WO2025137409A1 - Supercharged lipid nanodiscs and methods of use - Google Patents

Abstract

The disclosure provides a nanodisc comprising a lipid bilayer encircled by at least one membrane scaffold protein (MSP) linked at the N- and C-termini and conjugated to a supercharged protein located exterior to the lipid bilayer. The disclosure further provides a method of delivering a payload into a cell, the method comprising contacting the cell with the nanodisc comprising a payload, thereby delivering the payload to the cell.

Description

SUPERCHARGED LIPID NANODISCS AND METHODS OF USE

FIELD

[0001] The disclosure relates to lipid nanodiscs and use thereof for delivering a payload into a cell.

INCORPORATION BY REFERENCE OF RELATED APPLICATION

[0002] This application claims priority to U.S. Provisional Application No. 63/613,472 filed on December 21, 2023, the contents of which are incorporated in their entirety.

INCORPORATION BY REFERENCE OF ELECTRONICALLY SUBMITTED MATERIAL

[0003] This application contains, as a separate part of the disclosure, a Sequence Listing in computer-readable form which is incorporated by reference in its entirety and identified as follows: 59556_SeqListing.XML; Size: 6,650 bytes; Created: November 21, 2024.

GOVERNMENT SUPPORT CLAUSE

[0004] This invention was made with government support under GM 140920 awarded by the National Institutes of Health. The government has certain rights in the invention.

BACKGROUND

[0005] Lipid nanoparticles (LNPs) are potent vehicles for drug delivery by hijacking membrane fusion. However, current LNPs solely rely on ionizable lipids to fuse with endosomes, lacking the ability to directly target the plasma membrane. In addition, the fusion efficiency is limited and causes most payloads (e.g., siRNAs and mRNAs) to be trapped in the endosome. It remains challenging to directly deliver payload onto the plasma membrane using LNPs.

SUMMARY

[0006] The present disclosure provides a nanodisc comprising a lipid bilayer encircled by at least one membrane scaffold protein (MSP) linked at the N- and C-termini and conjugated to a supercharged protein located exterior to the lipid bilayer. In various aspects, the termini of the MSP and the supercharged protein are irreversibly conjugated. In various aspects, the termini of the MSP and the supercharged protein are conjugated via a connector, such as a connector utilizing a Catcher-Tag system. An example of the Catcher-Tag system is a SpyTag/SpyCatcher system, which is fused at each terminus of the MSP. In various aspects, the nanodisc further comprises a payload within the lipid bilaycr. Suitable payloads include, but arc not limited to, a protein, a nucleic acid, a small molecule drug, or any combination thereof. Optionally, the nanodisc of the present disclosure is about 11 nm or about 30 nm in diameter.

[0007] Optionally, the MSP is derived from apolipoprotein. For instance, in various aspects of the disclosure, the MSP is apolipoprotein Al or a derivative thereof.

[0008] In various aspects of the disclosure, the supercharged protein is a positively charged protein. Optionally, the supercharged protein has a net charge of at least +30 at pH 7.4. The disclosure contemplates a nanodisc wherein the supercharged protein is a positively charged green fluorescent protein (GFP), such as a monomeric GFP variant with a net charge of about +36 at pH of about 7.4. In various aspects of the disclosure, the supercharged protein is a positively charged GFP is derived from Aequorea victoria. The supercharged protein optionally comprises an amino acid sequence comprising at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1.

[0009] The disclosure also provides a method of delivering a payload into a cell, the method comprising contacting the cell with the nanodisc of the present disclosure, thereby delivering the payload to the cell. In embodiments, the payload is a cell membrane protein.

[0010] Additional embodiments and aspects of the presently disclosed compositions and methods are provided below. All headings are simply for organization and are not intended to limit the disclosure in any manner. The content of any individual section may be equally applicable to all sections.

BRIEF DESCRIPTION OF THE DRAWINGS

[0011] FIG. 1 illustrates an exemplary expression cassette encoding a fusion of a supercharged protein (here, supercharged green fluorescence protein (GFP)), a membrane scaffold protein (MSP), and a SpyTag/SpyCatcher system, as well as a schematic of a representative nanodisc comprising a lipid bilayer encircled by two MSPs linked at the N- and C- termini and conjugated to a supercharged protein (supercharged GFP) located exterior to the lipid bilayer. [0012] FIGs. 2A-2B illustrate the elution profiles of nanodiscs (NDs) (shown in black) and MSPs (shown in gray) only. Aiso is noted on y-axis, while elution volume is noted on the x-axis. FIG. 2A shows the elution profile of +36GFP-spDl NDs and +36GFP-spDl alone. FIG. 2B shows the elution profile of +36GFP-spNW30 NDs and +36GFP-spNW30 alone.

[0013] FIGs. 3A-3C illustrate the interaction between nanodiscs (NDs) and the electron microscopy (EM) images of the NDs. FIG. 3 A illustrates the interaction between +36GFP-spDl and -30GFP-spDl. FIGs. 3B-3C illustrate EM images of NDs made with +36GFP-spDl (FIG. 3B) and +36GFP-spNW30 (FIG. 3C).

[0014] FIG. 4 is a schematic representation of ND-mediated delivery of synthetic membrane cargo to the cell surface to enable optical control of membrane potential.

[0015] FIGs. 5A-5B are HEK293t cells treated with supercharged NDs harboring a red fluorescent membrane cargo in comparison with traditional NDs. FIG. 5A shows traditional NDs. FIG. 5B shows supercharged NDs made with +36GFP-spDl.

DETAILED DESCRIPTION

[0016] The present disclosure is based, at least in part, on the discovery that inclusion of a supercharged (e.g., positively charged) protein located exterior to the lipid bilayer of a nanodisc significantly increases delivery efficiency of the nanodisc into the cell. The disclosure provides a nanodisc comprising a lipid bilayer encircled by at least one membrane scaffold protein (MSP) linked at the N- and C-termini and conjugated to a supercharged protein located exterior to the lipid bilayer.

