecules are present in the LNP formulation at a dose of no more than about 10 milligram per kilogram (mg/kg, or mpk) body weight. In some embodiments, the nucleic acid molecules are present in the LNP formulation at a dose of no more than about 9 mg/kg, no more than about 8 mg/kg , no more than about 7 mg/kg, no more than about 6 mg/kg, no more than about 5 mg/kg, no more than about 4 mg/kg, no more than about 3 mg/kg, no more than about 2 mg/kg, no more than about 1 mg/kg, no more than about 0.5 mg/kg, no more than about 0.2 mg/kg, no more than about 0.1 mg/kg, no more than about 0.05 mg/kg, or no more than about 0.01 mg/kg. In some embodiments, In some embodiments, the nucleic acid molecules are present in the LNP formulation at a concentration of no more than about 5 milligram per milliliter (mg/mL). [00131] In some embodiments, In some embodiments, the nucleic acid molecules are present in the LNP formulation at a concentration of about 5, 4, 3, 2, 1 , 0.5, 0.2, or 0.1 milligram per milliliter (mg/mL), or of a range between (inclusive) any two of the foregoing values.

[00132] In some embodiments, In some embodiments, the nucleic acid molecules are present in the LNP formulation at a concentration of no more than about 5 milligram per milliliter (mg/mL). In some embodiments, the nucleic acid molecules are present in the LNP formulation at a concentration of no more than about 2 milligram per milliliter (mg/mL). In some embodiments, the nucleic acid molecules are present in the LNP formulation at a concentration of no more than about 1 milligram per milliliter (mg/mL). In some embodiments, the nucleic acid molecules are present in the LNP formulation at a concentration of no more than about 0.5 milligram per milliliter (mg/mL). In some embodiments, the nucleic acid molecules are present in the LNP formulation at a concentration of no more than about 0.1 milligram per milliliter (mg/mL).

[00133] In some embodiments, the nucleic acid molecules (e.g., mRNA) is present in LNP formulations at a concentration of about 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, 0.5, 0.2, or 0.1 microgram per milliliter (pg/mL), or of a range between (inclusive) any two of the foregoing values. In some embodiments, the nucleic acid molecules (e.g., mRNA) is present in LNP formulations at a concentration of no more than about 10, no more than about 9, no more than about 8, no more than about 7, no more than about 6, no more than about 5, no more than about 4, no more than about 3, no more than about 2, no more than about 1, no more than about 0.5, no more than about 0.2, no more than about 0.1 microgram per milliliter (μg/mL).

[00134] Any suitable dosage form of LNPs can be prepared for delivery, for example, via oral, rectal, vaginal, transmucosal, pulmonary including intratracheal or inhaled, or intestinal administration; parenteral delivery, including intramuscular, subcutaneous, intramedullary injections, as well as intrathecal, direct intraventricular, intravenous, intraperitoneal, intranasal, or intraocular injections.

[00135] In some embodiments, the subject comprises a mammal. In some embodiments, the mammal comprises a rodent, a non-human primate, or human. In some embodiments, the subject is a non-human primate. In some embodiments, the subject is a mouse or a rat.

[00136] In some embodiments, provided herein is a method for delivery of the LNPs to different cell types comprising contacting the cells with the lipid composition. In some embodiments of the method, the LNP comprises nucleic acid molecules (e.g., mRNA) assembled with a lipid composition as described in the present application, e.g., wherein the lipid composition comprises any of the head or tail groups disclosed herein.

[00137] In some embodiments, the contacting is ex vivo. In some embodiments, the contacting is in vitro. In some embodiments, the contacting is in vivo.

[00138] The LNP compositions and methods of the present disclosure may comprise a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers are well known in the art and include, for example, aqueous solutions such as water or physiologically buffered saline or other solvents or vehicles such as glycols, glycerol, oils such as olive oil, or injectable organic esters. In preferred embodiments, when such LNP compositions are for administration in animals, particularly for invasive routes of administration (i.e., routes, such as intravenous or intramuscular injection, that circumvent transport or diffusion through an epithelial barrier), the aqueous solution is pyrogen-free, or substantially pyrogen-free. The excipients can be chosen, for example, to effect delayed release of an agent or to selectively target one or more cells, tissues or organs. The composition can be in dosage unit form such as tablet, capsule (including sprinkle capsule and gelatin capsule), granule, lyophile for reconstitution, powder, solution, syrup, suppository, injection or the like. The composition can also be present in a transdermal delivery system, e.g., a skin patch. The composition can also be present in a solution suitable for topical administration, such as a lotion, cream, or ointment.

[00139] A pharmaceutically acceptable carrier can contain physiologically acceptable agents that act, for example, to stabilize, increase solubility or to increase the absorption of a lipid composition such as a lipid composition of the invention. Such physiologically acceptable agents include, for example, carbohydrates, such as glucose, sucrose or dextrans, antioxidants, such as ascorbic acid or glutathione, chelating agents, low molecular weight proteins or other stabilizers or excipients. The choice of a pharmaceutically acceptable carrier, including a physiologically acceptable agent, depends, for example, on the route of administration of the composition. The preparation or pharmaceutical composition can be a self-emulsifying drug delivery system or a self-microemulsifying drug delivery system. The pharmaceutical composition (preparation) also can be a liposome or other polymer matrix, which can have incorporated therein, for example, a lipidoid composition of the invention. Liposomes, for example, which comprise phospholipids or other lipids, are nontoxic, physiologically acceptable and metabolizable carriers that are relatively simple to make and administer.

[00140] The phrase "pharmaceutically acceptable" is employed herein to refer to those lipidoid compositions, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.

[00141] The phrase "pharmaceutically acceptable carrier" as used herein means a pharmaceutically acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient. Some examples of materials which can serve as pharmaceutically acceptable carriers include: (1) sugars, such as lactose, glucose, mannose, trehalose, and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) phosphate buffer solutions; and (21) other non-toxic compatible substances employed in pharmaceutical formulations.

[00142] The LNPs and lipid compositions can be administered parentally. The phrases “parenteral administration” and “administered parenterally” as used herein means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal and intr asternal injection and infusion. In some embodiments, the LNPs and lipid compositions provided herein are administered through parenteral routes (e.g., intravenous injection or intramuscular injection). LNPs and lipid compositions suitable for parenteral administration comprise one or more active lipid compositions in combination with one or more pharmaceutically acceptable sterile isotonic aqueous or nonaqueous solutions, dispersions, suspensions or emulsions, or sterile powders which may be reconstituted into sterile injectable solutions or dispersions just prior to use, which may contain antioxidants, buffers, bacteriostats, solutes which render the formulation isotonic with the blood of the intended recipient or suspending or thickening agents.

[00143] Examples of suitable aqueous and nonaqueous carriers that may be employed in the LNPs and lipid compositions of the invention include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.

[00144] These compositions may also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of the action of microorganisms may be ensured by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol sorbic acid, and the like. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like into the compositions. In addition, prolonged absorption of the injectable pharmaceutical form may be brought about by the inclusion of agents that delay absorption such as aluminum monostearate and gelatin.

