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WO2024187152A1 - Nanodiscs for use in removing lipid accumulation in the eye - Google Patents

Nanodiscs for use in removing lipid accumulation in the eye Download PDF

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Publication number
WO2024187152A1
WO2024187152A1 PCT/US2024/019239 US2024019239W WO2024187152A1 WO 2024187152 A1 WO2024187152 A1 WO 2024187152A1 US 2024019239 W US2024019239 W US 2024019239W WO 2024187152 A1 WO2024187152 A1 WO 2024187152A1
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WO
WIPO (PCT)
Prior art keywords
msp
phospholipid
pharmaceutical composition
dmpc
molar ratio
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
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PCT/US2024/019239
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French (fr)
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WO2024187152A9 (en
Inventor
Nehal N. MEHTA
Stephen G. Sligar
Mark A. Mclean
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Mobius Scientific Inc
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Mobius Scientific Inc
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Filing date
Publication date
Application filed by Mobius Scientific Inc filed Critical Mobius Scientific Inc
Priority to AU2024231998A priority Critical patent/AU2024231998A1/en
Priority to CN202480029998.XA priority patent/CN121079102A/en
Publication of WO2024187152A1 publication Critical patent/WO2024187152A1/en
Publication of WO2024187152A9 publication Critical patent/WO2024187152A9/en
Priority to TW113148821A priority patent/TW202532081A/en
Priority to PCT/US2024/060079 priority patent/WO2025129028A2/en
Anticipated expiration legal-status Critical
Pending legal-status Critical Current

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Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/1703—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • A61K38/1709—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0048—Eye, e.g. artificial tears
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/127—Synthetic bilayered vehicles, e.g. liposomes or liposomes with cholesterol as the only non-phosphatidyl surfactant
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P27/00—Drugs for disorders of the senses
    • A61P27/02—Ophthalmic agents

Definitions

  • the present disclosure generally relates to methods of removing lipid (e.g., cholesterol) or preventing lipid accumulation in the eye of an individual (e.g., human) and methods of preventing or treating an eye disease characterized by lipid accumulation in an individual.
  • the present disclosure also provides pharmaceutical compositions comprising a plurality of nanodiscs, and methods of using and making thereof.
  • Nanodiscs comprising a membrane scaffold protein (MSP) and a phospholipid, provide a native-like lipid environment for the incorporation of membrane proteins, and they have become a valuable platform for the study of membrane biophysics and for therapeutical applications.
  • MSP membrane scaffold protein
  • lipid e.g., cholesterol
  • AMD age-related macular degeneration
  • a method of removing lipid or preventing lipid accumulation in the eye of an individual comprising administering to the eye of the individual an effective amount of a pharmaceutical composition comprising a plurality of nanodiscs, wherein the nanodisc comprises a membrane scaffold protein (MSP) and a phospholipid.
  • MSP membrane scaffold protein
  • a method of preventing or treating an eye disease characterized by lipid accumulation in an individual comprising administering to the eye of the individual an effective amount of a pharmaceutical composition comprising a plurality of nanodiscs, wherein the nanodisc comprises an MSP and a phospholipid.
  • the MSP is selected from the group consisting of apolipoprotein A-I (ApoA-I), ApoA-II, ApoC, ApoE, ApoM, and a variant thereof. In some embodiments, the MSP is a variant of ApoA-I. In some embodiments, the MSP comprises one or more amphipathic helix sequences derived from ApoA-I. In some embodiments, the MSP comprises an N-terminus truncation relative to ApoA-I.
  • the MSP is selected from the group consisting of MSP1E1, MSP1E2, MSP1E3, MSP1, MSP1D1, MSP1E1D1, MSP1E2D1, MSP1E3D1, MSP2N2, and N-terminal His-tag portion removed variant thereof (His-tag removed variant).
  • the MSP is: (i) an MSP1D1 His-tag removed variant comprising the amino acid sequence of SEQ ID NO: 14; or (ii) an MSP1E3D1 His-tag removed variant comprising the amino acid sequence of SEQ ID NO: 15.
  • the phospholipid comprises DMPC, DPPC, DMPS, DSPC, POPC, POPS, PiP2, or a combination thereof.
  • the phospholipid is a phosphatidylcholine (PC).
  • the phospholipid is selected from the group consisting of DMPC, POPC, DPPC, DSPC, and a combination thereof.
  • at least about 85% of the plurality of nanodiscs in the pharmaceutical composition are fully-lipidated. In some embodiments, at least about 85% of the plurality of nanodiscs in the pharmaceutical composition are under-lipidated.
  • the molar ratio of the phospholipid to the MSP in the pharmaceutical composition is less than a phospholipid-MSP saturation ratio NS. In some embodiments, the molar ratio of the phospholipid to the MSP in the pharmaceutical composition is less than about 95% of the phospholipid-MSP saturation ratio N S . [0011] In some embodiments, the MSP is MSP1D1, MSP1, or His-tag removed variant thereof, and the molar ratio of the phospholipid to the MSP in the pharmaceutical composition is from about 10:1 to about 90:1.
  • the MSP is MSP1D1, MSP1, or His-tag removed variant thereof, wherein the phospholipid is POPC, and the molar ratio of POPC to the MSP in the pharmaceutical composition is from about 10:1 to about 65:1. In some embodiments, the MSP is MSP1D1 or His-tag removed variant thereof, and the molar ratio of POPC to the MSP in the pharmaceutical composition is about 65:1. In some embodiments, the MSP is MSP1D1 or His-tag removed variant thereof, the phospholipid is POPC, and the NS is about 62. In some embodiments, the molar ratio of POPC to the MSP in the pharmaceutical composition is less than about 58:1.
  • the molar ratio of POPC to the MSP in the pharmaceutical composition is about 50:1 or about 32.5:1.
  • the MSP is MSP1D1, MSP1, or His-tag removed variant thereof, wherein the phospholipid is DMPC, and the molar ratio of DMPC to the MSP in the pharmaceutical composition is from about 10:1 to about 85:1.
  • the MSP is MSP1D1 or His-tag removed variant thereof, the phospholipid is DMPC, and the molar ratio of DMPC to the MSP in the pharmaceutical composition is about 85:1.
  • the MSP is MSP1D1 or His-tag removed variant thereof, the phospholipid is DMPC, and the N S is about 81.
  • the molar ratio of DMPC to the MSP in the pharmaceutical composition is less than about 78:1.
  • the molar ratio of DMPC to the MSP in the pharmaceutical composition is about 65:1 to about 75:1.
  • the molar ratio of DMPC to the MSP in the pharmaceutical composition is from about 40:1 to about 60:1.
  • the molar ratio of DMPC to the MSP in the pharmaceutical composition is about 46:1 to about 55:1.
  • the MSP is MSP1 or His-tag removed variant thereof, and the molar ratio of DMPC to the MSP is about 80:1.
  • the MSP is MSP1D1, MSP1, or His-tag removed variant thereof, the phospholipid is DPPC, and the molar ratio of DPPC to the MSP in the ny-2692819 Attorney Docket No.: 283912000140 pharmaceutical composition is from about 10:1 to about 90:1.
  • the MSP is MSP1 or His-tag removed variant thereof, the phospholipid is DPPC, and the molar ratio of DPPC to the MSP in the pharmaceutical composition is about 90:1.
  • the MSP is MSP1D1 or His-tag removed variant thereof, the phospholipid is DPPC, and the N S is about 81. In some embodiments, the molar ratio of DPPC to the MSP in the pharmaceutical composition is less than about 75:1. [0014] In some embodiments, the MSP is MSP1E3D1, MSP1E3, or His-tag removed variant thereof, and the molar ratio of the phospholipid to the MSP in the pharmaceutical composition is from about 50:1 to about 200:1. In some embodiments, the MSP is MSP1E3 or His-tag removed variant thereof, wherein the phospholipid is POPC, and the molar ratio of POPC to the MSP in the pharmaceutical composition is about 130:1.
  • the MSP is MSP1E3D1 or His-tag removed variant thereof, the phospholipid is POPC, and the NS is about 126. In some embodiments, the molar ratio of POPC to the MSP in the pharmaceutical composition is less than about 118:1. In some embodiments, the MSP is MSP1E3D1 or His-tag removed variant thereof, the phospholipid is DMPC, and the NS is about 167. In some embodiments, the molar ratio of DMPC to the MSP in the pharmaceutical composition is less than about 158:1. In some embodiments, the molar ratio of DMPC to the MSP in the pharmaceutical composition is from about 50:1 to about 160:1.
  • the molar ratio of DMPC to the MSP in the pharmaceutical composition is from about 50:1 to about 100:1. In some embodiments, the molar ratio of DMPC to the MSP in the pharmaceutical composition is about 87:1 to about 96:1, about 58:1 to about 68:1, about 63:1, or about 62:1. In some embodiments, the MSP is MSP1E3 or His-tag removed variant thereof, the phospholipid is DMPC, and the molar ratio of DMPC to the MSP in the pharmaceutical composition is about 150:1.
  • the MSP is MSP1E3 or His-tag removed variant thereof, the phospholipid is DPPC, and the molar ratio of DPPC to the MSP in the pharmaceutical composition is about 170:1.
  • the MSP is MSP1E3D1 or His-tag removed variant thereof, the phospholipid is DPPC, and the N S is about 167.
  • the molar ratio of DPPC to the MSP in the pharmaceutical composition is less than about 158:1.
  • the MSP is MSP1E1D1 or His-tag removed variant thereof, the phospholipid is DMPC, and the NS is about 107.
  • the molar ratio of DMPC to the MSP in the pharmaceutical composition is less than about 100:1. In some ny-2692819 Attorney Docket No.: 283912000140 embodiments, the molar ratio of DMPC to the MSP in the pharmaceutical composition is from about 60:1 to about 65:1. [0016] In some embodiments, the nanodisc in the pharmaceutical composition is about 2 nm to about 20 nm in diameter. In some embodiments, the lipid for removal or prevention from accumulation in the eye is cholesterol. In some embodiments, the nanodisc does not comprise: (i) a cell-penetrating peptide (CPP); (ii) a target protein; and/or (iii) a prophylactic or therapeutic agent.
  • CPP cell-penetrating peptide
  • target protein a target protein
  • prophylactic or therapeutic agent a prophylactic or therapeutic agent
  • the nanodisc increases cholesterol efflux rate by at least about 2- fold compared to an untreated state, or the cholesterol efflux rate by ApoA-I or the MSP.