[0017] Nanodiscs are discoidal lipid bilayers (e.g., phospholipid bilayers) which are stabilized by two membrane scaffold proteins (MSPs), amphipathic helical protein belts which encircle the bilayer. Bayburt et al., Nano Letters 2.8 (2002): 853-856.; Denisov et al., Journal of the American Chemical Society 126.11 (2004): 3477-3487. The phospholipid associates as a bilayer domain while two molecules of MSP wrap around the edges of the discoidal structure in a beltlike configuration, one MSP covering the hydrophobic alkyl chains of each leaflet. Bayburt and Sligar. "Membrane protein assembly into Nanodiscs." FEBS Letters 584.9 (2010): 1721-1727. A nanodisc is illustrated in FIG. 1. [0018] Examples of phospholipids suitable for generating the lipid bilayer of a nanodisc include, but arc not limited to, phosphatidyl choline; phosphatidyl cthanolaminc; phosphatidyl inositol; dipalmitoyl-phosphatidylcholine; dimyristoyl phosphatidyl choline; l-palmitoyl-2- oleoyl-phosphatidyl choline; dihexanoyl phosphatidyl choline; dipalmitoyl phosphatidyl ethanolamine; dipalmitoyl phosphatidyl inositol; dimyristoyl phosphatidyl ethanolamine; dimyristoyl phosphatidyl inositol; dihexanoyl phosphatidyl ethanolamine; dihexanoyl phosphatidyl inositol; l-palmitoyl-2-oleoyl-phosphatidyl ethanolamine; l-palmitoyl-2-oleoyl- phosphatidyl inositol; and the like. Typically, the phospholipid contains two saturated fatty acids (e.g., from 6 to 20 carbon atoms) with a common “head” group, which can be uncharged, positively charged, negatively charged or zwitterionic. Examples of head groups include, but are not limited to, phosphatidyl choline, phosphatidyl ethanolamine, or phosphatidyl serine.

[0019] Nanodiscs may be generated in variety of sizes. The nanodisc of the present disclosure may be from about 5 nm to about 100 nm, from about 6 nm to about 60 nm, from about 8 nm to about 40 nm, from about 10 nm to about 35 nm, or from about 11 nm to about 30 nm in diameter, although the disclosure is not limited to any particular size. For example, in various aspects, the nanodisc is about 5 nm, about 10 nm, about 15 nm, about 20 nm, about 25 nm, about 30 nm, about 35 nm, about 40 nm, about 45 nm, about 50 nm, about 55 nm, about 60 nm, about 65 nm, about 70 nm, about 75 nm, about 80 nm, about 85 nm, about 90 nm, about 95 nm, or about 100 nm in diameter (or any range having the recited values as endpoints). In some embodiments, the nanodisc of the present disclosure is about 11 nm or about 30 nm in diameter. In some embodiments, the nanodisc of the present disclosure is about 11 nm in diameter. In some embodiments, the nanodisc of the present disclosure is about 30 nm in diameter. The diameter of a nanodisc may be determined using a variety of methods. In various aspects, the diameter of the nanodisc of the disclosure is measured by electron microscopy (EM).

[0020] The nanodisc is a non-covalent assembly of phospholipid and membrane scaffold proteins (MSP) wrapped around the periphery of the lipid bilayer. A variety of MSPs may be employed for assembling nanodiscs of different sizes to accommodate various pay loads (e.g., proteins with varying numbers of transmembrane domains). MSPs are generally a-helical- amphipathic proteins, optionally derived from apolipoprotein (e.g., human senrm apolipoprotein Al). Apolipoproteins are serum proteins that mediate carriage of cholesterol and other lipids in the serum. Such apolipoproteins form the protein components of lipoprotein particles such as HDL and low-density lipoprotein (LDL) in serum. In various aspects, the MSP is apolipoprotein Al or a derivative thereof. Apolipoprotein Al is a protein with roles in the transportation and metabolism of lipids and is the main protein component in high-density lipoprotein (HDL). In embodiments, the MSP is a His-tagged MSP protein. In embodiments, the MSP is an untagged MSP protein. MSPs are further described in, e.g., Grinkova et al., Protein Eng Des Sei. 2010 Nov; 23(11):843— 848; Sligar and Denisov, Protein Sci. 2021 Feb; 30(2):297-315; and U.S. Patent No. 7,592,008 each of which is incorporated by reference in its entirety and in particular with respect to the disclosure of nanodisc assembly and MSPs.

[0021] The nanodisc of the disclosure comprises at least one membrane scaffold protein (MSP) linked at the N- and C-termini and conjugated to a supercharged protein located exterior to the lipid bilayer. In various aspects, the nanodisc comprises two MSPs, although the disclosure also contemplates using three or more MSPs. The supercharged protein is a protein displaying an unusually high net positive or negative charge (e.g., a greater net positive or net negative charge compared with a parent protein, such as a protein which has not been modified to adjust the charge) and compatible with delivery through the cellular membrane. A representative method of generating a supercharged protein comprises substituting solvent- exposed residues on protein’s surface with either acidic or basic amino acids, thereby adjusting the overall charge of the protein. Supercharged proteins and methods of generating supercharged proteins are further described in Ma et al., Adv. Mater. 2020, 32, 1905309.