[00145] The present disclosure includes the use of pharmaceutically acceptable salts of the lipid compositions of the invention in the compositions and methods of the present invention. In some embodiments, contemplated salts of the invention include, but are not limited to, alkyl, dialkyl, trialkyl or tetra-alkyl ammonium salts. In some embodiments, contemplated salts of the invention include, but are not limited to, L-arginine, benenthamine, benzathine, betaine, calcium hydroxide, choline, deanol, diethanolamine, diethylamine, 2- (diethylamino)ethanol, ethanolamine, ethylenediamine, N-methylglucamine, hydrabamine, IH-imidazole, lithium, L-lysine, magnesium, 4-(2-hydroxyethyl)morpholine, piperazine, potassium, 1-(2-hydroxyethyl)pyrrolidine, sodium, triethanolamine, tromethamine, and zinc salts. In some embodiments, contemplated salts of the invention include, but are not limited to, Na, Ca, K, Mg, Zn or other metal salts. In some embodiments, contemplated salts of the invention include, but are not limited to, 1-hydroxy-2-naphthoic acid, 2,2-dichloroacetic acid, 2 -hydroxyethanesulfonic acid, 2-oxoglutaric acid, 4- acetamidobenzoic acid, 4-aminosalicylic acid, acetic acid, adipic acid, 1-ascorbic acid, 1-aspartic acid, benzenesulfonic acid, benzoic acid, (+)-camphoric acid, (+)-camphor-10-sulfonic acid, capric acid (decanoic acid), caproic acid (hexanoic acid), caprylic acid (octanoic acid), carbonic acid, cinnamic acid, citric acid, cyclamic acid, dodecylsulfuric acid, ethane- 1 ,2-disulfonic acid, ethanesulfonic acid, formic acid, fumaric acid, galactaric acid, gentisic acid, d glucoheptonic acid, d gluconic acid, d glucuronic acid, glutamic acid, glutaric acid, glycerophosphoric acid, glycolic acid, hippuric acid, hydrobromic acid, hydrochloric acid, isobutyric acid, lactic acid, lactobionic acid, lauric acid, maleic acid, 1-malic acid, malonic acid, mandelic acid, methanesulfonic acid , naphthalene- 1,5 -disulfonic acid, naphthalene-2-sulfonic acid, nicotinic acid, nitric acid, oleic acid, oxalic acid, palmitic acid, pamoic acid, phosphoric acid, proprionic acid, 1-pyroglutamic acid, salicylic acid, sebacic acid, stearic acid, succinic acid, sulfuric acid, 1 tartaric acid, thiocyanic acid, p-toluenesulfonic acid, trifluoroacetic acid, and undecylenic acid salts.

[00146] The pharmaceutically acceptable acid addition salts can also exist as various solvates, such as with water, methanol, ethanol, dimethylformamide, and the like. Mixtures of such solvates can also be prepared. The source of such solvate can be from the solvent of crystallization, inherent in the solvent of preparation or crystallization, or adventitious to such solvent.

[00147] Examples of pharmaceutically acceptable antioxidants include: (1) water- soluble antioxidants, such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite and the like; (2) oil-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol, and the like; and (3) metal-chelating agents, such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like. Definitions

[00148] Before the embodiments of the disclosure are described, it is to be understood that such embodiments are provided by way of example only, and that various alternatives to the embodiments of the disclosure described herein may be employed in practicing the disclosure. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the disclosure.

[00149] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, suitable methods and materials are described below. In case of conflict, the patent specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the disclosure.

[00150] In the context of the present application, the following terms have the meanings ascribed to them unless specified otherwise:

[00151] As used throughout the specification and claims, the terms “a”, “an” and “the” are generally used in the sense that they mean “at least one”, “at least a first”, “one or more” or “a plurality” of the referenced components or steps, except in instances wherein an upper limit is thereafter specifically stated. For example, a “cleavage sequence”, as used herein, means “at least a first cleavage sequence” but includes a plurality of cleavage sequences. The operable limits and parameters of combinations, as with the amounts of any single agent, will be known to those of ordinary skill in the art in light of the present application.

[00152] The terms “polypeptide”, “peptide”, and “protein” are used interchangeably herein to generally refer to polymers of amino acids of any length. The polymer may be linear or branched, it may comprise modified amino acids, and it may be interrupted by non-amino acids. The terms also encompass an amino acid polymer that has been modified, for example, by disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation, such as conjugation with a labeling component.

[00153] As used herein, the term “antibody” refers to an immunoglobulin (Ig) whether natural or partly or wholly synthetically produced. The term also covers any polypeptide or protein having a binding domain which is, or is homologous to, an antigen-binding domain. The term further includes “antigen-binding fragments” or “functional fragment thereof’, or “fragment of an antibody”, “antibody fragment”, “functional fragment of an antibody” and other interchangeable terms for similar binding fragments such as described below. An antibody includes, for example, monoclonal antibodies, chimeric antibodies, humanized antibodies, human antibodies, recombinant antibodies, chemically engineered antibodies, deimmunized antibodies, affinity-matured antibodies, multispecific antibodies (for example, bispecific antibodies and polyreactive antibodies), heteroconjugate antibodies, antibody fragments, and combinations thereof (e.g., a monoclonal antibody that is also deimmunized, a humanized antibody that is also deimmunized, etc.). An antibody can be, for example, murine, chimeric, humanized, heteroconjugate, bispecific, diabody, triabody, or tetrabody. The antigen binding fragment can include, for example, Fab’, F(ab’)2, Fab, Fv, rlgG, scFv, hcAbs (heavy chain antibodies), a single domain antibody, VHH, VNAR, sdAbs, or nanobody.

[00154] As used herein, the terms “treatment” or “treating,” or “palliating” or “ameliorating” are used interchangeably herein. These terms generally refer to an approach for obtaining beneficial or desired results including but not limited to a therapeutic benefit and/or a prophylactic benefit. By therapeutic benefit is meant eradication or amelioration of the underlying disorder being treated. Also, a therapeutic benefit is achieved with the eradication or amelioration of one or more of the physiological symptoms or improvement in one or more clinical parameters associated with the underlying disorder such that an improvement is observed in the subject, notwithstanding that the subject may still be afflicted with the underlying disorder. For prophylactic benefit, the compositions may be administered to a subject at risk of developing a particular disease, or to a subject reporting one or more of the physiological symptoms of a disease, even though a diagnosis of this disease may not have been made.

[00155] A “therapeutic effect” or “therapeutic benefit,” as used herein, generally refers to a physiologic effect, including but not limited to the mitigation, amelioration, or prevention of disease or an improvement in one or more clinical parameters associated with the underlying disorder in humans or other animals, or to otherwise enhance physical or mental wellbeing of humans or animals, resulting from administration of a polypeptide of the disclosure other than the ability to induce the production of an antibody against an antigenic epitope possessed by the biologically active protein. For prophylactic benefit, the compositions may be administered to a subject at risk of developing a particular disease, a recurrence of a former disease, condition or symptom of the disease, or to a subject reporting one or more of the physiological symptoms of a disease, even though a diagnosis of this disease may not have been made.

[00156] For the chemical groups and compound classes, the number of carbon atoms in the group or class is as indicated as follows: “Cn” defines the exact number (n) of carbon atoms in the group/class. “C≤n” defines the maximum number (n) of carbon atoms that can be in the group/class, with the minimum number as small as possible for the group/class in question, e.g., it is understood that the minimum number of carbon atoms in the group “alkenyl(C≤8)” or the class “alkene(C≤8)” is two. Compare with “alkoxy(C≤10)”, which designates alkoxy groups having from 1 to 10 carbon atoms. “Cm-n” or “Cm-Cn” defines both the minimum (m) and maximum number (n) of carbon atoms in the group. Thus, “C1-C10 alkyl” designates those alkyl groups having from 2 to 10 carbon atoms. These carbon number indicators may precede or follow the chemical groups or class it modifies and it may or may not be enclosed in parenthesis, without signifying any change in meaning. Thus, the terms “C5 olefin”, “C5- olefin”, “olefin(C5)”, and “olefines” are all synonymous.