  • the pharmaceutical composition is administered via intravitreal injection.
  • the eye disease characterized by lipid accumulation is selected from the group consisting of age-related macular degeneration (AMD), photoreceptor neurodegeneration, optic nerve atrophy, loss of acuity, hemianopia, visual agnosia, strabismus, retinal neurovascular disorder, lipid keratopathy, corneal lipidosis, and a combination thereof.
  • AMD age-related macular degeneration
  • the AMD is dry AMD.
  • the dry AMD is geographic atrophy (GA).
  • the nanodisc reduces lipid or prevents lipid accumulation in the eye of the individual by at least about 5%.
  • FIG. 1 displays an exemplary chromatogram of nanodisc preparation.
  • FIG. 2A displays a chromatogram of the formation of POPC MSP1D1 nanodiscs at different POPC to MSP1D1 ratios. The results indicate that a ratio of 65:1 forms a fully lipidated nanodisc.
  • FIG. 1 displays an exemplary chromatogram of nanodisc preparation.
  • FIG. 2A displays a chromatogram of the formation of POPC MSP1D1 nanodiscs at different POPC to MSP1D1 ratios. The results indicate that a ratio of 65:1 forms a fully lipidated nanodisc.
  • FIG. 3 shows the percent of cholesterol efflux from macrophages in vitro using various nanodiscs with different lipid to MSP ratios.
  • Lipid A refers to DMPC
  • Lipid B refers to POPC.
  • FIGs. 5A-5C shows the percent of cholesterol efflux from human retinal pigment epithelial (RPE) cells in vitro using a nanodisc with POPC to MSP1D1 ratio of 65:1 (MSP1D1 Lipid B), a nanodisc DMPC to MSP1D1 ratio of 85:1 (MSP1D1 Lipid A), and a MSP1D1 protein alone after 2 hours (FIG. 5A), 4 hours (FIG.
  • RPE retinal pigment epithelial
  • FIG. 6A and FIG.6B show fluorescence images following intravitreal delivery of a GFP-labeled exemplary nanodisc into the murine eye in vivo. From left to right, FIG.6A shows fluorescence images at 15 minutes, 30 minutes, 60 minutes, 6 hours, and 24 hours after delivery of a GFP-labeled exemplary nanodisc into the murine eye in vivo. The first row in FIG.6B, from left to right, shows fluorescence images at 7 minutes, 7 minutes, 10 minutes, 15 minutes, and 20 minutes after delivery of a GFP-labeled exemplary nanodisc in to the murine eye. The second row in FIG.
  • FIG. 7A is a schematic depicting schedule for intravitreal injection of fluorescent labeled exemplary nanodisc and tissue harvest.
  • FIG.7B shows representative images of immunostained retina sections taken from injected mice depicting retinal neurons (solid arrows)and fluorescent labeled exemplary nanodisc (blank arrows).
  • FIG.7B shows fluorescence images at 0 minutes, 15 minutes, 30 minutes, 1 hour, 6 hours and 24 hours after delivery of a fluorescent-labelled exemplary nanodisc in to the murine eye.
  • FIG.7B shows the fluorescence signals at retinal pigment epithelium (RPE), outer nuclear layer (ONL), and inner nuclear layer (INL).
  • RPE retinal pigment epithelium
  • ONL outer nuclear layer
  • INL inner nuclear layer
  • FIG.8A is a schematic depicting schedule for laser injury-induced choroidal neovascularization (CNV), intravitreal injection of the exemplary nanodisc, intravenous injection ny-2692819 Attorney Docket No.: 283912000140 of FITC Dextran, and tissue harvest.
  • CNV laser injury-induced choroidal neovascularization
  • FIG.8B shows representative confocal image of CNV area labeled by FITC Dextran and outlined in a circle following laser injury in the mouse.
  • FIG.9A shows a schematic depicting schedule for high-fat diet administration, intravitreal injections, baseline (T0) and follow-up (T1) imaging and tissue harvest for Abca1/g1-rod/-rod mice.
  • FIG.9A shows a schematic depicting schedule for high-fat diet administration, intravitreal injections, baseline (T0) and follow-up (T1) imaging and tissue harvest for Abca1/g1-rod/-rod mice.
  • FIG.9D shows representative images of hematoxylin and eosin-stained (H&E) sections of whole eyes (left) and retinas (right).
  • FIG.9E shows representative images of immunostained retina sections showing retinal neuron (solid arrows) and complement-3 (blank arrows) expression in the central (left) and mid-peripheral (right) retina.
  • FIG.9F shows representative baseline (top) and follow-up (bottom) optical coherence tomography (OCT) images of living mouse retina cross section showing incidence of (Vehicle) and absence of (Disc) inner/outer segment abnormalities (arrowhead in the bottom left image) and retinal pigment epithelium detachements (arrowhead in the bottom right image) in reference to baseline (asterisk and arrowhead in the top images).
  • OCT optical coherence tomography
  • FIG.10A shows a schematic depicting schedule for high-fat diet administration, intravitreal injections, baseline (T0) and follow-up (T1) imaging and blood collection, and tissue harvest for Abca1/g1-rod/-rod mice.
  • FIG.10B and FIG.10C show line graphs showing full- field scotopic (FIG.10B) and light bleach recovery (FIG.10C) response amplitudes and ratios in vehicle (‘Vehicle’) and the exemplary nanodisc-treated (‘Disc’) mouse groups.
  • the y-axis shows Amplitude ( ⁇ V).
  • FIG.10A shows a schematic depicting schedule for high-fat diet administration, intravitreal injections, baseline (T0) and follow-up (T1) imaging and blood collection, and tissue harvest for Abca1/g1-rod/-rod mice.
  • FIG.10B and FIG.10C show line graphs showing
  • FIG.11A shows one chromatogram of preparative size exclusion chromatography (SEC) of the exemplary DMPC/MSP1D1(-) nanodisc prepared at 30:1 ratio.
  • SEC preparative size exclusion chromatography
  • FIG.11B shows one chromatogram of preparative SEC of an exemplary DMPC/MSP1E3D1(-) nanodisc prepared at 50:1 ratio. A single highlighted peak (upper panel) was collected, which corresponds to an under-lipidated nanodisc with about 62 lipids per MSP1E3D1(-).
  • FIG.12 shows gas chromatography (GC) result for determining phospholipid to MSP ratio of the exemplary DMPC/MSP1D1(-) nanodisc prepared at 30:1 ratio.
  • 13A-13D show the correspondence between the phospholipid/MSP1D1(-) ratio in the preparation mixture and the final nanodisc for DMPC/MSP1D1(-) about 81:1 nanodisc (preparation mixture 80:1; final nanodisc 81 ⁇ 3 : 1) (FIG.13A), DMPC/MSP1D1(-) nanodisc about 75:1 (preparation mixture 55:1; final nanodisc 75 ⁇ 1.5 :1) (FIG.
  • FIGs. 13B DMPC/MSP1D1(-) about 51:1 nanodisc (preparation mixture 40:1; final nanodisc 51 ⁇ 1 :1) (FIG.13C), and DMPC/MSP1D1(-) about 51:1 nanodisc (preparation mixture 30:1; final nanodisc 51 ⁇ 1 :1) (FIG. 13D).
  • FIG. 14A-14C show the correspondence between the phospholipid/MSP1E3D1(-) ratio in the preparation mixture and the final nanodisc for DMPC/MSP1E3D1(-) about 93:1 nanodisc (preparation mixture 90:1; final nanodisc 93 ⁇ 3 :1) (FIG.14A), DMPC/MSP1E3D1(-) about 63:1 nanodisc (preparation mixture 60:1; final nanodisc 63 ⁇ 4 :1) (FIG.14B), and DMPC/MSP1E3D1(-) about 62:1 nanodisc (preparation mixture 50:1; final composition 62 ⁇ 0.5 :1) (FIG.14C). [0033] FIG.
  • 15A shows the cholesterol efflux assay results using various under-lipidated nanodiscs.
  • the phospholipid to MSP ratios were measured from the final nanodiscs (not preparation mixture).2.5 ⁇ g of samples were used.
  • (-) denotes the removal of the N-terminal His tag.
  • ny-2692819 Attorney Docket No.: 283912000140
  • FIG. 16 shows the SEC chromatogram of exemplary under-lipidated DMPC/MSP1D1(-) nanodiscs prepared at 30:1 ratio (final composition ratio was about 51:1) after freezing and thawing without cryoprotectant, compared to before-freezing. [0035] FIG.
  • 17 shows the cholesterol efflux results of exemplary under-lipidated DMPC:MSP1D1(-) nanodiscs prepared at 30:1 ratio (final ratio in the nanodiscs was about 51:1) before and after freezing and thawing, in comparison with controls including fully-lipidated DMPC:MSP1D1 nanodiscs prepared at 85:1 (final composition ratio was 83:1), HDL, and PBS solution.
  • the present application provides a method of removing lipid (e.g., cholesterol) or preventing lipid accumulation in the eye of an individual (e.g., human), and/or preventing or treating an eye disease characterized by lipid accumulation in an individual, comprising administering to the eye of the individual an effective amount of a pharmaceutical composition comprising a nanodisc (e.g., a plurality of nanodiscs), wherein the nanodisc comprises a membrane scaffold protein (MSP) and a phospholipid.
  • MSP membrane scaffold protein
  • the present invention is based, at least in part, on the surprising discovery that nanodiscs described herein i) are able to reach the posterior segment of the eye with excellent penetrance, ii) have good PK properties and do not dwell in the eye for an extended period of time (e.g., >24 hours); iii) can effectively remove lipids; iv) do not require the assistance of any targeting moiety or cell penetrating peptide (CPP); and v) is both effective and safe in treating eye disease characterized by lipid accumulation (e.g., age-related macular degeneration (AMD)) in vivo.
  • ALD age-related macular degeneration
  • the nanodiscs and methods described herein are therefore particularly helpful in treating a broad range of eye diseases characterized by lipid accumulation, including macular degeneration and atrophy in the eye.
  • the singular forms “a,” “an,” and “the” include the plural referents unless the context clearly indicates otherwise.
  • the term “about” indicates and encompasses an indicated value and a range above and below that value. In certain embodiments, the term “about” indicates the designated ny-2692819 Attorney Docket No.: 283912000140 value ⁇ 10%, ⁇ 5%, or ⁇ 1%. In certain embodiments, the term “about” indicates the designated value ⁇ one standard deviation of that value.