[0022] The supercharged protein is, in various aspects, less than or equal to about 50 kDa, although this is not required. In various aspects, the supercharged protein is albumin (e.g., bovine serum albumin or human serum albumin), a reporter protein (e.g., a fluorescent protein, such as green fluorescent protein (GFP), orange fluorescent protein, cyan fluorescent protein, yellow fluorescent protein, red fluorescent protein, far-red fluorescent protein, and nonfluorescent red protein), streptavidin, or avidin. Optionally, the supercharged protein is a GFP. Typical GFPs are derived from protein first isolated from the jellyfish Aequorea victoria. However, GFPs have been found in other organisms including corals, sea anemones, zoanithids, copepods, and lancelets. Cox, Guy, ed. Fundamentals of Fluorescence Imaging. CRC Press, 2019. GFPs are further described in, e.g., Prendergastand Mann., Biochemistry 17.17 (1978):3448-3453; and Tsien, Annual Review of Biochemistry 67.1 (1998):509-544. [0023] In embodiments, the supercharged protein is a positively charged protein. The phrase “positively charged protein” refers to any protein that exhibits a net charge of greater than about 0 when measured at pH 7.4. Positively charged proteins may have a net charge of about +1, about +2, about +3, about +4, about +5, about +6, about +7, about +8, about +9, about +10, about +11, about +12, about +13, about +14, about +15, about +16, about +17, about +18, about +19, about +20, about +21, about +22, about +23, about +24, about +25, about +26, about +27, about +28, about +29, or about +30. In the context of the disclosure, the supercharged protein optionally has a net charge of at least +30 at pH 7.4 (e.g., a net charge of about +31, about +32, about +33, about +34, about +35, about +36, about +37, about +38, about +39, about +40, about +41, about +42, about +43, about +44, about +45, about +46, about +47, about +48, about +49, about +50, about +51, about +52, about +53, about +54, about +55, about +56, about +57, about +58, about +59, about +60, about +61, about +62, about +63, about +64, about +65, about +66, about +67, about +68, about +69, about +70, about +71, about +72, about +73, about +74, about +75, about +76, about +77, about +78, about +79, about +80, about +81, about +82, about +83, about +84, about +85, about +86, about +87, about +88, about +89, about +90, about +91, about +92, about +93, about +94, about +95, about +96, about +97, about +98, about +99, about +100, or greater when measured at pH 7.4). In various aspects, streptavidin has a net charge of at least +30 at pH 7.4. In various aspects, streptavidin can function as the supercharged protein as described in the present disclosure. In various aspects, bovine serum albumin (BSA) has a net charge of at least +30 at pH 7.4. In various aspects, a BSA can function as the supercharged protein described in the present disclosure.

[0024] In various aspects, the supercharged protein is a fluorescent, positively charged protein, such as any of the fluorescent proteins described above. In various aspects, the supercharged protein is a green fluorescent protein (GFP), e.g., positively charged GFP. Optionally, the GFP is a monomeric cytoplasmic fluorescent protein that emits green fluorescent light upon exposure to the blue to ultraviolet (UV) spectral regions. Optionally, the positively charged GFP has a net charge of at least +30 at pH 7.4. To generate supercharged GFPs from traditional GFPs, solvent- exposed residues of the GFPs may be mutated to Lys and/or Arg. Supercharged GFPs and methods of generating supercharged GFPs are further described in, e.g., Lawrence et al., Supercharging proteins can impart unusual resilience." Journal of the American Chemical Society 129.33 (2007): 10110-10112. In various aspects, the positively charged GFP is a monomeric GFP variant with a net charge of about +36 at pH of about 7.4. Optionally, the positively charged GFP of the present disclosure is derived from Aequorea victoria. In various aspects of the disclosure, the GFP comprises an amino acid sequence comprising at least 80% sequence identity (e.g., at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, at least 98% sequence identity, at least 99% sequence identity, or 100% sequence identity) to the amino acid sequence set forth in SEQ ID NO: 1.

[0025] Amino acid sequences of representative supercharged GFP constructs are provided below:

[0026] Amino acid sequence of supercharged +36GFP:

ASKGERLFRGKVPILVELKGDVNGHKFSVRGKGKGDATRGKLTLKFICTTGKLPVPWPT LVTTLTYGVQCFSRYPKHMKRHDFFKSAMPKGYVQERTISFKKDGKYKTRAEVKFEGR TLVNRIKLKGRDFKEKGNILGHKLRYNFNSHKVYITADKRKNGIKAKFKIRHNVKDGSV QLADHYQQNTPIGRGPVLLPRNHYLSTRSKLSKDPKEKRDHMVLLEFVTAAGIKHGRDE RYK (SEQ ID NO: 1)

[0027] Amino acid sequence of +36GFP-spDl:

MGSSHHHHHHSSGLVPRGSHMASMTGGQQMGRGSASKGERLFRGKVPILVELKGDVN GHKFSVRGKGKGDATRGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPKHMKRH DFFKSAMPKGYVQERTISFKKDGKYKTRAEVKFEGRTLVNRIKLKGRDFKEKGNILGHK LRYNFNSHKVYITADKRKNGIKAKFKIRHNVKDGSVQLADHYQQNTPIGRGPVLLPRNH YLSTRSKLSKDPKEKRDHMVLLEFVTAAGIKHGRDERYKGAMVTTLSGLSGEQGPSGD MTTEEDSATHIKFSKRDEDGRELAGATMELRDSSGKTISTWISDGHVKDFYLYPGKYTF VETAAPDGYEVATAITFTVNEQGQVTVNGEATKGDAHTSTFSKLREQLGPVTQEFWDN LEKETEGLRQEMSKDLEEVKAKVQPYLDDFQKKWQEEMELYRQKVEPLRAELQEGAR QKLHELQEKLSPLGEEMRDRARAHVDALRTHLAPYSDELRQRLAARLEALKENGGARL AEYHAKATEHLSTLSEKAKPALEDLRQGLLPVLESFKVSFLSALEEYTKKLNTQVPTIVM VDAYKRYK (SEQ ID NO: 2)

[0028] Amino acid sequence of +36GFP-spNW30:

MGSSHHHHHHSSGLVPRGSHMASMTGGQQMGRGSASKGERLFRGKVPILVELKGDVN

GHKFSVRGKGKGDATRGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPKHMKRH DFFKSAMPKGYVQERTISFKKDGKYKTRAEVKFEGRTLVNRIKLKGRDFKEKGNILGHK LRYNFNSHKVYITADKRKNGIKAKFKIRHNVKDGSVQLADHYQQNTPIGRGPVLLPRNH YLSTRSKLSKDPKEKRDHMVLLEFVTAAGIKHGRDERYKENLYFQGGAMVTTLSGLSG