[00157] The term “saturated” when used to modify a compound or chemical group means the compound or chemical group has no carbon-carbon double and no carbon-carbon triple bonds, except as noted below. When the term is used to modify an atom, it means that the atom is not part of any double or triple bond. In the case of substituted versions of saturated groups, one or more carbon oxygen double bond or a carbon nitrogen double bond may be present. And when such a bond is present, then carbon-carbon double bonds that may occur as part of keto-enol tautomerism or imine/enamine tautomerism are not precluded. When the term “saturated” is used to modify a solution of a substance, it means that no more of that substance can dissolve in that solution.

[00158] The term “aliphatic” generally signifies that the compound or chemical group so modified is an acyclic or cyclic, but non-aromatic hydrocarbon compound or group. In aliphatic compounds/groups, the carbon atoms can be joined together in straight chains, branched chains, or non-aromatic rings (alicyclic). Aliphatic compounds/groups can be saturated, that is joined by single carbon-carbon bonds (alkanes/alkyl), or unsaturated, with one or more carbon-carbon double bonds (alkenes/alkenyl) or with one or more carbon-carbon triple bonds (alkynes/alkynyl). [00159] The term “aromatic” when used to modify a compound or a chemical group atom means the compound or chemical group contains a planar unsaturated ring of atoms that is stabilized by an interaction of the bonds forming the ring.

[00160] The term “alkyl” when used without the “substituted” modifier refers to a monovalent saturated aliphatic group with a carbon atom as the point of attachment, a linear or branched acyclic structure, and no atoms other than carbon and hydrogen. The groups -CH ₃ (Me), -CH ₂ CH ₃ (Et), -CH ₂ CH ₂ CH ₃ (n-Pr or propyl), -CH(CH ₃ )2 (i-Pr, ⁱ Pr or isopropyl), -CH ₂ CH ₂ CH ₂ CH (n-Bu), -CH(CH ₃ )CH ₂ CH ₃ (sec-butyl), -CH ₂ CH(CH ₃ ) ₂ (isobutyl), - C(CH ₃ ) ₃ (tert-butyl, t-butyl, t-Bu or ^t Bu), and -CH ₂ C(CH ₃ ) ₃ (neo-pentyl) are non-limiting examples of alkyl groups. The term “alkanediyl” when used without the “substituted” modifier refers to a divalent saturated aliphatic group, with one or two saturated carbon atom(s) as the point(s) of attachment, a linear or branched acyclic structure, no carbon-carbon double or triple bonds, and no atoms other than carbon and hydrogen. The groups -CH ₂ - (methylene), -CH ₂ CH ₂ -, -CH ₂ C(CH ₃ ) ₂ CH ₂ -, and -CH ₂ CH ₂ CH ₂ - are non-limiting examples of alkanediyl groups. An “alkane” refers to the class of compounds having the formula H-R, wherein R is alkyl as this term is defined above. When any of these terms is used with the “substituted” modifier one or more hydrogen atom has been independently replaced by -OH, -F, -Cl, -Br, -I, -NH ₂ , -NO ₂ , -CO ₂ H, -CO ₂ CH ₃ , -CN, -SH, -OCH ₃ , -OCH ₂ CH ₃ , -C(O)CH ₃ , -NHCH ₃ , -NHCH ₂ CH ₃ , -N(CH ₃ ) ₂ , -C(O)NH ₂ , -C(O)NHCH ₃ , -C(O)N(CH ₃ ) ₂ , -OC(O)CH ₃ , -NHC(O)CH ₃ , -S(O)2OH, or -S(O)2NH ₂ . The following groups are non-limiting examples of substituted alkyl groups: -CH ₂ OH, -CH ₂ CI, -CF ₃ , -CH ₂ CN, -CH ₂ C(O)OH, -CH ₂ C(O)OCH ₃ , -CH ₂ C(O)NH ₂ , -CH ₂ C(O)CH ₃ , -CH ₂ OCH ₃ , -CH ₂ OC(O)CH ₃ , -CH ₂ NH ₂ , -CH ₂ N(CH ₃ ) ₂ , and -CH ₂ CH ₂ CI. The term “haloalkyl” is a subset of substituted alkyl, in which the hydrogen atom replacement is limited to halo (i.e. -F, -Cl, -Br, or -I) such that no other atoms aside from carbon, hydrogen and halogen are present. The group, -CH ₂ CI is a non- limiting example of a haloalkyl. The term “fluoroalkyl” is a subset of substituted alkyl, in which the hydrogen atom replacement is limited to fluoro such that no other atoms aside from carbon, hydrogen and fluorine are present. The groups -CH ₂ F, -CF ₃ , and -CH ₂ CF ₃ are non- limiting examples of fluoroalkyl groups.

[00161] The term “cycloalkyl” when used without the “substituted” modifier refers to a monovalent saturated aliphatic group with a carbon atom as the point of attachment, the carbon atom forming part of one or more non-aromatic ring structures, no carbon-carbon double or triple bonds, and no atoms other than carbon and hydrogen. Non-limiting examples include: -CH(CH ₂ ) ₂ (cyclopropyl), cyclobutyl, cyclopentyl, or cyclohexyl (Cy). The term “cycloalkanediyl” when used without the “substituted” modifier refers to a divalent saturated aliphatic group with two carbon atoms as points of attachment, no carbon-carbon double or triple bonds, and no atoms other than carbon and hydrogen. The group is a non- limiting example of cycloalkanediyl group. A “cycloalkane” refers to the class of compounds having the formula H-R, wherein R is cycloalkyl as this term is defined above. When any of these terms is used with the “substituted” modifier one or more hydrogen atom has been independently replaced by -OH, -F, -Cl, -Br, -I, -NH ₂ , -NO ₂ , -CO ₂ H, -CO ₂ CH ₃ , -CN, -SH, -OCH ₃ , -OCH ₂ CH ₃ , -C(O)CH ₃ , -NHCH ₃ , -NHCH ₂ CH ₃ , -N(CH ₃ ) ₂ , -C(O)NH ₂ , -C(O)NHCH ₃ , -C(O)N(CH ₃ ) ₂ , -OC(O)CH ₃ , -NHC(O)CH ₃ , -S(O) ₂ OH, or -S(O) ₂ NH ₂ .

[00162] The term “alkenyl” when used without the “substituted” modifier refers to an monovalent unsaturated aliphatic group with a carbon atom as the point of attachment, a linear or branched, acyclic structure, at least one nonaromatic carbon-carbon double bond, no carbon- carbon triple bonds, and no atoms other than carbon and hydrogen. Non-limiting examples include: -CH=CH ₂ (vinyl), -CH=CHCH ₃ , -CH=CHCH ₂ CH ₃ , -CH ₂ CH=CH ₂ (allyl), -CH ₂ CH=CHCH ₃ , and -CH=CHCH=CH ₂ . The term “alkenediyl” when used without the “substituted” modifier refers to a divalent unsaturated aliphatic group, with two carbon atoms as points of attachment, a linear or branched, a linear or branched acyclic structure, at least one nonaromatic carbon-carbon double bond, no carbon-carbon triple bonds, and no atoms other than carbon and hydrogen. The groups -CH=CH-, -CH=C(CH ₃ )CH ₂ -, -CH=CHCH ₂ -, and -CH ₂ CH=CHCH ₂ - are non-limiting examples of alkenediyl groups. It is noted that while the alkenediyl group is aliphatic, once connected at both ends, this group is not precluded from forming part of an aromatic structure. The terms “alkene” and “olefin” are synonymous and refer to the class of compounds having the formula H-R, wherein R is alkenyl as this term is defined above. Similarly, the terms “terminal alkene” and “a-olefin” are synonymous and refer to an alkene having just one carbon-carbon double bond, wherein that bond is part of a vinyl group at an end of the molecule. When any of these terms are used with the “substituted” modifier one or more hydrogen atom has been independently replaced by -OH, -F, -Cl, -Br, -I, -NH ₂ , -NO ₂ , -CO ₂ H, -CO ₂ CH ₃ , -CN, -SH, -OCH ₃ , -OCH ₂ CH ₃ , -C(O)CH ₃ , -NHCH ₃ , -NHCH ₂ CH ₃ , -N(CH ₃ ) ₂ , -C(O)NH ₂ , -C(O)NHCH ₃ , -C(O)N(CH ₃ ) ₂ , -OC(O)CH ₃ , -NHC(O)CH ₃ , -S(O) ₂ OH, or -S(O) ₂ NH ₂ . The groups -CH=CHF, -CH=CHCl and -CH=CHBr are non-limiting examples of substituted alkenyl groups.