  • phrases “effective amount” and “pharmaceutically effective amount” refer to a sufficient amount of an agent to provide the desired biological result. That result can be reduction (e.g., reducing at least about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or 100%) and/or alleviation of the signs, symptoms, or causes of a disease or disorder, or any other desired alteration of a biological system.
  • An appropriate effective amount in any individual case may be determined by one of ordinary skill in the art using routine experimentation.
  • the terms “patient,” “subject,” “individual,” and the like are used interchangeably herein, and refer to any animal, in some embodiments a mammal, and in some embodiments a human, having a complement system, including a human in need of therapy for, or susceptible to, a condition or its sequelae.
  • the individual may include, for example, dogs, cats, pigs, cows, sheep, goats, horses, rats, rabbits, hamsters, guinea pigs, monkeys, mice, and humans.
  • the individual is a human.
  • treatment typically refers to clinical intervention designed to alter the natural course of the individual or cell being treated during the course of clinical pathology.
  • Desirable effects of treatment include decreasing the rate of disease progression, ameliorating or palliating the disease state, and remission or improved prognosis.
  • an individual is successfully “treated” if one or more symptoms associated with disease or disorder are mitigated or eliminated, including, but not limited to, decreasing the frequency and/or severity of a sign and/or symptoms resulting from the disease, increasing the quality of life of those suffering from the disease, decreasing the dose of other medications required to treat the disease, and/or prolonging survival of individuals.
  • Treatment may be prophylactic (to prevent or delay the onset of the disease, or to prevent the manifestation of clinical or subclinical symptoms thereof) or therapeutic suppression or alleviation of symptoms after the manifestation of the disease.
  • percent (%) amino acid sequence identity and “homology” with respect to a peptide, polypeptide or antibody sequence are defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the specific peptide or polypeptide sequence, after aligning the sequences and introducing gaps, if ny-2692819 Attorney Docket No.: 283912000140 necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity.
  • Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN or MEGALIGN TM (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared.
  • “amphipathic” molecules refer to molecules that have both hydrophilic and hydrophobic properties. Examples include phospholipids, cholesterol, glycolipids and fatty acids.
  • embodiments of the invention described herein include “consisting of” and/or “consisting essentially of” embodiments. II.
  • lipid e.g., cholesterol
  • lipid accumulation in the eye of an individual e.g., human
  • administering to the eye of the individual an effective amount of a pharmaceutical composition comprising a nanodisc, wherein the nanodisc comprises an MSP and a phospholipid.
  • a method of removing lipid or preventing lipid accumulation in the eye of an individual comprising administering to the eye of the individual an effective amount of a pharmaceutical composition comprising a plurality of nanodiscs, wherein the nanodisc comprises a membrane scaffold protein (MSP) and a phospholipid.
  • MSP membrane scaffold protein
  • the MSP is a variant of ApoA-I.
  • the MSP is selected from the group consisting of MSP1E1, MSP1E2, MSP1E3, MSP1, MSP1D1, MSP1E1D1, MSP1E2D1, MSP1E3D1, MSP2N2, and N-terminal His-tag portion removed variant thereof (His-tag removed variant).
  • the MSP is selected from the group consisting of MSP1E1, MSP1E2, MSP1E3, MSP1, MSP1D1, MSP1D2, and MSPE3D1.
  • the MSP is (i) an MSP1D1 His-tag removed variant comprising the amino acid sequence of SEQ ID NO: 14; or (ii) an MSP1E3D1 His-tag removed variant comprising the amino acid sequence of SEQ ID NO: ny-2692819 Attorney Docket No.: 283912000140 15.
  • the phospholipid comprises DMPC, DPPC, DMPS, DSPC, POPC, POPS, PiP2, or a combination thereof.
  • the phospholipid is a PC, such as selected from the group consisting of DMPC, POPC, DPPC, DSPC, and a combination thereof.
  • a method of removing lipid or preventing lipid accumulation in the eye of an individual comprising administering to the eye of the individual an effective amount of a pharmaceutical composition comprising a plurality of nanodiscs, wherein at least about 85% of the plurality of nanodiscs in the pharmaceutical composition are fully-lipidated.
  • a method of removing lipid or preventing lipid accumulation in the eye of an individual comprising administering to the eye of the individual an effective amount of a pharmaceutical composition comprising a plurality of nanodiscs, wherein at least about 85% of the plurality of nanodiscs in the pharmaceutical composition are under-lipidated.
  • the molar ratio of the phospholipid to the MSP in the pharmaceutical composition is less than a phospholipid-MSP saturation ratio N S .
  • the molar ratio of the phospholipid to the MSP in the pharmaceutical composition is less than about 95% of the phospholipid-MSP saturation ratio NS.
  • the MSP is MSP1D1, MSP1, or His-tag removed variant thereof, and the molar ratio of the phospholipid to the MSP in the pharmaceutical composition is from about 10:1 to about 90:1. In some embodiments, the MSP is MSP1D1 or MSP1, and the molar ratio of phospholipid to MSP is from about 10:1 to about 90:1. In some embodiments, the MSP is MSP1D1, MSP1, or His-tag removed variant thereof, the phospholipid is POPC, and the molar ratio of POPC to the MSP in the pharmaceutical composition is from about 10:1 to about 65:1.
  • the MSP is MSP1D1 or MSP1
  • the phospholipid is POPC
  • the molar ratio of POPC to MSP1D1 or MSP1 is from about 10:1 to about 65:1.
  • the MSP is MSP1D1
  • the phospholipid is POPC
  • the molar ratio of POPC to MSP1D1 is about 50:1 or about 32.5:1.
  • the MSP is MSP1 or MSP1D1
  • the phospholipid is POPC
  • the molar ratio of POPC to MSP1 or MSP1D1 is about 65:1.
  • the MSP is MSP1D1 or His-tag removed variant thereof, the phospholipid is POPC, and the molar ratio of POPC to the MSP in the pharmaceutical composition is about 65:1.
  • the MSP is MSP1D1 or His-tag removed variant thereof, the phospholipid is POPC, and the NS is about 62.
  • the MSP is MSP1D1 or His-tag removed variant thereof, the phospholipid is POPC, the molar ratio of POPC to MSP1D1 ny-2692819 Attorney Docket No.: 283912000140 or His-tag removed variant thereof in the pharmaceutical composition is less than about 58:1.
  • the MSP is MSP1D1 or His-tag removed variant thereof
  • the phospholipid is POPC
  • the molar ratio of POPC to MSP1D1 or His-tag removed variant thereof in the pharmaceutical composition is about 50:1 or about 32.5:1.
  • the MSP is MSP1D1, MSP1, or His-tag removed variant thereof
  • the phospholipid is DMPC, wherein the molar ratio of DMPC to the MSP in the pharmaceutical composition is from about 10:1 to about 85:1.
  • the MSP is MSP1D1 or MSP1
  • the phospholipid is DMPC
  • the molar ratio of DMPC to MSP1D1 or MSP1 is from about 10:1 to about 85:1.
  • the MSP is MSP1D1 or His-tag removed variant thereof, the phospholipid is DMPC, and the molar ratio of DMPC to the MSP in the pharmaceutical composition is about 85:1.
  • the MSP is MSP1D1
  • the phospholipid is DMPC
  • the molar ratio of DMPC to MSP1D1 is about 85:1.
  • the molar ratio of DMPC to MSP1D1 in the pharmaceutical composition is about 30:1 to about 80:1, such as any of about 40:1 to about 60:1, about 46:1 to about 55:1, about 51:1, about 55:1, or about 50:1.
  • the MSP is MSP1D1 or His-tag removed variant thereof, the phospholipid is DMPC, and the NS is about 81.
  • the MSP is MSP1
  • the phospholipid is DMPC
  • the molar ratio of DMPC to MSP1 is about 80:1.
  • the MSP is MSP1D1 or His-tag removed variant thereof, the phospholipid is DMPC
  • the molar ratio of DMPC to the MSP in the pharmaceutical composition is from about 40:1 to about 60:1, such as about 46:1 to about 55:1, about 51:1, about 55:1, or about 50:1.
  • the MSP is MSP1D1, MSP1, or His-tag removed variant thereof, wherein the phospholipid is DPPC, and the molar ratio of DPPC to the MSP1D1, MSP1, or His-tag removed variant thereof in the pharmaceutical composition is from about 10:1 to about 90:1.
  • the MSP is MSP1 or His-tag removed variant thereof, the phospholipid is DPPC, and the molar ratio of DPPC to the MSP1D1, MSP1, or His-tag removed variant thereof in the pharmaceutical composition is about 90:1.
  • the MSP is MSP1D1 or His-tag removed variant thereof, the phospholipid is DPPC, and the N S is about 81.
  • the molar ratio of DPPC to the MSP1D1 or His-tag removed variant thereof in the pharmaceutical composition is less than about 75:1.
  • the MSP is MSP1
  • the phospholipid is DPPC
  • the molar ratio of DPPC to MSP1 is about 90:1.
  • ny-2692819 Attorney Docket No.: 283912000140 [0050]
  • the MSP is MSP1E3, and the molar ratio of phospholipid to MSP1E3 (e.g., in the nanodisc, or in the pharmaceutical composition) is from about 100:1 to about 200:1.
  • the phospholipid is POPC, the MSP is MSP1E3, and the molar ratio of POPC to MSP1E3 is about 130:1.
  • the phospholipid is DMPC, the MSP is MSP1E3, and the molar ratio of DMPC to MSP1E3 (e.g., in the nanodisc, or in the pharmaceutical composition) is about 150:1.
  • the phospholipid is DPPC, the MSP is MSP1E3, and the molar ratio of DPPC to MSP1E3 (e.g., in the nanodisc, or in the pharmaceutical composition) is about 170:1.
  • the MSP is MSP1E3D1 or His-tag removed variant thereof
  • the phospholipid is DMPC
  • the molar ratio of DMPC to the MSP in the pharmaceutical composition is about 87:1 to about 96:1, about 58:1 to about 68:1, about 63:1, or about 62:1.
  • the nanodisc comprises DMPC and MSP1D1, such as in a molar ratio of about 85:1.
  • the nanodisc comprises DMPC and MSP1D1, optionally wherein the molar ratio of DMPC to MSP1D1in the pharmaceutical composition is about 46:1 to about 55:1, about 51:1, about 55:1, or about 50:1.