EQGPSGDMTTEEDSATHIKFSKRDEDGRELAGATMELRDSSGKTISTWISDGHVKDFYL

YPGKYTFVETAAPDGYEVATAITFTVNEQGQVTVNGEATKGDAHTSTFSKLREQLGPVT

QEFWDNLEKETEGLRQEMSKDLEEVKAKVQPYLDDFQKKWQEEMELYRQKVEPLRAE

LQEGARQKLHELQEKLSPLGEEMRDRARAHVDALRTHLAPYSDELRQRLAARLEALKE

NGGARLAEYHAKATEHLSTLSEKAKPALEDLRQGLLPVLESFKVSFLSALEEYTKKLNT

QGTPVTQEFWDNLEKETEGLRQEMSKDLEEVKAKVQPYLDDFQKKWQEEMELYRQKV

EPLRAELQEGARQKLHELQEKLSPLGEEMRDRARAHVDALRTHLAPYSDELRQRLAAR

LEALKENGGARLAEYHAKATEHLSTLSEKAKPALEDLRQGLLPVLESFKVSFLSALEEY

TKKLNTQGTPVTQEFWDNLEKETEGLRQEMSKDLEEVKAKVQPYLDDFQKKWQEEME

LYRQKVEPLRAELQEGARQKLHELQEKLSPLGEEMRDRARAHVDALRTHLAPYSDELR QRLAARLEALKENGGARLAEYHAKATEHLSTLSEKAKPALEDLRQGLLPVLESFKVSFL SALEEYTKKLNTQLPGTGAAALEVPTIVMVDAYKRYK (SEQ ID NO: 3)

[0029] Amino acid sequence of -30GFP-spDl:

MGSSHHHHHHSSGLVPRGSHMASMTGGQQMGRGSSKGEELFDGVVPILVELDGDVNG

HEFSVRGEGEGDATEGELTLKFICTTGELPVPWPTLVTTLTYGVQCFSDYPDHMDQHDF

FKSAMPEGYVQERTISFKDDGTYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEY

NFNSHDVYITADKQENGIKAEFEIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDDHYLS

TESALSKDPNEDRDHMVLLEFVTAAGIDHGMDELYKGAMVTTLSGLSGEQGPSGDMTT

EEDSATHIKFSKRDEDGRELAGATMELRDSSGKTISTWISDGHVKDFYLYPGKYTFVET

AAPDGYEVATAITFTVNEQGQVTVNGEATKGDAHTSTFSKLREQLGPVTQEFWDNLEK

ETEGLRQEMSKDLEEVKAKVQPYLDDFQKKWQEEMELYRQKVEPLRAELQEGARQKL

HELQEKLSPLGEEMRDRARAHVDALRTHLAPYSDELRQRLAARLEALKENGGARLAEY HAKATEHLSTLSEKAKPALEDLRQGLLPVLESFKVSFLSALEEYTKKLNTQVPTIVMVD AYKRYK (SEQ ID NO: 4)

[0030] The MSP and the supercharged protein are conjugated such that the MSP(s) encircles the lipid bilayer and is linked to the supercharged protein on the exterior of the nanodisc. In various aspects, the MSP and the supercharged protein are irreversibly conjugated. Any means of conjugation is permitted in the context of the disclosure such that the supercharged protein is linked to the MSP. Examples of conjugations include cysteine disulfide cross-linking, isopeptide bonding, and the like. In various aspects, the MSP and the supercharged protein may be conjugated via a connector, i.e., a moiety that operably links two biomolecules to form a complex (e.g., an irreversible complex). An example of a connector system for conjugating, e.g., proteins, is a Catcher-Tag system. A Catcher-Tag system takes advantage of conjugation mediated by a covalent isopeptide bond that forms autocatalytically between a “Catcher” and a “Tag.” Potential advantages of Catcher-Tag systems include, but are not limited to, use of mild reaction conditions to achieve the conjugation, which can be spontaneous and efficient. Examples of Catcher-Tag systems include SpyCatcher/SpyTag, SpyStapler/SpyTag, Spyligase/SpyTag, and SdyCatcher/SdyTag. Catcher-Tag systems are further described in, e.g., Fan and Aranko, “Catcher/Tag Toolbox: Biomolecular Click-Reactions for Protein Engineering Beyond Genetics,” ChemBioChem, 2023, e202300600, 1-13 (doi.org/10.1002/cbic.202300600); and Hatlem et al., Int. J. Mol. Sci. 2019, 20(9), 2129.

[0031] Optionally, the MSP and supercharged protein is conjugated using a SpyTag/SpyCatcher system, e.g., the supercharged protein is conjugated to the MSP via a SpyTag/SpyCatcher system fused at the termini of the MSP. The peptide SpyTag (13 amino acids) spontaneously reacts with the protein SpyCatcher (12.3 kDa) to form an intermolecular isopeptide bond between the pair. Zakeri et al. "Peptide tag forming a rapid covalent bond to a protein, through engineering a bacterial adhesin." Proceedings of the National Academy of Sciences 109.12 (2012): E690-E697. A representative construct is illustrated in Figure 1, wherein a SpyCatcher moiety is fused at one terminus of the MSP and a SpyTag moiety is fused at the other terminus. In various embodiments, the SpyCatcher is at the N-terminal of the MSP and the SpyTag is at the C-terminal of the MSP. The supercharged protein is conjugated to the MSP by way of the components of the SpyCatcher/SpyTag system. In various embodiments, the SpyCatcher moiety is fused to the supercharged protein.

[0032] In embodiments, the nanodisc of the present disclosure further comprises a payload within the lipid bilayer. In various aspects, the payload is not fused to the lipid bilayer, MSP, or supercharged protein. For instance, the payload is not fused to the supercharged protein in some aspects of the disclosure. Thus, the design of the nanodisc may be independent of the specific payload to be carried by the nanodisc. Put another way, the selection of supercharged protein and MSP and the construction of a fusion protein comprising the supercharged protein and MSP need not be dependent on the particular payload of interest. The payload for delivery through the plasma membrane is optionally located within the lipid bilayer of the nanodisc, i.e., not fully exposed externally to the environment or attached to the exterior of the nanodisc.