[00163] The term “alkynyl” when used without the “substituted” modifier refers to a monovalent unsaturated aliphatic group with a carbon atom as the point of attachment, a linear or branched acyclic structure, at least one carbon-carbon triple bond, and no atoms other than carbon and hydrogen. As used herein, the term alkynyl does not preclude the presence of one or more non-aromatic carbon-carbon double bonds. The groups -C=CH, -C=CCH ₃ , and -CH ₂ C=CCH ₃ are non-limiting examples of alkynyl groups. An “alkyne” refers to the class of compounds having the formula H-R, wherein R is alkynyl. When any of these terms are used with the “substituted” modifier one or more hydrogen atom has been independently replaced by -OH, -F, -Cl, -Br, -I, -NH ₂ , -NO ₂ , -CO ₂ H, -CO ₂ CH ₃ , -CN, -SH, -OCH ₃ , -OCH ₂ CH ₃ , -C(O)CH ₃ , -NHCH ₃ , -NHCH ₂ CH ₃ , -N(CH ₃ ) ₂ , -C(O)NH ₂ , -C(O)NHCH ₃ , -C(O)N(CH ₃ ) ₂ , -OC(O)CH ₃ , -NHC(O)CH ₃ , -S(O) ₂ OH, or -S(O) ₂ NH ₂ .

[00164] The term “aryl” when used without the “substituted” modifier refers to a monovalent unsaturated aromatic group with an aromatic carbon atom as the point of attachment, the carbon atom forming part of a one or more six-membered aromatic ring structure, wherein the ring atoms are all carbon, and wherein the group consists of no atoms other than carbon and hydrogen. If more than one ring is present, the rings may be fused or unfused. As used herein, the term does not preclude the presence of one or more alkyl or aralkyl groups (carbon number limitation permitting) attached to the first aromatic ring or any additional aromatic ring present. Non-limiting examples of aryl groups include phenyl (Ph), methylphenyl, (dimethyl)phenyl, -C ₆ H ₄ CH ₂ CH ₃ (ethylphenyl), naphthyl, and a monovalent group derived from biphenyl. The term “arenediyl” when used without the “substituted” modifier refers to a divalent aromatic group with two aromatic carbon atoms as points of attachment, the carbon atoms forming part of one or more six-membered aromatic ring structure(s) wherein the ring atoms are all carbon, and wherein the monovalent group consists of no atoms other than carbon and hydrogen. As used herein, the term does not preclude the presence of one or more alkyl, aryl or aralkyl groups (carbon number limitation permitting) attached to the first aromatic ring or any additional aromatic ring present. If more than one ring is present, the rings may be fused or unfused. Unfused rings may be connected via one or more of the following: a covalent bond, alkanediyl, or alkenediyl groups (carbon number limitation permitting). Non-limiting examples of arenediyl groups include:

[00165] The term “aralkyl” when used without the “substituted” modifier refers to the monovalent group -alkanediyl-aryl, in which the terms alkanediyl and aryl are each used in a manner consistent with the definitions provided above. Non-limiting examples are: phenylmethyl (benzyl, Bn) and 2-phenyl-ethyl. When the term aralkyl is used with the “substituted” modifier one or more hydrogen atom from the alkanediyl and/or the aryl group has been independently replaced by -OH, -F, -Cl, -Br, -I, -NH ₂ , -NO ₂ , -CO ₂ H, -CO ₂ CH ₃ , -CN, -SH, -OCH ₃ , -OCH ₂ CH ₃ , -C(O)CH ₃ , -NHCH ₃ , -NHCH ₂ CH ₃ , -N(CH ₃ ) ₂ , -C(O)NH ₂ , -C(O)NHCH ₃ , -C(O)N(CH ₃ ) ₂ , -OC(O)CH ₃ , -NHC(O)CH ₃ , -S(O) ₂ OH, or -S(O) ₂ NH ₂ . Non- limiting examples of substituted aralkyls are: (3-chlorophenyl)-methyl, and 2-chloro-2-phenyl- eth-l-yl.

[00166] The term “hetero” when used to modify a compound or chemical group means the compound or chemical group has at least an atom that is not carbon, for example, N, O, S, Se, P, Si, B, or any other heteroatom. For example, a heteroaliphatic can be any aliphatic moiety containing at least one heteroatom selected from N, O, P, B, S, Si, Sb, Al, Sn, As, Se, and Ge. A heterocycle can be any ring containing a ring atom that is not carbon. A heterocycle can be substituted with any number of substituents, for example, alkyl groups and halogen atoms. A heterocycle can be aromatic (heteroaryl) or non-aromatic. Non-limiting examples of heterocycles include pyrrole, pyrrolidine, pyridine, piperidine, succinamide, maleimide, morpholine, imidazole, thiophene, furan, tetrahydrofuran, pyran, and tetrahydropyran.

[00167] The term “heteroaryl” when used without the “substituted” modifier refers to a monovalent aromatic group with an aromatic carbon atom or nitrogen atom as the point of attachment, the carbon atom or nitrogen atom forming part of one or more aromatic ring structures wherein at least one of the ring atoms is nitrogen, oxygen or sulfur, and wherein the heteroaryl group consists of no atoms other than carbon, hydrogen, aromatic nitrogen, aromatic oxygen and aromatic sulfur. Heteroaryl rings may contain 1, 2, 3, or 4 ring atoms selected from are nitrogen, oxygen, and sulfur. If more than one ring is present, the rings may be fused or unfused. As used herein, the term does not preclude the presence of one or more alkyl, aryl, and/or aralkyl groups (carbon number limitation permitting) attached to the aromatic ring or aromatic ring system. Non-limiting examples of heteroaryl groups include furanyl, imidazolyl, indolyl, indazolyl (Im), isoxazolyl, methylpyridinyl, oxazolyl, phenylpyridinyl, pyridinyl (pyridyl), pyrrolyl, pyrimidinyl, pyrazinyl, quinolyl, quinazolyl, quinoxalinyl, triazinyl, tetrazolyl, thiazolyl, thienyl, and triazolyl. The term ‘ W-heteroaryl” refers to a heteroaryl group with a nitrogen atom as the point of attachment. The term “heteroarenediyl” when used without the “substituted” modifier refers to an divalent aromatic group, with two aromatic carbon atoms, two aromatic nitrogen atoms, or one aromatic carbon atom and one aromatic nitrogen atom as the two points of attachment, the atoms forming part of one or more aromatic ring structure(s) wherein at least one of the ring atoms is nitrogen, oxygen or sulfur, and wherein the divalent group consists of no atoms other than carbon, hydrogen, aromatic nitrogen, aromatic oxygen and aromatic sulfur. If more than one ring is present, the rings may be fused or unfused. Unfused rings may be connected via one or more of the following: a covalent bond, alkanediyl, or alkenediyl groups (carbon number limitation permitting). As used herein, the term does not preclude the presence of one or more alkyl, aryl, and/or aralkyl groups (carbon number limitation permitting) attached to the aromatic ring or aromatic ring system. Non- limiting examples of heteroarenediyl groups include:

[00168] The term “heterocycloalkyl” when used without the “substituted” modifier refers to a monovalent non-aromatic group with a carbon atom or nitrogen atom as the point of attachment, the carbon atom or nitrogen atom forming part of one or more non-aromatic ring structures wherein at least one of the ring atoms is nitrogen, oxygen or sulfur, and wherein the heterocycloalkyl group consists of no atoms other than carbon, hydrogen, nitrogen, oxygen and sulfur. Heterocycloalkyl rings may contain 1, 2, 3, or 4 ring atoms selected from nitrogen, oxygen, or sulfur. If more than one ring is present, the rings may be fused or unfused. As used herein, the term does not preclude the presence of one or more alkyl groups (carbon number limitation permitting) attached to the ring or ring system. Also, the term does not preclude the presence of one or more double bonds in the ring or ring system, provided that the resulting group remains non-aromatic. Non-limiting examples of heterocycloalkyl groups include aziridinyl, azetidinyl, pyrrolidinyl, piperidinyl, piperazinyl, morpholinyl, thiomorpholinyl, tetrahydrofuranyl, tetrahydrothiofuranyl, tetrahydropyranyl, pyranyl, oxiranyl, and oxetanyl. The term ‘W-heterocycloalkyl” refers to a heterocycloalkyl group with a nitrogen atom as the point of attachment. N-pyrrolidinyl is an example of such a group. The term “heterocycloalkanediyl” when used without the “substituted” modifier refers to a divalent cyclic group, with two carbon atoms, two nitrogen atoms, or one carbon atom and one nitrogen atom as the two points of attachment, the atoms forming part of one or more ring structure(s) wherein at least one of the ring atoms is nitrogen, oxygen or sulfur, and wherein the divalent group consists of no atoms other than carbon, hydrogen, nitrogen, oxygen and sulfur. If more than one ring is present, the rings may be fused or unfused. Unfused rings may be connected via one or more of the following: a covalent bond, alkanediyl, or alkenediyl groups (carbon number limitation permitting). As used herein, the term does not preclude the presence of one or more alkyl groups (carbon number limitation permitting) attached to the ring or ring system. Also, the term does not preclude the presence of one or more double bonds in the ring or ring system, provided that the resulting group remains non-aromatic. Non-limiting examples of heterocycloalkanediyl groups include:

[00169] When these terms are used with the “substituted” modifier one or more hydrogen atom has been independently replaced by -OH, -F, -Cl, -Br, -I, -NH ₂ , -NO ₂ , -CO ₂ H, -CO ₂ CH ₃ , -CN, -SH, -OCH ₃ , -OCH ₂ CH ₃ , -C(O)CH ₃ , -NHCH ₃ , -NHCH ₂ CH ₃ , -N(CH ₃ ) ₂ , -C(O)NH ₂ , -C(O)NHCH ₃ , -C(O)N(CH ₃ ) ₂ , -OC(O)CH ₃ , -NHC(O)CH ₃ , -S(O) ₂ OH, or -S(O) ₂ NH ₂ .

[00170] The term “acyl” when used without the “substituted” modifier refers to the group -C(O)R, in which R is a hydrogen, alkyl, cycloalkyl, alkenyl, aryl, aralkyl or heteroaryl, as those terms are defined above. The groups, -CHO, -C(O)CH ₃ (acetyl, Ac), -C(O)CH ₂ CH ₃ , -C(O)CH ₂ CH ₂ CH ₃ , -C(O)CH(CH ₃ ) ₂ , -C(O)CH(CH ₂ ) ₂ , -C(O)C ₆ H ₅ , -C(O)C ₆ H ₄ CH ₃ , -C(O)CH ₂ C ₆ H ₅ , -C(O)(imidazolyl) are non-limiting examples of acyl groups. A “thioacyl” is defined in an analogous manner, except that the oxygen atom of the group -C(O)R has been replaced with a sulfur atom, -C(S)R. The term “aldehyde” corresponds to an alkane, as defined above, wherein at least one of the hydrogen atoms has been replaced with a -CHO group. When any of these terms are used with the “substituted” modifier one or more hydrogen atom (including a hydrogen atom directly attached to the carbon atom of the carbonyl or thiocarbonyl group, if any) has been independently replaced by -OH, -F, -Cl, -Br, -I, -NH ₂ , -NO ₂ , -CO ₂ H, -CO ₂ CH ₃ , -CN, -SH, -OCH ₃ , -OCH ₂ CH ₃ , -C(O)CH ₃ , -NHCH ₃ , -NHCH ₂ CH ₃ , -N(CH ₃ ) ₂ , -C(O)NH ₂ , -C(O)NHCH ₃ , -C(O)N(CH ₃ )2, -OC(O)CH ₃ , -NHC(O)CH ₃ , -S(O) ₂ OH, or -S(O) ₂ NH ₂ . The groups, -C(O)CH ₂ CF ₃ , -CO ₂ H (carboxyl), -CO ₂ CH ₃ (methylcarboxyl), -CO ₂ CH ₂ CH ₃ , -C(O)NH ₂ (carbamoyl), and -CON(CH ₃ ) ₂ , are non-limiting examples of substituted acyl groups.

[00171] The term “alkoxy” when used without the “substituted” modifier refers to the group -OR, in which R is an alkyl, as that term is defined above. Non-limiting examples include: -OCH ₃ (methoxy), -OCH ₂ CH ₃ (ethoxy), -OCH ₂ CH ₂ CH ₃ , -OCH(CH ₃ ) ₂ (isopropoxy), -OC(CH ₃ ) ₃ (tert-butoxy), -OCH(CH ₂ ) ₂ , -O-cyclopentyl, and -O-cyclohexyl. The terms “cycloalkoxy”, “alkenyloxy”, “alkynyloxy”, “aryloxy”, “aralkoxy”, “heteroaryloxy”, “heterocycloalkoxy”, and “acyloxy”, when used without the “substituted” modifier, refers to groups, defined as -OR, in which R is cycloalkyl, alkenyl, alkynyl, aryl, aralkyl, heteroaryl, heterocycloalkyl, and acyl, respectively. The term “alkoxydiyl” refers to the divalent group -O-alkanediyl-, -O-alkanediyl-O-, or -alkanediyl-O-alkanediyl-. The term “alkylthio” and “acylthio” when used without the “substituted” modifier refers to the group -SR, in which R is an alkyl and acyl, respectively. The term “alcohol” corresponds to an alkane, as defined above, wherein at least one of the hydrogen atoms has been replaced with a hydroxy group. The term “ether” corresponds to an alkane, as defined above, wherein at least one of the hydrogen atoms has been replaced with an alkoxy group. When any of these terms is used with the “substituted” modifier one or more hydrogen atom has been independently replaced by -OH, -F, -Cl, -Br, -I, -NH ₂ , -NO ₂ , -CO ₂ H, -CO ₂ CH ₃ , -CN, -SH, -OCH ₃ , -OCH ₂ CH ₃ , -C(O)CH ₃ , -NHCH ₃ , -NHCH ₂ CH ₃ , -N(CH ₃ ) ₂ , -C(O)NH ₂ , -C(O)NHCH ₃ , -C(O)N(CH ₃ ) ₂ , -OC(O)CH ₃ , -NHC(O)CH ₃ , -S(O) ₂ OH, or -S(O) ₂ NH ₂ .

[00172] The term “alkylamino” when used without the “substituted” modifier refers to the group -NHR, in which R is an alkyl, as that term is defined above. Non-limiting examples include: -NHCH ₃ and -NHCH ₂ CH ₃ .