  • a method of removing lipid (e.g., cholesterol) or preventing lipid accumulation in the eye of an individual (e.g., human), comprising administering to the eye of the individual an effective amount of a pharmaceutical composition comprising a plurality of nanodiscs, wherein the nanodisc comprises DMPC and MSP1D1, and the molar ratio of DMPC and MSP1D1 in the pharmaceutical composition is about 46:1 to about 55:1, about 51:1, about 55:1, or about 50:1.
  • the nanodisc comprises POPC and MSP1D1, such as in a molar ratio of about 65:1, about 50:1, or about 32.5:1.
  • a method of removing lipid (e.g., cholesterol) or preventing lipid accumulation in the eye of an individual (e.g., human), comprising administering to the eye of the individual an effective amount of a pharmaceutical composition comprising a nanodisc, wherein the nanodisc comprises DMPC and MSP1D1 at a molar ratio of about 85:1, about 51:1, about 55:1, or about 50:1.
  • the nanodisc comprises DMPC and MSP1E3D1 and the molar ratio of DMPC and MSP1E3D1 in the pharmaceutical composition is about 87:1 to about 96:1, about 58:1 to about 68:1, about 63:1, or about 62:1.
  • the molar ratio of DMPC to MSP1E3D1 in the pharmaceutical composition is about 60:1 to about 65:1.
  • a method of removing lipid (e.g., cholesterol) or preventing lipid accumulation in the eye of an individual (e.g., human) comprising administering to the eye of the individual an effective amount of a pharmaceutical composition comprising a plurality of nanodiscs, wherein the nanodisc comprises DMPC and MSP1E3D1, and the molar ratio of DMPC and MSP1D1E3 in the pharmaceutical composition is about 58:1 to about 68:1, about 63:1, or about 62:1.
  • a method of removing lipid (e.g., cholesterol) or preventing lipid accumulation in the eye of an individual (e.g., human), comprising administering to the eye of the individual an effective amount of a pharmaceutical composition comprising a plurality of nanodiscs, wherein the nanodisc comprises POPC and MSP1D1 (e.g., SEQ ID NO: 3) at a molar ratio of about 65:1, about 50:1, or about 32.5:1.
  • lipid e.g., cholesterol
  • MSP1D1 e.g., SEQ ID NO: 3
  • a method of removing lipid (e.g., cholesterol) or preventing lipid accumulation in the eye of an individual (e.g., human), comprising administering to the eye of the individual an effective amount of a pharmaceutical composition comprising a plurality of nanodiscs, wherein the nanodisc comprises DMPC and MSP1D1 (e.g., SEQ ID NO: 3) or His-tag removed variant thereof (e.g., SEQ ID NO: 14), and wherein the molar ratio of DMPC and MSP1D1 (e.g., SEQ ID NO: 3) or His-tag removed variant thereof (e.g., SEQ ID NO: 14) in the pharmaceutical composition is about 46:1 to about 55:1, about 51:1, about 55:1, or about 50:1.
  • DMPC and MSP1D1 e.g., SEQ ID NO: 3
  • His-tag removed variant thereof e.g., SEQ ID NO: 14
  • a method of removing lipid (e.g., cholesterol) or preventing lipid accumulation in the eye of an individual (e.g., human), comprising administering to the eye of the individual an effective amount of a pharmaceutical composition comprising a plurality of nanodiscs, wherein the nanodisc comprises DMPC and MSP1E3D1 (e.g., SEQ ID NO: 8) or His-tag removed variant thereof (e.g., SEQ ID NO: 15), and the molar ratio of DMPC and MSP1E3D1 (e.g., SEQ ID NO: 8) or His-tag removed variant thereof (e.g., SEQ ID NO: 15) in the pharmaceutical composition is about 58:1 to about 68:1, about 63:1, or about 62:1.
  • DMPC and MSP1E3D1 e.g., SEQ ID NO: 8
  • His-tag removed variant thereof e.g., SEQ ID NO: 15
  • the lipid for removal or prevention from accumulation in the eye is cholesterol.
  • the nanodisc is administered intravitreally.
  • the nanodisc is about 2 nm to about 20 nm, such as about 5 nm to about 20 nm or ny-2692819 Attorney Docket No.: 283912000140 about 10 nm to about 20 nm in diameter.
  • the nanodisc does not comprise a CPP.
  • the nanodisc does not comprise a target protein.
  • the nanodisc does not comprise a prophylactic or therapeutic agent.
  • the nanodisc increases cholesterol efflux rate by at least about 2-fold (e.g., at least about any of 2.5, 3, 4, 5, 10, 15-fold, or more) compared to an untreated state, or the cholesterol efflux rate by ApoA-I or the MSP.
  • the nanodisc reduces lipid or prevents lipid accumulation in the eye of the individual by at least about 5% (e.g., at least about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%).
  • a method of preventing or treating an eye disease characterized by lipid (e.g., cholesterol) accumulation in an individual (e.g., human) comprising administering to the eye of the individual an effective amount of a pharmaceutical composition comprising a plurality of nanodiscs, wherein the nanodisc comprises an MSP and a phospholipid.
  • the MSP is a variant of ApoA-I.
  • the MSP is selected from the group consisting of MSP1E1, MSP1E2, MSP1E3, MSP1, MSP1D1, MSP1E1D1, MSP1E2D1, MSP1E3D1, MSP2N2, and N-terminal His-tag portion removed variant thereof (His-tag removed variant).
  • the MSP is selected from the group consisting of MSP1E1, MSP1E2, MSP1E3, MSP1, MSP1D1, MSP1D2, and MSPE3D1.
  • the MSP is (i) an MSP1D1 His-tag removed variant comprising the amino acid sequence of SEQ ID NO: 14; or (ii) an MSP1E3D1 His-tag removed variant comprising the amino acid sequence of SEQ ID NO: 15.
  • the phospholipid comprises DMPC, DPPC, DMPS, DSPC, POPC, POPS, PiP2, or a combination thereof.
  • the phospholipid is a PC, such as selected from the group consisting of DMPC, POPC, DPPC, DSPC, and a combination thereof.
  • lipid e.g., cholesterol
  • a pharmaceutical composition comprising a nanodisc, wherein at least about 85% of the plurality of nanodiscs in ny-2692819 Attorney Docket No.: 283912000140 the pharmaceutical composition are fully-lipidated.
  • the molar ratio of the phospholipid to the MSP in the pharmaceutical composition is less than a phospholipid-MSP saturation ratio NS.
  • the molar ratio of the phospholipid to the MSP in the pharmaceutical composition is less than about 95% of the phospholipid-MSP saturation ratio NS.
  • the MSP is MSP1D1, MSP1, or His-tag removed variant thereof, and the molar ratio of the phospholipid to the MSP in the pharmaceutical composition is from about 10:1 to about 90:1. In some embodiments, the MSP is MSP1D1 or MSP1, and the molar ratio of phospholipid to MSP is from about 10:1 to about 90:1. In some embodiments, the MSP is MSP1D1, MSP1, or His-tag removed variant thereof, the phospholipid is POPC, and the molar ratio of POPC to the MSP in the pharmaceutical composition is from about 10:1 to about 65:1.
  • the phospholipid is POPC, and the molar ratio of POPC to MSP is from about 10:1 to about 65:1.
  • the MSP is MSP1D1
  • the phospholipid is POPC
  • the molar ratio of POPC to MSP1D1 is about 50:1 or about 32.5:1.
  • the MSP is MSP1 or MSP1D1
  • the phospholipid is POPC
  • the molar ratio of POPC to MSP1 or MSP1D1 is about 65:1.
  • the MSP is MSP1D1 or His- tag removed variant thereof
  • the phospholipid is POPC
  • the molar ratio of POPC to the MSP in the pharmaceutical composition is about 65:1.
  • the MSP is MSP1D1 or His-tag removed variant thereof
  • the phospholipid is POPC
  • the NS is about 62.
  • the molar ratio of POPC to the MSP1D1 or His-tag removed variant thereof in the pharmaceutical composition is less than about 58:1.
  • the molar ratio of POPC to the MSP1D1 or His-tag removed variant thereof in the pharmaceutical composition is about 50:1 or about 32.5:1.
  • the MSP is MSP1D1, MSP1, or His-tag removed variant thereof
  • the phospholipid is DMPC, wherein the molar ratio of DMPC to the MSP in the pharmaceutical composition is from about 10:1 to about 85:1.
  • the MSP is MSP1D1, MSP1, or His-tag removed variant thereof, the phospholipid is DMPC, and the molar ny-2692819 Attorney Docket No.: 283912000140 ratio of DMPC to MSP is from about 10:1 to about 85:1.
  • the MSP is MSP1D1 or His-tag removed variant thereof, the phospholipid is DMPC, and the molar ratio of DMPC to the MSP in the pharmaceutical composition is about 85:1.
  • the MSP is MSP1D1, the phospholipid is DMPC, and the molar ratio of DMPC to MSP1D1 is about 85:1.
  • the molar ratio of DMPC to MSP1D1 in the pharmaceutical composition is about 30:1 to about 80:1, such as any of about 40:1 to about 60:1, about 46:1 to about 55:1, about 51:1, about 55:1, or about 50:1.
  • the MSP is MSP1D1 or His-tag removed variant thereof
  • the phospholipid is DMPC
  • the NS is about 81.
  • the MSP is MSP1
  • the molar ratio of DMPC to MSP1 is about 80:1.
  • the MSP is MSP1D1
  • the phospholipid is DMPC
  • the molar ratio of DMPC to MSP1D1 in the pharmaceutical composition is about 80:1, about 51:1, about 55:1, or about 50:1.
  • the MSP is MSP1D1, MSP1, or His-tag removed variant thereof
  • the phospholipid is DMPC
  • the molar ratio of DMPC to the MSP in the pharmaceutical composition is less than about 78:1.
  • the molar ratio of DMPC to the MSP in the pharmaceutical composition is about 65:1 to about 75:1.
  • the molar ratio of DMPC to the MSP1D1, MSP1, or His-tag removed variant thereof in the pharmaceutical composition is from about 40:1 to about 60:1. In some embodiments, the molar ratio of DMPC to the MSP in the pharmaceutical composition is about 46:1 to about 55:1, about 51:1, about 55:1, or about 50:1. [0063] In some embodiments, the MSP is MSP1D1, MSP1, or His-tag removed variant thereof, wherein the phospholipid is DPPC, and the molar ratio of DPPC to the MSP in the pharmaceutical composition is from about 10:1 to about 90:1.