[0033] The payload may be, for example, a protein, a nucleic acid (e.g., DNA or RNA, such as an mRNA or an siRNA), a small molecule drug, or any combination thereof, although the disclosure is not limited to these representative examples. An example of suitable payload for the nanodisc of the disclosure is a genome editing reagent. In another representative example, the payload is a cell membrane protein. In various aspects, the payload is a G protein-coupled receptor (GPCR), oxidoreductase, transferase, hydrolase, a synthetic ion channel, an immune recognition receptor, a cell-adhesion peptide, a reactive lipid, a membrane associated glycan, or a small molecule.

[0034] The nanodisc of the disclosure is accompanied by technical advantages. For instance, in various aspects of the disclosure, the efficiency of payload delivery mediated by the nanodisc is at least 100-fold higher compared to nanodiscs which do not comprise a supercharged protein (e.g., supercharged GFP). Delivery efficiency may be determined using cell-free and/or cellbased assays.

[0035] The nanodisc of the disclosure provides an efficient tool for, e.g., delivering a payload (e.g., biomolecule) of interest to or through a lipid membrane, such a cell membrane. In various aspects, the nanodisc is capable of fusing with a target membrane, which allows, e.g., modification of a cell surface with synthetic ion channels, receptors, and lipids. In addition, the nanodisc can bind toxic extracellular proteins and promote their clearance through fusing with immune cells.

[0036] In various aspects, the nanodisc demonstrates improved fusion with lipid membranes (e.g., liposomes) compared to constructs which do not comprise a supercharged protein. For instance, the nanodisc of the disclosure optionally demonstrates increased fusion efficiency (e.g., fusion efficiency is improved by at least 2 fold, by at least 3 fold, by at least 4 fold, by at least 5 fold, by at least 6 fold, by at least 7 fold, by at least 8 fold, by at least 9 fold, or by at least 10 fold) with liposomes reconstituted in in vitro and/or in vivo assays compared to nanodiscs lacking a supercharged protein. Alternatively, or in addition, the nanodisc of the disclosure may enhance delivery of payload into the cells by at least 2 fold, by at least 3 fold, by at least 4 fold, by at least 5 fold, by at least 6 fold, by at least 7 fold, by at least 8 fold, by at least 9 fold, or by at least 10 fold compared to nanodiscs which do not comprise a supercharged protein. Pay load delivery may be improved in vitro or in vivo. Any cell type is contemplated, including mammalian cells (e.g., embryonic stem cells), bacterial cells (E. coli cells), and the like.

[0037] The disclosure further provides methods of using the nanodiscs of the disclosure. For example, the disclosure provides a method of delivering a payload into a cell, the method comprising contacting the cell with the nanodisc of the present disclosure comprising payload. The contacting may occur in vitro or in vivo.

[0038] The disclosure also provides a method of inhibiting expression of a nucleic acid in a cell, the method comprising contacting the cell with the nanodisc of the disclosure, wherein the payload is an expression inhibitor that targets the nucleic acid such that expression is inhibited. Examples of expression inhibitors which modulate nucleic acid expression include siRNA, miRNA, and gene editing (e.g., CRISPR/Cas) system components.

[0039] Additionally, the disclosure provides a method of treating, preventing, or delaying progression of a disease or condition in a subject in need thereof, the method comprising administering to the subject an effective amount of the nanodisc comprising a payload which alleviates or inhibits one or more symptoms or underlying causes of the disease or condition. In embodiments, the disease or condition is associated with expression of a protein, and the nanodisc comprises a payload that induces targeted protein degradation or targets a nucleic acid encoding the protein to reduce the expression of the protein.

[0040] Throughout this specification and claims, the word “comprise” or variations such as “comprises” or “comprising,” will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers.

[0041] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the disclosure. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed within the disclosure, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure.

[0042] As used herein and in the appended claims, the singular forms "a," "and," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a lipid nanoparticle (LNP)" includes a plurality of lipid nanoparticles (LNPs) and equivalents thereof known to those skilled in the art, and so forth.

[0043] The term “about” signifies not more or less than 10 percent of the stipulated amount. Thus, a diameter of about 11 nm may be interpreted to be inclusive of 9.9 nm to 12.1 nm.

[0044] As will be apparent to those of skill in the art upon reading this disclosure, each of the individual aspects described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any of the other several aspects without departing from the scope or spirit of the present disclosure. Any recited method can be carried out in the order of events recited or in any other order which is logically possible. This is intended to provide support for all such combinations.

EXAMPLES

[0045] The examples are offered for illustrative purposes only and are not intended to limit the scope of the present invention in any way.

[0046] The following examples describe representative materials and methods for generating the nanodisc of the present disclosure, as well as further characterize various features of the nanodisc of the present disclosure.

Example 1 - Production and characterization of nanodiscs

[0047] Super positively charged (+36)-GFP was genetically fused with spMSPIDl that generates circularized nanodiscs about 11 nm in diameter. Because of the covalent bond formed between SpyCatcher and SpyTag in spMSPIDl , the resulting nanodisc framework is firmly circularized, thereby exhibiting excellent yields, stability, and homogeneity useful for both structural and functional characterizations of membrane proteins. The resulting fused protein, (+36)-GFP-spMSPlDl, was well expressed and monodisperse. Next, reconstitution experiments were performed using PC lipids to determine if (+36)-GFP-spMSPlDl is still able to form nanodiscs. Using size-exclusion chromatography (SEC), the protein was eluted at 17 ml as 66 kDa proteins. In contrast, reconstituted nanodiscs were eluted as 15.5 ml, indicating two copies of the scaffold proteins with 110 copies of lipids. Quantitative measurements using fluorescent lipids also revealed 100 molecules of lipids per nanodisc. These nanodiscs also were characterized through negative stain electron microscopy (EM), which revealed the formation of 11 nm size discoidal particles. Finally, the possibility to functionalize nanodiscs using super negatively charged (-30)-GFP was explored. The scaffold protein (-30)-GFP-spMSPlDl is also monodisperse and could form the typical discoidal nanodisc structure. Together, these data suggest that conjugation with supercharged proteins (here, supercharged GFPs) does not interfere with the framework of circularized nanodiscs.