[00173] The term “dialkylamino” when used without the “substituted” modifier refers to the group -NRR', in which R and R' can be the same or different alkyl groups, or R and R' can be taken together to represent an alkanediyl. Non-limiting examples of dialkylamino groups include: -N(CH ₃ ) ₂ and -N(CH ₃ )(CH ₂ CH ₃ ). The terms “cycloalkylamino”, “alkenylamino”, “alkynylamino”, “arylamino”, “aralkylamino”, “heteroarylamino”, “heterocycloalkylamino”, “alkoxy amino”, and “alkylsulfonylamino” when used without the “substituted” modifier, refers to groups, defined as -NHR, in which R is cycloalkyl, alkenyl, alkynyl, aryl, aralkyl, heteroaryl, heterocycloalkyl, alkoxy, and alkylsulfonyl, respectively. A non-limiting example of an arylamino group is -NHC ₆ H ₅ . The term “alkylaminodiyl” refers to the divalent group -NH-alkanediyl-, -NH-alkanediyl-NH-, or -alkanediyl-NH-alkanediyl-. The term “amido” (acylamino), when used without the “substituted” modifier, refers to the group -NHR, in which R is acyl, as that term is defined above. A non-limiting example of an amido group is -NHC(O)CH ₃ . The term “alkylimino” when used without the “substituted” modifier refers to the divalent group =NR, in which R is an alkyl, as that term is defined above. When any of these terms is used with the “substituted” modifier one or more hydrogen atom attached to a carbon atom has been independently replaced by -OH, -F, -Cl, -Br, -I, -NH ₂ , -NO ₂ , -CO ₂ H, -CO ₂ CH ₃ , -CN, -SH, -OCH ₃ , -OCH ₂ CH ₃ , -C(O)CH ₃ , -NHCH ₃ , -NHCH ₂ CH ₃ , -N(CH ₃ ) ₂ , -C(O)NH ₂ , -C(O)NHCH ₃ , -C(O)N(CH ₃ ) ₂ , -OC(O)CH ₃ , -NHC(O)CH ₃ , -S(O) ₂ OH, or -S(O) ₂ NH ₂ . The groups -NHC(O)OCH ₃ and -NHC(O)NHCH ₃ are non-limiting examples of substituted amido groups. [00174] The term “substituted” refers to moieties having substituents replacing a hydrogen on one or more carbons of the backbone. It will be understood that “substitution” or “substituted with” includes the implicit proviso that such substitution is in accordance with permitted valence of the substituted atom and the substituent, and that the substitution results in a stable compound, e.g., which does not spontaneously undergo transformation such as by rearrangement, cyclization, elimination, etc. As used herein, the term “substituted” is contemplated to include all permissible substituents of organic compounds. In a broad aspect, the permissible substituents include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, aromatic and non-aromatic substituents of organic compounds. The permissible substituents can be one or more and the same or different for appropriate organic compounds. For purposes of this invention, the heteroatoms such as nitrogen may have hydrogen substituents and/or any permissible substituents of organic compounds described herein which satisfy the valences of the heteroatoms. Substituents can include any substituents described herein, for example, a halogen, a hydroxyl, a carbonyl (such as a carboxyl, an alkoxycarbonyl, a formyl, or an acyl), a thiocarbonyl (such as a thioester, a thioacetate, or a thioformate), an alkoxyl, a phosphoryl, a phosphate, a phosphonate, a phosphinate, an amino, an amido, an amidine, an imine, a cyano, a nitro, an azido, a sulfhydryl, an alkylthio, a sulfate, a sulfonate, a sulfamoyl, a sulfonamido, a sulfonyl, a heterocyclyl, an aralkyl, or an aromatic or heteroaromatic moiety. It will be understood by those skilled in the art that the moieties substituted on the hydrocarbon chain can themselves be substituted, if appropriate. Unless specified otherwise, aliphatic, heteroaliphatic, oxyaliphatic, alkyl, alkylene, alkenyl, alkenylene, alkynyl, alkynylene, cycloalkyl, cycloalkylene, cycloalkenyl, cycloalkenylene, cycloalkynyl, cycloalkynylene, hydroxyalkyl, heterocycloalkyl, heterocycloalkylene, heterocycloalkenyl, heterocycloalkenylene, aryl, and heteroaryl mentioned herein include both substituted and unsubstituted moieties.

[00175] Unless otherwise indicated, all numbers expressing quantities of ingredients, reaction conditions, and so forth used in the specification and claims are to be understood as being modified in all instances by the term “about.” Accordingly, unless indicated to the contrary, the numerical parameters set forth in the present specification and attached claims are approximations that can vary depending upon the desired properties sought to be obtained by the present application. Generally, the term “about,” as used herein when referring to a measurable value such as an amount of weight, time, dose, etc. is meant to encompass in one example variations of ± 20% or ± 10%, in another example ± 5%, in another example ± 3%, in another example ± 1%, and in yet another example ± 0.1% from the specified amount, as such variations are appropriate to perform the disclosed method.

[00176] As used in this application, the term “average molecular weight” refers to the relationship between the number of moles of each polymer species and the molar mass of that species. In particular, each polymer molecule may have different levels of polymerization and thus a different molar mass. The average molecular weight can be used to represent the molecular weight of a plurality of polymer molecules. Average molecular weight is typically synonymous with average molar mass. In particular, there are three major types of average molecular weight: number average molar mass, weight (mass) average molar mass, and Z- average molar mass. In the context of this application, unless otherwise specified, the average molecular weight represents either the number average molar mass or weight average molar mass of the formula. In some embodiments, the average molecular weight is the number average molar mass. In some embodiments, the average molecular weight may be used to describe a PEG component present in a lipid.

[00177] The terms “comprise,” “have” and “include” are open-ended linking verbs. Any forms or tenses of one or more of these verbs, such as “comprises,” “comprising,” “has,” “having,” “includes” and “including,” are also open-ended. For example, any method that “comprises,” “has” or “includes” one or more steps is not limited to possessing only those one or more steps and also covers other unlisted steps.

[00178] The term “effective,” as that term is used in the specification and/or claims, means adequate to accomplish a desired, expected, or intended result. “Effective amount,” “Therapeutically effective amount” or “pharmaceutically effective amount” when used in the context of treating a patient or subject with a compound means that amount of the compound which, when administered to a subject or patient for treating a disease, is sufficient to effect such treatment for the disease.

[00179] As used herein, the term “subject” refers to a living mammalian organism, such as a human, monkey, cow, sheep, goat, dog, cat, mouse, rat, guinea pig, or transgenic species thereof. In certain embodiments, the patient or subject is a primate (e.g., non-human primate). In certain embodiments, the patient or subject is a human. Non-limiting examples of human subjects are adults, juveniles, infants and fetuses.

[00180] The term “assemble” or “assembled,” as used herein, in context of delivery of a payload to target cell(s) generally refers to covalent or non-covalent interaction(s) or association(s), for example, such that a therapeutic or prophylactic agent be complexed with or encapsulated in a lipid composition.

[00181] As used herein, the term “lipid composition” generally refers to a composition comprising lipid compound(s), including but not limited to, a lipoplex, a liposome, a lipid particle. Examples of lipid compositions include suspensions, emulsions, and vesicular compositions.

[00182] As generally used herein “pharmaceutically acceptable” refers to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues, organs, and/or bodily fluids of human beings and animals without excessive toxicity, irritation, allergic response, or other problems or complications commensurate with a reasonable benefit/risk ratio.