  • the MSP is MSP1 or His-tag removed variant thereof, the phospholipid is DPPC, and the molar ratio of DPPC to the MSP in the pharmaceutical composition is about 90:1.
  • the MSP is MSP1D1 or His-tag removed variant thereof, the phospholipid is DPPC, and the N S is about 81.
  • the molar ratio of DPPC to the MSP in the pharmaceutical composition is less than about 75:1.
  • the MSP is MSP1, the phospholipid is DPPC, and the molar ratio of DPPC to MSP1 is about 90:1.
  • the MSP is MSP1E3D1, MSP1E3, or His-tag removed variant thereof, and the molar ratio of the phospholipid to the MSP in the pharmaceutical composition is from about 50:1 to about 200:1.
  • the MSP is MSP1E3, and the molar ratio ny-2692819 Attorney Docket No.: 283912000140 of phospholipid to MSP1E3 (e.g., in the nanodisc, or in the pharmaceutical composition) is from about 100:1 to about 200:1.
  • the phospholipid is POPC, and the molar ratio of POPC to MSP1E3 (e.g., in the nanodisc, or in the pharmaceutical composition) is about 130:1.
  • the MSP is MSP1E3 or His-tag removed variant thereof, the phospholipid is POPC, and the molar ratio of POPC to the MSP1E3D1, or His-tag removed variant thereof in the pharmaceutical composition is about 130:1.
  • the MSP is MSP1E3D1 or His-tag removed variant thereof, the phospholipid is POPC, and the NS is about 126.
  • the molar ratio of POPC to the MSP1E3D1, or His-tag removed variant thereof in the pharmaceutical composition is less than about 118:1.
  • the phospholipid is DMPC, the MSP is MSP1E3, and the molar ratio of DMPC to MSP1E3 is about 150:1.
  • the phospholipid is DPPC
  • the MSP is MSP1E3 or His-tag removed variant thereof
  • the molar ratio of DPPC to MSP1E3 is about 170:1.
  • the MSP is MSP1E3D1 or His-tag removed variant thereof
  • the phospholipid is DMPC
  • the molar ratio of DMPC to the MSP in the pharmaceutical composition is about 87:1 to about 96:1, about 58:1 to about 68:1, about 63:1, or about 62:1.
  • the nanodisc comprises DMPC and MSP1D1, such as in a molar ratio of about 85:1.
  • the nanodisc comprises DMPC and MSP1D1 (e.g., SEQ ID NO: 3) or His-tag removed variant thereof (e.g., SEQ ID NO: 14), optionally wherein the molar ratio of DMPC to MSP1D1 (e.g., SEQ ID NO: 3) or His-tag removed variant thereof (e.g., SEQ ID NO: 14) in the pharmaceutical composition is about 46:1 to about 55:1, about 51:1, about 55:1, or about 50:1.
  • MSP1D1 e.g., SEQ ID NO: 3
  • His-tag removed variant thereof e.g., SEQ ID NO: 14
  • a method of preventing or treating an eye disease characterized by lipid (e.g., cholesterol) accumulation in the eye of an individual comprising administering to the eye of the individual an effective amount of a pharmaceutical composition comprising a plurality of nanodiscs, wherein the nanodisc comprises DMPC and MSP1D1 (e.g., SEQ ID NO: 3) or His-tag removed variant thereof (e.g., SEQ ID NO: 14), and the molar ratio of DMPC and MSP1D1 (e.g., SEQ ID NO: 3) or His-tag removed variant thereof (e.g., SEQ ID NO: 14) in the pharmaceutical composition is about 46:1 to about 55:1, about 51:1, about 55:1, or about 50:1.
  • DMPC and MSP1D1 e.g., SEQ ID NO: 3
  • His-tag removed variant thereof e.g., SEQ ID NO: 14
  • the nanodisc comprises POPC and MSP1D1 (e.g., SEQ ID NO: 3), such as in a molar ratio of about 65:1, about 50:1, or about 32.5:1.
  • lipid e.g., cholesterol
  • the nanodisc comprises DMPC and MSP1D1 (e.g., SEQ ID NO: 3) at a molar ratio of about 85:1, about 51:1, about 55:1, or about 50:1.
  • the nanodisc comprises DMPC and MSP1E3D1 (e.g., SEQ ID NO: 8) or His-tag removed variant thereof (e.g., SEQ ID NO: 15) and the molar ratio of DMPC and MSP1E3D1 (e.g., SEQ ID NO: 8) or His-tag removed variant thereof (e.g., SEQ ID NO: 15)in the pharmaceutical composition is about 87:1 to about 96:1, about 58:1 to about 68:1, about 63:1, or about 62:1.
  • a method of preventing or treating an eye disease characterized by lipid (e.g., cholesterol) accumulation in the eye of an individual comprising administering to the eye of the individual an effective amount of a pharmaceutical composition comprising a plurality of nanodiscs, wherein the nanodisc comprises DMPC and MSP1E3D1 (e.g., SEQ ID NO: 8) or His-tag removed variant thereof (e.g., SEQ ID NO: 15), and the molar ratio of DMPC and MSP1E3D1 (e.g., SEQ ID NO: 8) or His-tag removed variant thereof (e.g., SEQ ID NO: 15) in the pharmaceutical composition is about 58:1 to about 68:1, about 63:1, or about 62:1.
  • DMPC and MSP1E3D1 e.g., SEQ ID NO: 8
  • His-tag removed variant thereof e.g., SEQ ID NO: 15
  • a method of preventing or treating an eye disease characterized by lipid (e.g., cholesterol) accumulation in the eye of an individual comprising administering to the eye of the individual an effective amount of a pharmaceutical composition comprising a nanodisc, wherein the nanodisc comprises POPC and MSP1D1 (e.g., SEQ ID NO: 3) at a molar ratio of about 65:1, about 50:1, or about 32.5:1.
  • lipid e.g., cholesterol
  • MSP1D1 e.g., SEQ ID NO: 3
  • a method of preventing or treating an eye disease characterized by lipid (e.g., cholesterol) accumulation in the eye of an individual comprising administering to the eye of the individual an effective amount of a pharmaceutical composition comprising a plurality of nanodiscs, wherein the nanodisc comprises DMPC and MSP1D1 (e.g., SEQ ID NO: 3) or His-tag removed variant thereof (e.g., SEQ ID NO: 14), and the molar ratio of DMPC and MSP1D1 (e.g., SEQ ID NO: 3) or His-tag removed variant thereof (e.g., SEQ ID NO: 14) in the pharmaceutical composition is about 46:1 to about 55:1, about 51:1, about 55:1, or about 50:1.
  • DMPC and MSP1D1 e.g., SEQ ID NO: 3
  • His-tag removed variant thereof e.g., SEQ ID NO: 14
  • DMPC and MSP1E3D1 e.g., SEQ ID NO: 8
  • His-tag removed variant thereof e.g., SEQ ID NO: 15
  • the pharmaceutical composition is about 58:1 to about
  • the lipid for removal or prevention from accumulation in the eye is cholesterol.
  • the eye disease characterized by lipid accumulation is photoreceptor neurodegeneration, a retinal neurovascular disorder, age-related macular degeneration (AMD), or atrophy.
  • AMD age-related macular degeneration
  • the eye disease characterized by lipid accumulation is AMD, such as dry AMD.
  • the dry AMD is geographical atrophy (GA).
  • the eye disease characterized by lipid accumulation is wet AMD.
  • the eye disease is choroidal neovascularization (CNV).
  • the nanodisc is administered intravitreally.
  • the nanodisc is about 2 nm to about 20 nm, such as any of about 5 nm to about 20 nm or about 10 nm to about 20 nm in diameter. In some embodiments, the nanodisc does not comprise a CPP. In some embodiments, the nanodisc does not comprise a target protein. In some embodiments, the nanodisc does not comprise a prophylactic or therapeutic agent. In some embodiments, the nanodisc increases cholesterol efflux rate by at least about 2-fold (e.g., at least about any of 2.5, 3, 4, 5, 10, 15-fold, or more) compared to an untreated state, or the cholesterol efflux rate by ApoA-I or the MSP.
  • 2-fold e.g., at least about any of 2.5, 3, 4, 5, 10, 15-fold, or more
  • the nanodisc reduces lipid or prevents lipid accumulation in the eye of the individual by at least about 5% (e.g., at least about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%).
  • a method of preventing or treating an AMD in the eye of an individual comprising administering to the eye of the individual an effective amount of a pharmaceutical composition comprising a nanodisc, wherein the nanodisc comprises an MSP and a phospholipid.
  • a method of preventing or treating an AMD in the eye of an individual comprising administering to the eye of the individual an effective amount of a pharmaceutical composition ny-2692819 Attorney Docket No.: 283912000140 comprising a nanodisc, wherein the nanodisc comprises DMPC and MSP1D1 (e.g., SEQ ID NO: 3) at a molar ratio of about 85:1.
  • a pharmaceutical composition ny-2692819 Attorney Docket No.: 283912000140 comprising a nanodisc, wherein the nanodisc comprises DMPC and MSP1D1 (e.g., SEQ ID NO: 3) at a molar ratio of about 85:1.
  • a method of preventing or treating an AMD in the eye of an individual comprising administering to the eye of the individual an effective amount of a pharmaceutical composition comprising a plurality of nanodiscs, wherein the nanodisc comprises DMPC and MSP1D1 (e.g., SEQ ID NO: 3) or His- tag removed variant thereof (e.g., SEQ ID NO: 14), and the molar ratio of DMPC and MSP1D1 (e.g., SEQ ID NO: 3) or His-tag removed variant thereof (e.g., SEQ ID NO: 14) in the pharmaceutical composition is about 46:1 to about 55:1, about 51:1, about 55:1, or about 50:1.
  • DMPC and MSP1D1 e.g., SEQ ID NO: 3
  • His-tag removed variant thereof e.g., SEQ ID NO: 14
  • a method of preventing or treating an AMD in the eye of an individual comprising administering to the eye of the individual an effective amount of a pharmaceutical composition comprising a plurality of nanodiscs, wherein the nanodisc comprises DMPC and MSP1E3D1 (e.g., SEQ ID NO: 8) or His-tag removed variant thereof (e.g., SEQ ID NO: 15), and the molar ratio of DMPC and MSP1E3D1 (e.g., SEQ ID NO: 8) or His-tag removed variant thereof (e.g., SEQ ID NO: 15) in the pharmaceutical composition is about 58:1 to about 68:1, about 63:1, or about 62:1.