[0048] To explore whether supercharged GFPs mediate the formation of nanodisc oligomers when functionally exposed on the surface of nanodiscs, (+36)-GFP-spMSPlDl and (-30)-GFP- spMSPlDl nanodiscs were incubated together, which resulted in formation of large and aggregated particles in a salt dependent manner, confirming that supercharged GFPs are free to bind each other through electrostatic interactions. The observations suggest that the function of supercharged GFPs was correctly maintained in the nanodisc framework, and they are readily accessible to engage with other molecules and proteins.

[0049] Different sizes of nanodiscs with increased loading capacities were studied. (+36)- GFP was fused onto MSP1E3 and spNW30 that form non-circularized 13 and circularized 30 nm nanodiscs, respectively. Interestingly, only (+36)-GFP-spNW30 maintained the ability to form nanodiscs with the correct diameters as determined by SEC and negative stain EM. Nanodiscs formed with (+36)-GFP-E3 were larger than expected and much more polydisperse. In addition, the yield of (+36)-GFP-E3 was significantly lower than the circularized (+36)-GFP-spMSPlDl and (+36)-GFP-spNW30.

[0050] Next, the ability of supercharged nanodiscs to fuse with liposomes in reconstituted assays was assessed. To do so, the classic membrane fusion assays developed for the characterization of soluble N-ethylmaleimide-sensitive factor activating protein receptor (SNARE) proteins that mediate vesicular transport were employed. To monitor the fusion of lipid bilayers, nanodiscs were prepared with a lipid mixture harboring 0.5% NBD-PE and Rho- PE. When embedded in the same nanodiscs, nitrobenzoxadiazole (NBD) fluorescence was quenched by Rho-PE because of FRET. However, fusion with target membranes results in the dispersion of NBD-PE and Rho-PE, thus dequenching of NBD fluorescence. Using this assay, it was found that (+36)-GFP-spMSPlDl nanodiscs readily fused with liposomes. The fusion reaction is sensitive to high concentrations of NaCl, suggesting that it is driven by the electrostatic interaction of (+36)-GFP and phospholipids. In contrast, nanodiscs formed with regular spMSPIDl or (-30)-GFP did not show detectable fusion activities. To prevent nanodiscs fusing with each other, all nanodiscs were kept in high salt buffer, so that they only became fusogenic when incubated with target membranes at physiology relevant conditions.

[0051] Next, a content mixing assay was utilized to determine if the fusion of nanodiscs with liposomes is hemifusion or full fusion. For this purpose, liposomes were encapsulated with starburst dendrimer (SBD) that is self-quenched. Release of SBD from fusion pores formed between nanodiscs and liposomes will cause its dequenching, as observed using (+36)-GFP- spMSPlDl nanodiscs. Again, regular spMSPIDl nanodiscs did not show any detectable fusion activity. In addition, SBD release was also not observed using purified (+36)-GFP-spMSPlDl below 30 pM. Low levels of SBD leakage at higher protein concentrations were observed, probably due to membrane disruption by protein crowding on lipid bilayers. Thus, the observed content mixing activity with nanodiscs at 10-50 nM concentrations is likely through membrane fusion, not due to membrane leakage caused by (+36)-GFP-spMSPlDl.

[0052] The robust fusion activities of supercharged nanodiscs led to the investigation of the molecular basis of membrane fusion mediated by (+36)-GFP-spMSPlDl. Most fusogens in nature can bridge two opposing membranes and locally disrupt lipid bilayers. The ability of (+36)-GFP-spMSPlDl to cluster and remodel membranes was characterized. Using light scattering assay, (+36)-GFP-spMSPlDl caused the aggregation of vesicles at low salt conditions which was reversed using high salt buffers. In addition, (+36)-GFP-spMSPlDl alone resulted in membrane deformation of small and giant unilamellar vesicles to various degrees at 1 pM - 10 pM concentrations, consistent with the typical features of membrane fusogens.

[0053] Nanodiscs formed using (+36)-GFP-E3 and (+36)-GFP-spNW30 also were characterized. The data showed that (+36)-GFP-spNW30 still exhibited robust fusion activities, whereas (+36)-GFP-E3 was not able to drive membrane fusion. Without wishing to be bound by any particular theory, it is believed that the rigid framework of circularized nanodiscs formed by (+36)-GFP-spMSPlDl and (+36)-GFP-spNW30 allows for the optimal positioning of (+36)- GFP to bind target membranes and catalyze the fusion with the lipid bilayer encased in nanodiscs. In contrast, the non-circularized nanodiscs enclosed by (+36)-GFP-E3 might have a more flexible structure that causes the improper placement of (+36)-GFP between two opposing membranes and thus not able to drive fusion. To determine whether and how long the fusion pore is open, dithiothreitol (DTT) quenching experiments were utilized. The data showed that the pore was eventually all closed and membranes in nanodiscs were merged with liposomes after fusion, consistent with the results from the content mixing assay showing that these fusion reactions are full fusion, not hemifused intermediate states.

[0054] Finally, the diameter of fusion pores formed by supercharged nanodiscs was characterized. Surprisingly, both (+36)-GFP-spMSPlDl and (+36)-GFP-spNW30 nanodiscs are not able to release 40 kDa fluorescent dextrans, indicating that the resulting fusion pores are relatively small, distinct from pores formed using fusogenic transmembrane proteins. To gain further insights into pore sizes and dynamics, electrical recordings were employed to characterize the properties of individual fusion pores. Consistently, the results showed that fusion pores formed by (+36)-GFP-spMSPlDl and (+36)-GFP-spNW30 are less than 0.5 nm in diameter and rapidly closed within a few minutes. Nevertheless, the robust fusion activities observed using (+36)-GFP-spMSPlDl and (+36)-GFP-spNW30 establish the basis for their application in membrane engineering of targeted cells.