[00183] “Pharmaceutically acceptable salts” means salts of compounds of the present application which are pharmaceutically acceptable, as defined above, and which possess the desired pharmacological activity. Such salts include acid addition salts formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like; or with organic acids such as 1,2-ethanedisulfonic acid, 2-hydroxyethanesulfonic acid, 2-naphthalenesulfonic acid, 3 -phenylpropionic acid, 4,4'-methylenebis(3-hydroxy-2-ene- 1-carboxylic acid), 4-methylbicyclo[2.2.2]oct-2-ene-l-carboxylic acid, acetic acid, aliphatic mono- and dicarboxylic acids, aliphatic sulfuric acids, aromatic sulfuric acids, benzenesulfonic acid, benzoic acid, camphorsulfonic acid, carbonic acid, cinnamic acid, citric acid, cyclopentanepropionic acid, ethanesulfonic acid, fumaric acid, glucoheptonic acid, gluconic acid, glutamic acid, glycolic acid, heptanoic acid, hexanoic acid, hydroxynaphthoic acid, lactic acid, laurylsulfuric acid, maleic acid, malic acid, malonic acid, mandelic acid, methanesulfonic acid, muconic acid, o-(4-hydroxybenzoyl)benzoic acid, oxalic acid, p-chlorobenzenesulfonic acid, phenyl-substituted alkanoic acids, propionic acid, p-toluenesulfonic acid, pyruvic acid, salicylic acid, stearic acid, succinic acid, tartaric acid, tertiarybutylacetic acid, trimethylacetic acid, and the like. Pharmaceutically acceptable salts also include base addition salts which may be formed when acidic protons present are capable of reacting with inorganic or organic bases. Acceptable inorganic bases include sodium hydroxide, sodium carbonate, potassium hydroxide, aluminum hydroxide and calcium hydroxide. Acceptable organic bases include ethanolamine, diethanolamine, triethanolamine, tromethamine, A-methylglucamine and the like. It should be recognized that the particular anion or cation forming a part of any salt of this disclosure is not critical, so long as the salt, as a whole, is pharmacologically acceptable. Additional examples of pharmaceutically acceptable salts and their methods of preparation and use are presented in Handbook of Pharmaceutical Salts: Properties, and Use (P. H. Stahl & C. G. Wermuth eds., Verlag Helvetica Chimica Acta, 2002).

[00184] The term “pharmaceutically acceptable carrier,” as used herein means a pharmaceutically-acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material, involved in carrying or transporting a chemical agent.

[00185] The term “helper lipid” as used in this disclosure refers to a lipid that contributes to the stability or delivery efficacy of a lipid composition. A helper lipid can be a zwitterionic lipid, such as a phospholipid. A helper lipid can be phosphatidylcholine, distearoylphosphatidylcholine, dioleoylphosphatidylethanolamine, 1 ,2-dioleoyl-sn-glycero-3- phosphoethanolamine (DOPE) or l,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC). In some cases, the term “helper lipid” refers to phospholipids or other zwitterionic lipids in the LNP composition. In some cases, when describing the formulation of an LNP using weight ratios of the lipid components (e.g., lipidoid, steroid, helper lipid, and polymer conjugated lipid), a helper lipid refers to a phospholipid or another zwitterionic lipid. For example, the weight ratio of the lipidoid/steroid/helper lipid/polymer conjugated lipid is about 4/ 1/1/1. “Helper lipid” can refer to any class of lipid molecules that improves the particle stability and fluidity of lipid nanoparticles (LNP). Several classes of molecules can be used as helper lipids such as phospholipids (e.g., phosphoethanolamine, phosphocholine), zwitterionic lipids, steroid derivatives, and polymer conjugated lipids (e.g., PEGylated lipid). Representative helper lipids include cholesterol, l,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1 ,2-dioleoyl- sn-glycero-3-phosphocholine (DOPC), Phosphatidylcholine (PC), Methoxy- Polyethyleneglycol (MW 2k)-distearoylphosphatidylethanolamine (mPEG2k-DSPE), and 1,2- dimyristoyl-rac-glycero-3 -methoxypolyethylene gly col-2000 (DMG-PEG2k) .

EXAMPLES

[00186] The following examples are provided to further illustrate some embodiments of the present disclosure but are not intended to limit the scope of the disclosure; it will be understood by their exemplary nature that other procedures, methodologies, or techniques known to those skilled in the art may alternatively be used.

Example 1. mRNA library construction

[00187] A library of nucleic acid molecules (e.g., mRNA molecules) comprising nucleic acid sequences (e.g., mRNA sequences) encoding a reporter and a plurality of different peptidyl barcodes can be constructed. The reporter protein can be an intracellular protein, a membrane bound protein, or a secreted protein. FIG. 1A shows the design of a representative mRNA sequence encoding an intracellular reporter protein. FIG. 1B shows the design of an mRNA encoding a secreted protein. The reporter protein could be firefly luciferase, beta-galactosidase, or another enzyme that can be quantified using enzyme activity assay. The nucleic acid sequence encoding the reporter protein also encodes a peptidyl barcode (e.g., peptide tag), such as His, Flag, GST tags, or any other protein sequence will be introduced to generate the fusion protein. The tags can be specifically detected using a capture molecule, for example, a binding ligand or an antibody capable of binding to the tag. As depicted in FIG. 1A, in a mRNA production vector or gene fragment with proprietary 5’UTR and 3’UTRs, a tag sequence, which can be either peptide sequences such as His, Flag and GST tags or any other protein sequences, is introduced immediately after the start codon ATG or before the stop codon TAA, TAG or TGA of a reporter gene such as firefly luciferase or fluorescence proteins. As depicted in FIG. 1B, signaling peptide sequence is introduced immediately after the start codon ATG of a reporter gene such as firefly luciferase or fluorescence proteins. A tag sequence, which can be either peptide sequences such as His, Flag and GST tags or any other protein sequences, is introduced immediately before the stop codon TAA, TAG or TGA of the reporter gene.

[00188] mRNAs are produced through in vitro transcription (IVT), as depicted in FIG.2. Three types of DNAs are used as templates for IVT. The upper panel shows an enzymatically linearized plasmid with either T7 or Sp6 promotor and a desired insert. The middle panel shows PCR products with either T7 or Sp6 promoter sequence designed in the 5’ primer and oligo(dT120) introduced in the 3’ primer. The lower panel shows a synthetic gene fragment, which includes a T7 or Sp6 promoter sequence, an insert, and poly(A120) tail. The above templates are transcribed into multiple mRNAs by RNA polymerase when supplied with ATP, CTP, GTP, Nl-Methyl-Pseudouridine and CleanCap (AG) and incubated at 37°C for two hours. Immediately after IVT, mRNA reactions are purified with oligo(dT) affinity columns. The eluted fractions with mRNA contents measured by Nanodrop are combined and go through concentration and buffer exchange to ImM sodium citrate (pH 6.4) on a Formulatrix pPULSE TFF machine. The final purified mRNAs are aliquoted and kept at -80°C.

[00189] The qualities of mRNAs are assessed with Fragment Analyzer and Western Blot analysis. In Fragment Analyzer, only those mRNAs that generate a single sharp peak with correct sizes are selected. mRNAs combined with Lipofectamine MessengerMax (Invitrogen) are transfected into HEK293 cells seeded into 6-well plates the day before. 24 hours post transfection, cell pellets are collected, and cell lysates are prepared in 1% TritonX-100- containing lysis buffer. BCA method is used to quantitate protein concentration in each cell lysate. Equal amount of protein from each sample is loaded into precast 4-20% gradient SDS PAGE mini-gel. Run the gel at 200 volts for about one hour or until the loading dye reaches the bottom of the gel. Perform membrane transferring in an Invitrogen iblot 2 gel transfer device. Block the membrane with 5% non-fat milk in 1 x TBST washing buffer. Incubate the membrane with anti-Tag primary antibodies diluted in 1 x TBST at 4°C overnight. Wash the membrane with 1 x TBST three times, each time for about 5 minutes. Incubate the membrane with HRP-conjugated second antibody diluted in 5% non-fat milking at room temperature for one hour. WesternSure® PREMIUM Chemiluminescent Substrate is used for detecting. After adding substrate, the membrane is scanned with LI-COR C-DiGit® Blot Scanner.