  • DMPC and MSP1E3D1 e.g., SEQ ID NO: 8
  • His-tag removed variant thereof e.g., SEQ ID NO: 15
  • a method of preventing or treating an AMD in the eye of an individual comprising administering to the eye of the individual an effective amount of a pharmaceutical composition comprising a nanodisc, wherein the nanodisc comprises POPC and MSP1D1 (e.g., SEQ ID NO: 3) at a molar ratio of about 65:1, about 50:1, or about 32.5:1.
  • the AMD is dry AMD.
  • the dry AMD is GA.
  • the AMD is wet AMD.
  • the wet AMD comprises CNV.
  • the method removes lipid (e.g., cholesterol) or prevents lipid (e.g., cholesterol) accumulation in the eye of the individual.
  • the nanodisc is administered intravitreally.
  • the nanodisc is about 2 nm to about 20 nm, such as any of about 5 nm to about 20 nm or about 10 nm to about 20 nm in diameter.
  • the nanodisc does not comprise a CPP.
  • the nanodisc does not comprise a target protein.
  • the nanodisc does not comprise a prophylactic or therapeutic agent.
  • the nanodisc increases cholesterol efflux rate by at least about 2-fold (e.g., at least about any of 2.5, 3, 4, 5, 10, 15-fold, or more) compared to an untreated state, or the cholesterol efflux rate by ApoA-I or the MSP.
  • the nanodisc reduces lipid or prevents lipid accumulation in the eye of the individual by at least about 5% (e.g., at least about any of ny-2692819 Attorney Docket No.: 283912000140 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%).
  • the AMD is GA
  • the nanodisc reduces the total area of GA lesion by at least about 5% (e.g., at least about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%).
  • the nanodisc slows the progression of the total area of GA lesions in the eye of the individual by at least about 5% (e.g., at least about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%).
  • the total area of GA lesion is determined by autofluorescence imaging.
  • the lipid for removal or prevention from accumulation in the eye is selected from the group consisting of fats, sterols, phospholipids and any combination thereof.
  • the lipid comprises cholesterol esters (ChEs), wax esters (WEs), diesters, triacylglycerol (TG), free cholesterol, free fatty acids (FAs), (O-acyl)- ⁇ -hydroxy fatty acids (OAHFAs), or any combination thereof.
  • the lipid is a fatty acid or its derivative (including tri-, di-, mono-glycerides, and phospholipids).
  • the lipid for removal or prevention from accumulation in the eye comprises two or more of the lipids described herein.
  • the lipid for removal or prevention from accumulation in the eye is cholesterol.
  • the eye diseases characterized by lipid accumulation is selected from the group consisting of AMD, photoreceptor neurodegeneration, optic nerve atrophy, loss of acuity, hemianopia, visual agnosia, strabismus, retinal neurovascular disorder, lipid keratopathy, corneal lipidosis, and any combination thereof.
  • exemplary eye diseases characterized by lipid accumulation include, but are not limited to, AMD including dry AMD (atrophic AMD) and wet AMD (neovascular AMD), choroidal neovascularization (CNV), retinal angiomatous proliferation (RAP), retinal neovascularization (RNV), juvenile macular degeneration (e.g., Stargardt disease), macular telangiectasia, maculopathy (e.g., age- related maculopathy (ARM) and diabetic maculopathy (DMP), including partial ischemic DMP), macular edema (e.g., diabetic macular edema (DME), including clinically significant DME, focal DME and diffuse DME, Irvine-Gass syndrome (postoperative macular edema), and macular edema following RVO, including central RVO and branch RVO), retinopathy (e.g., diabetic retinopathy (DR), including in patients with DME, proliferative vitreoretinopathy (DR), prolife
  • the eye disease characterized by lipid accumulation is photoreceptor neurodegeneration or retinal neurovascular disorder.
  • the eye disease characterized by lipid accumulation is AMD.
  • the AMD is dry AMD. Dry AMD is generally characterized by a buildup of yellowish deposits called drusen beneath the retina and typically affects vision in both eyes, although vision loss often occurs in one eye before the other. A more advanced stage of dry AMD is known as geographic atrophy (GA), in which areas of the macula waste away (atrophy), resulting in severe vision loss. [0078] Dry AMD can progress into wet AMD. Wet AMD is generally characterized by the growth of leaky blood vessels underneath the macula.
  • the eye disease characterized by lipid accumulation is wet AMD.
  • the eye disease characterized by lipid accumulation is choroidal neovascularization (CNV).
  • the wet AMD comprises CNV and/or RAP.
  • the methods for removing lipid or preventing lipid accumulation, or for treating an eye disease characterized by lipid accumulation described here can achieve one or more following effects: i) removing (e.g., at least about any of 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100%) lipid (e.g., cholesterol) in the eye; ii) increasing (e.g., at least about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 1- fold, 2-fold, 2.5-fold, 5-fold, 10-fold, or more) the efflux rate of the lipid (e.g., cholesterol) in the eye; iii) reducing (e.g., reducing at least about any of 5%, 10%, 20%, 30%, 40%, 50%, 60%, ny-2692819 Attorney Docket No.: 283912000140 70%, 80%, 90%, 95%, or 100%) the likelihood of the occurrence of lipid accumulation and
  • the eye disease characterized by lipid accumulation is GA
  • the method of preventing or treating GA achieves one or more following effects: i) reducing lipid (e.g., cholesterol) or preventing lipid accumulation in the eye of the individual by at least about 5% (e.g., at least about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100%); ii) reducing the total area of GA lesions in the eye of the individual by at least about 5% (such as by at least about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100%); and iii) slows the progression of the total area of GA lesions in the eye of the individual by at least about 5% (such as at least about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100%).
  • the total area of GA lesions can be determined by any methods known in the art, such as autofluorescence imaging.
  • the autofluorescence is fundus autofluorescence (FAF).
  • FAF fundus autofluorescence
  • a method of reducing and/or slowing the progress of total area of GA lesions in the eye of an individual comprising administering to the eye of the individual an effective amount of a pharmaceutical composition comprising a nanodisc, wherein the nanodisc comprises DMPC and MSP1D1 (e.g., SEQ ID NO: 3) at a molar ratio of about 85:1.
  • a pharmaceutical composition comprising a nanodisc, wherein the nanodisc comprises DMPC and MSP1D1 (e.g., SEQ ID NO: 3) at a molar ratio of about 85:1.
  • a method of reducing and/or slowing the progress of total area of GA lesions in the eye of an individual comprising administering to the eye of the individual an effective amount of a pharmaceutical composition comprising a plurality of nanodiscs, wherein the nanodisc comprises DMPC and MSP1D1 (e.g., SEQ ID NO: ny-2692819 Attorney Docket No.: 283912000140 3) or His-tag removed variant thereof (e.g., SEQ ID NO: 14), and the molar ratio of DMPC and MSP1D1 (e.g., SEQ ID NO: 3) or His-tag removed variant thereof (e.g., SEQ ID NO: 14) in the pharmaceutical composition is about 46:1 to about 55:1, about 51:1, about 55:1, or about 50:1.
  • DMPC and MSP1D1 e.g., SEQ ID NO: ny-2692819 Attorney Docket No.: 283912000140
  • His-tag removed variant thereof e.g., S
  • a method of reducing and/or slowing the progress of total area of GA lesions in the eye of an individual comprising administering to the eye of the individual an effective amount of a pharmaceutical composition comprising a plurality of nanodiscs, wherein the nanodisc comprises DMPC and MSP1E3D1 (e.g., SEQ ID NO: 8) or His- tag removed variant thereof (e.g., SEQ ID NO: 15), and the molar ratio of DMPC and MSP1E3D1 (e.g., SEQ ID NO: 8) or His-tag removed variant thereof (e.g., SEQ ID NO: 15) in the pharmaceutical composition is about 58:1 to about 68:1, about 63:1, or about 62:1.
  • DMPC and MSP1E3D1 e.g., SEQ ID NO: 8
  • His-tag removed variant thereof e.g., SEQ ID NO: 15
  • a method of reducing and/or slowing the progress of total area of GA lesions in the eye of an individual comprising administering to the eye of the individual an effective amount of a pharmaceutical composition comprising a nanodisc, wherein the nanodisc comprises POPC and MSP1D1 (e.g., SEQ ID NO: 3) at a molar ratio of about 65:1, about 50:1, or about 32.5:1.
  • the method removes lipid (e.g., cholesterol) or prevents lipid (e.g., cholesterol) accumulation in the eye of the individual.
  • the nanodisc is administered intravitreally.
  • the nanodisc is about 2 nm to about 20 nm, such as any of about 5 nm to about 20 nm or about 10 nm to about 20 nm in diameter. In some embodiments, the nanodisc does not comprise a CPP. In some embodiments, the nanodisc does not comprise a target protein. In some embodiments, the nanodisc does not comprise a prophylactic or therapeutic agent. In some embodiments, the nanodisc increases cholesterol efflux rate by at least about 2-fold (e.g., at least about any of 2.5, 3, 4, 5, 10, 15-fold, or more) compared to an untreated state, or the cholesterol efflux rate by ApoA-I or the MSP.
  • 2-fold e.g., at least about any of 2.5, 3, 4, 5, 10, 15-fold, or more
  • the nanodisc reduces lipid or prevents lipid accumulation in the eye of the individual by at least about 5% (e.g., at least about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%). In some embodiments, the nanodisc reduces the total area of GA lesion by at least about 5% (e.g., at least about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%). In some embodiments, the nanodisc slows the progression of the total area of GA lesions in the eye of the individual by at least about 5% (e.g., at least about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%).
  • the total area of GA lesion is determined by autofluorescence imaging. ny-2692819 Attorney Docket No.: 283912000140 [0082]
  • the methods described herein inhibits (e.g., inhibiting at least about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100%) neovascularization in the eye of an individual suffering from AMD.
  • the method delays (e.g., delaying at least about any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 18, 24, 36, or more months) the development of AMD or CNV.
  • the method treats and/or reduces (e.g., reducing at least about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100%) the risk of developing retinal neovascularization (RNV).
  • the method treats and/or reduces (e.g., reducing at least about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100%) the risk of developing retinal angiomatous proliferation (RAP).