Example 2 - Functional reconstitution of membrane proteins in supercharged nanodiscs

[0055] For the purpose of delivering proteins directly onto cell surface using supercharged nanodiscs, the payloads need to be maintained in functional states. To evaluate the potential interference of (+36)-GFP in conformational dynamics of protein payload, a prototype ATP- binding cassette transporter, MalFGK . that mediates maltose uptake into bacteria was employed as payload. It is known that the ATPase activity of MalFGK? is highly sensitive to its local environment and is regulated by maltose binding protein (MBP) and maltose. MalFGBG was reconstituted into (+36)-GFP-spMSPlDl nanodiscs and its ATPase activity was measured in comparison with the transporter isolated in detergents or reconstituted in proteoliposomes. The results showed that MalFGIG embedded in (+36)-GFP-spMSPlDl nanodiscs exhibited low ATPase activities and is significantly stimulated by MBP and maltose, similar to the results obtained from proteoliposomes. In contrast, MalFGKo in detergents showed much higher endogenous ATPase activities and is only modest responsive to MBP and maltose.

[0056] The compatibility of supercharged nanodiscs for the reconstitution of GPCRs also was assayed. To this end, A2aR was labeled with F19 and incorporated into nanodiscs of the disclosure. The dynamics of A2aR were recorded and compared with results obtained from previous studies, showing almost identical conformational landscape. The data suggested that supercharged nanodiscs can maintain membrane proteins in functional states.

[0057] The ability of supercharged nanodiscs to deliver ion channels was also tested.

Engineered KcsA channels/Epx2 pore was reconstituted in (+36)-GFP-spMSPlDl nanodiscs and added into a planar lipid bilayer system. These nanodiscs rapidly fused with the planar lipid bilayer and gave rise to stable channels as shown by electrical recordings. The observed pore sizes and dynamics are in line with previous studies using liposome-based approaches. The data suggest that correct functional states of membrane proteins is maintained in supercharged nanodiscs.

Example 3 - Delivery of membrane proteins onto cell surface using supercharged nanodiscs

[0058] Fusion of supercharged nanodiscs with target membranes was examined. Nanodiscs were prepared harboring NBD-PE and Rho-PE as described above. Fusion of these nanodiscs with HEK293T cells were evaluated by Fluorescence- activated cell sorting (FACs). Results showed that that the FRET efficiencies between NBD and Rho in nanodiscs were drastically decreased upon incubation with HEK293T cells at 37 °C. In contrast, cells incubated with nanodiscs at 4 °C still exhibited significant FRET, indicating that nanodiscs were bound to cell surface but unable to fuse. Moreover, the FRET efficiency on single cells after fusion using fluorescence life-time measurement on a con-focal microscope was examined. The data demonstrated that the lifetime of NBD-PE and Rho-PE were drastically changed, showing the disappearance of FRET over the time course of fusion, in agreement with the FACs measurements.

[0059] A study was performed to confirm that ion channels can be delivered onto cell surface through membrane fusion by supercharged nanodiscs. A synthetic ion channel was incorporated into nanodiscs and incubated with cells. After allowing fusion to take place, the passage of potassium ions was monitored to confirm that KcsA channels were inserted into target cells. The results showed robust potassium currents as detected using optical and electrical measurements. Moreover, supercharged nanodiscs are much less toxic than LNPs formed using the positively charged lipid DOTAP that caused profound cell death and deformation, by which patch-clamp recordings of the plasma membrane after the delivery of ion channels cannot be successfully performed.

[0060] The nanodiscs described herein were also useful for delivering lipids and receptors. Using supercharged nanodiscs, cholesterol was rapidly delivered onto neurons and caused the aggregation of amyloid precursor proteins on the plasma membrane. In addition, cell adhesion GPCRs were delivered onto cultured neurons and rapidly induced synapse formation. Thus, the results suggest that supercharged nanodiscs are appropriate vehicles to deliver a spectrum of payloads onto the targeted cell membranes.

Example 4 - Potentiation of mRN A delivery using supercharged nanodiscs

[0061] (+36)-GFP-spMSPlDl was incubated with nanodiscs encapsulating Luciferase (Luc) mRNA, and the efficacy of delivery was evaluated by quantifying the expression of Luc. The expression of Luc in HEK293T and Jeko cells was increased by 2-4 fold in the presence of (+36)-GFP-spMSPlDl. Interestingly, the enhancement by (+36)-GFP-spMSPlDl exhibited an effect, as higher concentrations showed much lower stimulation. Detailed characterizations showed that (+36)-GFP-spMSPlDl at high protein concentrations caused aggregation of nanodiscs and might also disrupt their stability in vivo, thus inhibiting the efficacy of mRNA delivery.

[0062] The increased mR A-nanodisc delivery by (+36)-GFP-spMSPlDl could either increase endocytic uptake or mRNA release into the cytoplasm. To examine these possibilities, nanodiscs were loaded with Dil dye and their uptake into HEK293T cells was monitored using FACs. The results showed that the stimulation effect in mRNA expression exceed the increased uptake of Dil-labeled nanodiscs. Thus, supercharged nanodiscs promote mRNA delivery by facilitating both endocytic uptake and payload release.

[0063] Finally, to determine whether supercharged nanodiscs can stimulate mRNA delivery in animal models, nanodiscs harboring Luc mRNAs together with (+36)-GFP-spMSPlDl were injected into Balb/C mice. The results showed a 2-fold increase in the expression of Luc in mice. Without wishing to be bound by any particular theory, it is believed that, because of the faster fusion with the nearby cells, the expression of Luc mRNAs in the presence of (+36)-GFP- spMSPlDl is much more specific in muscle cells than using nanodiscs without a supercharged protein. The utility of (+36)-GFP-spMSPlDl for mRNA vaccines was tested and the stimulation in bNAb expression was observed. The results suggest that supercharged MSPs are also useful for potentiating in vivo mRNA delivery.