Example 2. mRNA and LNP nanocomplexation:

[00190] The lipid library are synthesized as per our previous reports. All test lipids were synthesized through a solvent free Michael Addition reaction between an amine head and an alkyl- acrylate lipid tail.

[00191] In the cases of homogenous tailed lipids, which comprises one kind of hydrophobic tails, the aliphatic amine head (3,3’-Diamino-N-methyldipropylamine ) and the corresponding acrylate tail were mixed at 1 to 5 molar ratio in Teflon-lined glass screw-top vials at 70 °C for 48 h. The crude products were purified using a Teledyne Isco Chromatography system using the mobile phase of methanol/DCM. The purified lipidiods were characterized by electrospray ionization mass spectrometry (ESI-MS).

[00192] In the cases of asymmetric heterogenous lipids, the amine head was mixed with the major lipid tail at 1 to 3.5 molar ratio. After 48 hours stirring at 70 °C, three tailed lipids were purified similarly and reacted with the fourth tail subsequently at 1: 1.5 molar ratio. The final products were isolated and verified by ESI-MS.

[00193] In the cases of symmetric heterogenous lipids, hoc protected amine head was reacted with one lipid tail at 1 to 2.5 molar ratio. After mixing at 70 °C for 48 hours, the half assembled lipid was purified, hoc -protection group was removed by trifluoroacetic acid in DCM, and the half lipid was further reacted with the second lipid tail to afford the full heterogenous but symmetric lipid product.

[00194] LNPs are prepared using a NanoAssemblr microfluidic system (Precision Nanosystems). Briefly, synthetic lipids (partial list shown in Figure 3), cholesterol, phospholipids (DSPC, DOPE, and DOPC), and PEGylated lipid, such as methoxypolyethylene glycol (DMG-PEG2000) are dissolved in 100% ethanol at molar ratios of 50/38.5/10/1.5 at a final lipidoids concentration of 10 mg/mL. Though we choose this molar ratio as a starting point, other ratio or other excipients may be used to finely tune the formulation. The lipid solution is then mixed with an acidic sodium acetate buffer containing mRNA (0.45 mg/mL, pH 4.0) by using the NanoAssemblr microfluidic system. The resulting LNP is dialyzed against PBS (pH 7.4, 10 mM) overnight at 4 °C. mRNA are mixed at the appropriate weight ratio in sodium acetate buffer (25 mM, pH 5.2). The mRNA solution and the lipid solution are each injected into the NanoAssemblr microfluidic device at a ratio of 3:1, and the device resulted in the rapid mixing of the two components and thus the self-assembly of LNPs. Formulations are further dialyzed against PBS (10 mM, pH 7.4) in dialysis cassettes overnight at 4°C. The particle size of formulations are measured by dynamic light scattering (DLS) using a ZetaPALS DLS machine (Brookhaven Instruments). RNA encapsulation efficiency was characterized by Ribogreen assay. FIGs. 3A-3I show non-limiting exemplary synthetic lipids that can be used for the mRNA library delivery described herein.

Example 3. In vivo multiplex screening of LNP formulations using mRNA library encoding reporter with various peptidyl barcodes

[00195] The mixed mRNA/LNP formulation is administered into animals such as mice, rats, rabbits, or monkeys through different routes such as intravenous (IV), subcutaneous (SC), intramuscular (IM) or intradermal (ID). FIG. 4 depicts a schematic illustration of exemplary multiplex screening of LNP formulations in vivo. Six hours post injection, animal organs such as liver, lung, spleen, kidney, brain, muscle, heart, bone marrow, fat are harvested. To screen organ specific delivery of LNPs, LNPs comprising mRNA encoding a cytosolic reporter protein and peptidyl barcodes were used. Tissues are digested, and whole cell tissue lysates are prepared. To screen tissue cell type specific delivery of LNPs, tissues are digested into single- cell suspensions. Subgroup of cells are sorted by FACS and lysed. To screen LNP for secretary protein production, the mRNA encoding reporter with secretary peptide (FIG. 1B) can be used. In this case, the blood is withdrawn and analyzed using sandwich ELISA (FIG. 5) with tag- specific capturing molecules, such as anti-tag antibodies.

Example 4. High throughput screening assay using sandwich ELISA.

[00196] A black Costar 96-well plate with flat bottom is coated with different anti-tag antibodies (lOOng/ml) in 100 pl coating solution at 4°C overnight (FIG. 5 which depicts a schematic illustration of an exemplary sandwich ELISA assay for high throughput screening). Wash the plate and block it with blocking buffer (200 pl/well) at room temperature for one hour. The plate is washed three times with washing buffer, loaded with cell lysates prepared from different organs, and incubated at room temperature for 1 hour. The plate is washed three times and loaded with luciferase substrate luciferin (100 pl/well). Luminescence is measured immediately on a spectrometer. To prove the concept of this high throughput screening method, three different tissue targeting LNPs were formulated with 3 different tagged FLuc mRNAs, i.e. liver targeting LNP1 is formulated with FLuc mRNA-Flag (LNPl-FLuc-Flag), lung targeting LNP2 is formulated with FLuc mRNA-his (LNP2-FLuc-his) and spleen targeting LNP3 is formulated with FLuc mRNA-HA (LNP3-FLuc-HA). For LNP characterization, size and polydispersity index (PDI) were determined by dynamic light scattering using a Zetasizer Nano ZS. 10 pL of LNP suspension was transferred to a low-volume cuvette containing 900 pL of Ca ²⁺ and Mg ²⁺ free PBS at pH 7.4. Refractive index was set to 1.14 and temperature was set to 25 °C. Encapsulation efficiency (EE%) was measured using a fluorescence plate-based assay employing the Ribogreen reagent (Invitrogen) as per PNI Ribogreen assay protocol 16. This assay measures the quantity of mRNA in samples with intact LNPs to determine the quantity of unencapsulated RNA as well as in LNP samples disrupted by triton X-100 to measure the total RNA. EE% is calculated as the difference between the total RNA and the unencapsulated RNA divided by the total RNA. Formulations and results from characterization assays are listed in TABLE 3 here.

00197] The mRNA quality (FIG. 6A and FIG. 6B) and the characteristics of LNP- FLuc-tag formulations (as depicted in FIGs. 3A-3I) were analyzed. The individual or mixed LNP-tagged mRNA were injected into mice. Liver, lung and spleen tissues were collected 6 hours post injection. Tissues were lysed, and total luminescence was measured by direct adding of luciferase substrate luciferin. Sanwich ELISA was performed as previously described. Luciferase protein expression in tissue lysates is calculated against a standard curve generated using recombinant luciferase protein, followed by normalization to protein concentration in tissue lysates. As shown in FIG. 6C, the total luciferase protein expression patterns for each individual LNP-FLuc-tag and the mixed LNP formulations were the same as expected. The further quantitation of FLuc-Flag protein expression in both individual and mixed LNPs treated groups confirmed that mRNA-tagl protein were expressed in both liver and spleen tissues, which were consistent with the previous results (FIG. 6D). The detection of FLuc-his (FIG. 6E) and FLuc-HA (FIG. 6F) protein expression showed that the respective luciferase activity was observed dominantly in its target tissue and very weak protein expression in spleens for FLuc-his or lungs for FLuc-HA.