  • RAP retinal angiomatous proliferation
  • the method removes lipid (e.g., cholesterol) ny-2692819 Attorney Docket No.: 283912000140 or prevents lipid (e.g., cholesterol) accumulation in the eye of the individual.
  • the nanodisc is administered intravitreally.
  • the nanodisc is about about 2 nm to about 20 nm, such as any of about 5 nm to about 20 nm or about 10 nm to about 20 nm in diameter.
  • the nanodisc does not comprise a CPP.
  • the nanodisc does not comprise a target protein.
  • the nanodisc does not comprise a prophylactic or therapeutic agent.
  • the nanodisc increases cholesterol efflux rate by at least about 2-fold (e.g., at least about any of 2.5, 3, 4, 5, 10, 15-fold, or more) compared to an untreated state, or the cholesterol efflux rate by ApoA-I or the MSP.
  • the nanodisc reduces lipid or prevents lipid accumulation in the eye of the individual by at least about 5% (e.g., at least about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%).
  • the individual is suffering from AMD and is at risk of or suffering from RAP.
  • the individual is suffering from AMD and is at risk of or suffering from CNV.
  • the individual is suffering from AMD and is at risk of or suffering from RNV.
  • the individual has been identified as having one or more genetic polymorphisms that increases the risk of AMD.
  • the method of preventing or treating an eye disease characterized by lipid accumulation (e.g., AMD) described herein further comprises determining whether the individual has a genetic polymorphism that increases the risk of AMD.
  • the individual has injury at the Bruch’s membrane.
  • the individual has deficient Abca1 and/or Abcg1 cholesterol efflux transporters in rod photoreceptors.
  • the individual has at least one of RPE disruption and hypertransmission, ellipsoid zone abnormalities, inner retinal subsidence, and hyperreflective foci.
  • the individual has at least one of inner segment/outer segment (IS/OS) junction abnormalities and RPE disruptions including pigment epithelial detachments (PED).
  • the individual has been on a high-fat diet.
  • a method of improving vision impairment and/or reducing the likelihood of vision loss associated with an eye disease characterized by lipid accumulation in the eye in an individual comprising administering to the eye of the individual an effective amount of a pharmaceutical composition comprising a nanodisc, wherein the nanodisc comprises an MSP and a phospholipid.
  • the nanodisc ny-2692819 Attorney Docket No.: 283912000140 comprises DMPC and MSP1D1 (e.g., SEQ ID NO: 3), such as in a molar ratio of about 85:1.
  • a method of improving vision impairment and/or reducing the likelihood of vision loss associated with an eye disease characterized by lipid accumulation in the eye in an individual comprising administering to the eye of the individual an effective amount of a pharmaceutical composition comprising a plurality of nanodiscs, wherein the nanodisc comprises DMPC and MSP1D1 (e.g., SEQ ID NO: 3) or His-tag removed variant thereof (e.g., SEQ ID NO: 14), and the molar ratio of DMPC and MSP1D1 (e.g., SEQ ID NO: 3) or His-tag removed variant thereof (e.g., SEQ ID NO: 14) in the pharmaceutical composition is about 46:1 to about 55:1, about 50:1, about 51:1, or about 55:1.
  • DMPC and MSP1D1 e.g., SEQ ID NO: 3
  • His-tag removed variant thereof e.g., SEQ ID NO: 14
  • a method of improving vision impairment and/or reducing the likelihood of vision loss associated with an eye disease characterized by lipid accumulation in the eye in an individual comprising administering to the eye of the individual an effective amount of a pharmaceutical composition comprising a plurality of nanodiscs, wherein the nanodisc comprises DMPC and MSP1E3D1 (e.g., SEQ ID NO: 8) or His-tag removed variant thereof (e.g., SEQ ID NO: 15), and the molar ratio of DMPC and MSP1E3D1 (e.g., SEQ ID NO: 8) or His-tag removed variant thereof (e.g., SEQ ID NO: 15) in the pharmaceutical composition is about 58:1 to about 68:1, about 63:1, or about 62:1.
  • DMPC and MSP1E3D1 e.g., SEQ ID NO: 8
  • His-tag removed variant thereof e.g., SEQ ID NO: 15
  • the nanodisc comprises POPC and MSP1D1 (e.g., SEQ ID NO: 3), such as in a molar ratio of about 65:1, about 50:1, or about 32.5:1.
  • the method provided herein improves vision impairment and/or reduces the likelihood of vision loss associated with eye diseases characterized by lipid accumulation in the eye by at least about 5%, such as by at least about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100%.
  • the vision impairment and/or vision loss can be associated with any of the eye diseases described herein.
  • the vision impairment or loss is associated with atrophic AMD including non-central or/and central GA or neovascular AMD including types 1, 2 or/and 3 NV, or the vision improvement can occur in a subject with atrophic AMD or neovascular AMD.
  • the nanodisc ny-2692819 Attorney Docket No.: 283912000140 comprises DMPC and MSP1D1 (e.g., SEQ ID NO: 3), such as in a molar ratio of about 85:1.
  • a method of improving the normal luminance best- corrected visual acuity (NL-BCVA) score and/or the low luminance best-corrected visual acuity (LL-BCVA) score of an individual comprising administering to the eye of the individual an effective amount of a pharmaceutical composition comprising a plurality of nanodiscs, wherein the nanodisc comprises DMPC and MSP1D1 (e.g., SEQ ID NO: 3) or His- tag removed variant thereof (e.g., SEQ ID NO: 14), and the molar ratio of DMPC and MSP1D1 (e.g., SEQ ID NO: 3) or His-tag removed variant thereof (e.g., SEQ ID NO: 14) in the pharmaceutical composition is about 46:1 to about 55:1, about 51:1, about 55:1, or about 50:1.
  • DMPC and MSP1D1 e.g., SEQ ID NO: 3
  • His-tag removed variant thereof e.g., SEQ ID NO: 14
  • a method of improving the normal luminance best- corrected visual acuity (NL-BCVA) score and/or the low luminance best-corrected visual acuity (LL-BCVA) score of an individual comprising administering to the eye of the individual an effective amount of a pharmaceutical composition comprising a plurality of nanodiscs, wherein the nanodisc comprises DMPC and MSP1E3D1 (e.g., SEQ ID NO: 8) or His- tag removed variant thereof (e.g., SEQ ID NO: 15), and the molar ratio of DMPC and MSP1E3D1 (e.g., SEQ ID NO: 8) or His-tag removed variant thereof (e.g., SEQ ID NO: 15) in the pharmaceutical composition is about 58:1 to about 68:1, about 63:1, or about 62:1.
  • DMPC and MSP1E3D1 e.g., SEQ ID NO: 8
  • His-tag removed variant thereof e.g., SEQ ID NO: 15
  • the nanodisc comprises POPC and MSP1D1 (e.g., SEQ ID NO: 3), such as in a molar ratio of about 65:1, about 50:1, or about 32.5:1.
  • the method provided herein improves the NL-BCVA score and/or the LL-BCVA score of the individual by at least about 5%, such as by at least about any of 10%, 20%, 30%, 40%, 50%,60%, 70%, 80%, 90%, 95%, or 100%.
  • the NL-BCVA score and/or LL-BCVA score can be determined by any known methods in the art.
  • any of the methods of i) removing lipid or preventing lipid accumulation, ii) preventing or treating an eye disease characterized by lipid accumulation, iii) reducing and/or slowing the progress of total area of GA lesions, iv) inhibiting neovascularization, v) delaying the development of AMD or CNV, vi) treating and/or reducing the risk of developing RNV and/or RAP, vii) improving vision impairment and/or reducing the likelihood of vision loss associated with an eye disease characterized by lipid accumulation, and viii) improving the NL-BCVA score and/or the LL-BCVA score mentioned herein is compared to a baseline state before treatment (or untreated state), a natural progression without any ny-2692819 Attorney Docket No.: 283912000140 treatment, a treatment with placebo, or a treatment with ApoA-I or the MSP protein (e.g., MSP1D1) alone.
  • the efficacy of any of the methods provided herein is compared to a treatment with HDL alone.
  • the efficacy of any of the methods provided herein may be determined by any method known in the art.
  • the efficacy of any of the methods provided herein may be determined by measuring photoreceptor and RPE function, optionally by full-field scotopic and light bleach recovery ERG diagnostics.
  • the efficacy of any of the methods provided herein may be determined by measuring anatomical changes linked to AMD progression such as RPE disruption and hypertransmission, ellipsoid zone abnormalities, inner retinal subsidence, and hyperreflective foci, optionally by noninvasive imaging such as optical coherence tomography (OCT).
  • OCT optical coherence tomography
  • the efficacy of any of the methods provided herein may be determined by measuring visual function or cumulative incidence of retinal lesions, optionally by full-field scotopic and light bleach recovery ERG diagnostics.
  • the efficacy of any of the methods provided herein may be determined by examining potential changes in individual lipid species in the eye such as cholesteryl ester (CE) composition, optionally by lipidomics analyses.
  • CE cholesteryl ester
  • any of the methods provided herein is safe and leads to minimal side effects, wherein the safety of the methods can be determined by any method known in the art.
  • any of the methods provided herein does not lead to significant retinal inflammation or differences in retinal integrity.
  • the retinal inflammation or differences in retinal integrity may be measured by examining retinal histology.
  • any of the methods provided herein does not exacerbate expected complement signaling in individuals suffering dry AMD.
  • the complement signaling may be determined by retina section immunostaining.
  • Nanodisc refers to a nanocomposite resembling a disc in which a lipid bilayer comprising a phospholipid is surrounded by a stabilizing belt comprising an MSP.
  • the nanodisc comprises DMPC and MSP1D1, optionally in a molar ratio ny-2692819 Attorney Docket No.: 283912000140 (e.g., the molar ratio of DMPC and MSP1D1 in the pharmaceutical composition) of about 85:1.
  • the nanodisc comprises DMPC and MSP1D1, and the molar ratio of DMPC and MSP1D1 in the pharmaceutical composition is about 46:1 to about 55:1.
  • the nanodisc comprises DMPC and MSP1E3D1, and the molar ratio of DMPC and MSP1D1E3 in the pharmaceutical composition is about 58:1 to about 68:1, about 63:1, or about 62:1.
  • the nanodisc comprises POPC and MSP1D1, optionally in a molar ratio (e.g., the molar ratio of POPC and MSP1D1 in the pharmaceutical composition) of about 65:1, or about 50:1, or about 32.5:1.