Discussion

[0064] The present disclosure demonstrates the development of supercharged LNPs through the engineering of MSPs. Without wishing to be bound by any particular theory, these LNPs are highly fusogenic by virtue of their electrostatic interactions with phospholipids. Furthermore, supercharged LNPs of the disclosure can rapidly deliver membrane proteins onto the cell surface and potentiate the release of mRNAs by hijacking membrane fusion. Additionally, supercharged LNPs do not disrupt the targeted membranes, which is an advantage over other approaches resulting in leakage and/or lysis. Membrane leakage using the (+36)-GFP-MSPlDl protein was not observed even at 10 pM - 30 pM concentrations. Further, the present disclosure demonstrates the utility of these LNPs for several distinct families of membrane protein complexes and macromolecules with different structures and chemical properties.

[0065] The high efficiency of supercharged LNPs in fusing with cell surface is useful for membrane engineering. For example, synthetic membrane proteins can now be rapidly delivered onto the target cells for reprogramming. The present disclosure demonstrates the advantage of rapidly delivering synthetic membrane proteins onto the target cells using synthetic ion channels. These channels are beneficial for probing the mechanism of channel gating in cell-free systems. However, it was generally not feasible to characterize these channels in cells because they are chemically synthesized in vitro. The approach described herein allows characterization not previously available. The small amount of lipids in the supercharged LNPs and the nanoscale transient fusion pores leave little interference to the targeted membranes and cellular homeostasis, much less toxic than previous DOTAP-bearing liposome methods. In addition, the delivery of many other non-natural existing molecules onto the cell surface can be imaged directly, thereby allowing for orthogonal investigations using the vast chemical space untapped for cell biology in previous studies. [0066] Another representative use of supercharge LNPs described herein is mRNA delivery. The findings described herein suggest that the amphipathic helixes of, e.g., ApoAl can rapidly associate with LNPs and reprogram their interactions with cells. The increased mRNA delivery by the supercharged LNPs manifests accelerated membrane fusion at the cell surface and endosomes.

[0067] All references, including publications, patent applications, and patents, cited herein are hereby incorporated by reference to the same extent as if each reference were individually and specifically indicated to be incorporated by reference and were set forth in its entirety herein.

[0068] As used herein, the singular forms "a," "and," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a lipid nanoparticle (LNP)" includes a plurality of lipid nanoparticles (LNPs) and equivalents thereof known to those skilled in the art, and so forth. The term “about” signifies not more or less than 10 percent of the stipulated amount. Thus, a diameter of about 11 nm may be interpreted to be inclusive of 9.9 nm to 12.1 nm. It should be understood that, while various embodiments in the specification are presented using “comprising” language, under various circumstances, a related embodiment may also be described using “consisting of’ or “consisting essentially of’ language. The disclosure contemplates embodiments described as “comprising” a feature to include embodiments which “consist of’ or “consist essentially of’ the feature. The use of any and all examples, or exemplary language (e.g., "such as") provided herein, is intended merely to better illuminate the disclosure and does not pose a limitation on the scope of the disclosure unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the disclosure.

[0069] It should also be understood that when describing a range of values, the disclosure contemplates individual values found within the range. In any of the ranges described herein, the endpoints of the range are included in the range. However, the description also contemplates the same ranges in which the lower and/or the higher endpoint is excluded.

[0070] As will be apparent to those of skill in the art upon reading this disclosure, each of the individual aspects described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any of the other several aspects. Any recited method can be carried out in the order of steps recited or in any other order which is logically possible. This is intended to provide support for all such combinations.

[0071] Preferred embodiments of this disclosure are described herein. Variations of those preferred embodiments may become apparent to those of ordinary skill in the art upon reading the foregoing description. This disclosure includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the disclosure unless otherwise indicated herein or otherwise clearly contradicted by context. Also, only such limitations which are described herein as critical to the invention should be viewed as such; variations of the invention lacking limitations which have not been described herein as critical are intended as aspects of the invention.

Claims

CLAIMS What is claimed is:

1. A nanodisc comprising a lipid bilayer encircled by at least one membrane scaffold protein (MSP) linked at the N- and C-termini and conjugated to a supercharged protein located exterior to the lipid bilayer.

2. The nanodisc of claim 1, wherein the termini of the MSP and the supercharged protein are irreversibly conjugated.

3. The nanodisc of claim 1 or 2, wherein the termini of the MSP and the supercharged protein are conjugated via a connector.

4. The nanodisc of claim 3, wherein the connector utilizes a Catcher-Tag system.

5. The nanodisc of any one of claims 1-4, wherein the supercharged protein is conjugated to the MSP via a SpyTag/SpyCatcher system fused at each terminus of the MSP.

6. The nanodisc of any one of claims 1-5, wherein the MSP is derived from apolipoprotein.

7. The nanodisc of claim 6, wherein the MSP is apolipoprotein Al or a derivative thereof.

8. The nanodisc of any one of claims 1-7, wherein the supercharged protein is a positively charged protein.

9. The nanodisc of claim 8, wherein the supercharged protein has a net charge of at least +30 at pH 7.4.

10. The nanodisc of claim 8 or 9, wherein the supercharged protein is a positively charged green fluorescent protein (GFP).

11 . The nanodisc of claim 10, wherein the supercharged protein is a monomeric GFP variant with a net charge of about +36 at pH of about 7.4.

12. The nanodisc of claim 10 or 11, wherein the supercharged protein is a positively charged GFP is derived from Aequorea victoria.

13. The nanodisc of any one of claims 10-12, wherein the supercharged protein comprises an amino acid sequence comprising at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1.

14. The nanodisc of any one of claims 1-13, which is about 11 nm or about 30 nm in diameter.

15. The nanodisc of any one of claims 1-14, further comprising a pay load within the lipid bilayer.

16. The nanodisc of claim 15, wherein the payload is a protein, a nucleic acid, a small molecule drug, or any combination thereof.

17. The nanodisc of any one of claims 1-16, wherein the efficiency of payload delivery mediated the nanodisc is at least 100-fold higher compared to nanodiscs which do not comprise supercharged GFP based on cell-free and/or cell-based assays.

18. A method of delivering a pay load into a cell, the method comprising contacting the cell with the nanodisc of any one of claims 1-17, thereby delivering the payload to the cell.

19. The method of claim 18, wherein the payload is a cell membrane protein.