  • the nanodiscs have an average diameter of about 2 nm to about 20 nm, such as any of about 5 nm to about 20 nm, about 5 nm to about 15 nm, about 2 nm to about 15 nm, about 2 nm to about 10 nm, about 10 nm to about 20 nm, or about 10 nm to about 15 nm.
  • the polydispersity index of the size of the nanodics in the pharmaceutical composition is less than about 0.2.
  • the polydispersity index of the phospholipid content of the nanodics in the pharmaceutical composition is less than about 0.2.
  • the nanodisc does not comprise a CPP.
  • the CPP is selected from the group consisting of penetratin, polyarginine (R8), LL-37, transportan, Pep-1, and membrane translocating sequence (MTS).
  • the nanodisc does not comprise a target protein.
  • the nanodisc does not comprise a prophylactic or therapeutic agent.
  • MSP Membrane scaffold protein
  • the MSP is selected from the group consisting of apolipoprotein A-I (ApoA-I), ApoA-II, ApoC, ApoE, ApoM, and a variant thereof.
  • the MSP is a variant of ApoA-I.
  • Apolipoproteins play an important role in maintaining the structural integrity and solubility of lipoproteins, in lipoprotein receptor recognition, and in the regulation of certain enzymes in lipoprotein metabolism.
  • Main types of apolipoproteins include, but are not limited to, apolipoprotein A (Apo-A) such as Apo-AI, Apo-A2, Apo-A4, and Apo-A5, apolipoprotein B (Apo-B), such as Apo-B48 and Apo B-100, apolipoprotein C (Apo-C), such as ApoC-I, apo ApoC-II, apo ApoC-III, and ApoC-IV, apolipoprotein D (Apo-D), apolipoprotein E (Apo-E), and apolipoprotein M (ApoM).
  • Apo-A apolipoprotein A
  • Apo-B apolipoprotein B
  • Apo-B a
  • Apolipoproteins can be from any organism, including but not limited to, human, cows, horses, sheep, monkeys, baboons, goats, rabbits, dogs, hedgehogs, badgers, ny-2692819 Attorney Docket No.: 283912000140 mice, rats, cats, guinea pigs, hamsters, duck, chicken, salmon, and eel.
  • the apolipoprotein is human apolipoprotein.
  • the MSP comprises a variant of an apolipoprotein selected from the group consisting of ApoA-I, ApoA-II, Apo-C, Apo-E, Apo-M, and any combination thereof.
  • the MSP further comprises a fragment derived from a natural apolipoprotein selected from the group consisting of ApoA-I, ApoA-II, Apo-C, Apo-E, and Apo- M.
  • the MSP comprises a mutation (e.g., insertion, deletion, substitution, or any combination thereof) compared to a naturally existing apolipoprotein (e.g., ApoA-I).
  • the MSP comprises one or more cysteine (Cys) substitution.
  • the mutation e.g., Cys substitution
  • the mutation is at the N terminus, at the C terminus, and/or among the amphipathic helices of the MSP.
  • the Cys substitution is in one of the helixes of the MSP.
  • the cysteine may be linked to an imaging group, optionally a fluorescent group (e.g., GFP, RFP, YFP).
  • the cysteine in the MSP may be linked to an imaging group, optionally a fluorescent group (e.g., fluorescein, rhodamine, or other common luminescent small molecules).
  • the MSP is fused with a fluorescent protein such as GFP, RFP, or YFP.
  • the MSP comprises a tag, such a tag commonly used in protein expression and/or purification.
  • the tag is a polyhistidine, c-Myc, FLAG, biotin, or any combination thereof.
  • the MSP comprises an enzymatic cleavage site, e.g., Tobacco Etch Protease (TEV) recognition site (boxed in Table 1).
  • TEV Tobacco Etch Protease
  • the MSP comprises a His-tag.
  • the MSP does not comprise a His-tag.
  • the symbol “(-)” as used herein refers to an MSP with the His-tag removed.
  • the His-tag is followed by a TEV protease recognition site.
  • the MSP is derived from a naturally occurring ApoA-I, e.g., human ApoA-I.
  • TEV protease recognition site sequence is boxed in Table 1.
  • ApoA-I is the major protein component of HDL particles in plasma. The protein enables efflux of fat molecules by accepting fats within cells for transport elsewhere, such as back to LDL particles or to the liver for excretion.
  • Human ApoA-I contains an N-terminal globular domain (GLOB) made up of about 43 residues followed by 10 amphipathic helices (H1- ny-2692819 Attorney Docket No.: 283912000140 H10) made up of 11, 22, or 24 residues each. Seven of the helices are 22 amino acids in length, one helix (H10) is 24 amino acids in length, while two helices (H3, H9) are 11 amino acids in length. The helices are separated by a glycine or proline. The sequences of different domains are indicated for human ApoA-I in Table 1.
  • a Full-length human ApoA-I comprises from N’ to C’: GLOB-H1-H2-H3-H4-H5-H6-H7-H8-H9-H10.
  • Human ApoA-I comprises the amino acid sequence of SEQ ID NO: 1.
  • the GLOB domain comprises the amino acid sequence of DEPPQSPWDRVKDLATVYVDVLKDSGRDYVSQFEGSALGKQLN (SEQ ID NO: 19).
  • ApoA-1 can be from any organism.
  • ApoA-1 is derived from animals selected from the group consisting of human, cows, horses, sheep, monkeys, baboons, goats, rabbits, dogs, hedgehogs, badgers, mice, rats, cats, guinea pigs, hamsters, duck, chicken, salmon, eel, and any combination thereof.
  • ApoA-1 is a human ApoA-1.
  • ApoA-1 comprises (or consists of, or consists essentially of) the amino acid sequence of SEQ ID NO: 1.
  • the MSP is a variant of ApoA-I.
  • the MSP has at least about 70% (e.g., at least about any of 80%, 85%, 90%, 95%, 99%, or higher) sequence identity to SEQ ID NO: 1.
  • MSPs containing alternate amphipathic helical polypeptide sequences can be generated by adding or removing one or more helical segments (e.g., H1, H2, etc.) or partial helical segments (e.g., removing partial H1 sequence of ApoA-I to arrive at H(0.5)), or adding, removing, or substituting amino acid residues, based off an ApoA-I (e.g., human ApoA-I) protein.
  • an MSP can be derived from ApoA-I (e.g., human ApoA-I) by removing the GLOB domain.
  • the MSP can be derived from ApoA-I (e.g., human ApoA-I) by adding one (e.g., H4), two (e.g., H4 and H5), three (e.g., H4-H6), or more helical segments.
  • H4 e.g., human ApoA-I
  • Exemplary MSPs and their contained domains and sequences are indicated in Table 1. Other MSPs with variations relative to those in Table 1 can also be used herein.
  • ny-2692819 Attorney Docket No.: 283912000140 Table 1.
  • the MSP comprises an N-terminus truncation relative to ApoA-I.
  • the truncation comprises deleting 1, 2, 3, 4, 5, 10, 15, 20, 30, 40, 50, 60, 100, or more consecutive amino acids from the N-terminus of ApoA-I.
  • the truncation comprises deleting about 10-70 amino acids from the N-terminus of ApoA-I.
  • the MSP is MSP1, MSP1D1, or MSP1D2.
  • the truncation comprises deleting about 10-70 amino acids from the N-terminus of ApoA-I.
  • the N-terminus GLOB domain of ApoA-I is removed in the MSP.
  • the first 11 amino acids of H1 of ApoA-I is removed in the MSP, resulting in a H(0.5) helical portion of STFSKLREQLG (SEQ ID NO: 20) (see H(0.5) in Table 1).
  • both the N-terminus GLOB domain and the first 11 amino acids of H1 of ApoA-I is removed in the MSP.
  • the MSP comprises an amino acid sequence of MGHHHHHHHDYDIPTTENLYFQG (SEQ ID NO: 21) at the N-terminus, hereinafter also referred to as “His-tag” or “His-tag portion.”
  • the TEV protease recognition site is indicated by boxing.
  • the MSP does not comprise the N-terminal His-tag portion (his- tag removed).
  • the N-terminal His-tag (SEQ ID NO: 21) is removed from the MSP by cleaving with the TEV protease.
  • cleavage at the TEV protease ny-2692819 Attorney Docket No.: 283912000140 recognition site may result in a residual glycine (G) residue at the N-terminus of the resulting MSP sequence.
  • MSP1D1(-) (SEQ ID NO: 14) is obtained by cleaving MSP1D1 (SEQ ID NO: 3) with TEV protease
  • MSP1E3D1(-) SEQ ID NO: 15
  • cleaving MSP1E3D1 SEQ ID NO: 8 with TEV protease.
  • MSPs wherein the indicated His-tag (SEQ ID NO: 21) is not removed, the N-terminal methionine residue will be often removed during heterologous expression, e.g., if expressed in a bacteria such as E. coli.
  • the MSP is MSP1.
  • MSP1 comprises the amino acid sequence of SEQ ID NO: 2.
  • MSP1 is derived from the sequence of ApoA-1, but without the globular N-terminal domain of native ApoA-1.
  • the MSP has at least about 70% (e.g., at least about any of 80%, 85%, 90%, 95%, 99%, or 100%) sequence identity to MSP1 (e.g., SEQ ID NO: 2).
  • the MSP is MSP1D1.
  • MSP1D1 is derived from the sequence of ApoA-1, but without the globular N-terminal domain of native ApoA-1 and removing 11 amino acids from the first helix labeled H1 in the parent ApoA-I, generating the half helix labeled H(0.5) and MSP1D1.
  • MSP1D1 comprises the amino acid sequence of SEQ ID NO: 3.
  • the MSP has at least about 70% (e.g., at least about any of 80%, 85%, 90%, 95%, 99%, or 100%) sequence identity to SEQ ID NO: 3.
  • the MSP is a variant of MSP1D1 that does not comprise the N-terminal His- tag portion (his-tag removed variant) (hereinafter referred to as MSP1D1(-)), which comprises the amino acid sequence of SEQ ID NO: 14.
  • MSP1D1(-) which comprises the amino acid sequence of SEQ ID NO: 14.
  • the MSP has at least about 70% (e.g., at least about any of 80%, 85%, 90%, 95%, 99%, or 100%) sequence identity to SEQ ID NO: 4.
  • the MSP comprises one or more (e.g., 1, 2, 3, or more) additional amphipathic helix sequences relative to ApoA-I.
  • each additional amphipathic helix sequence is independently derived from any of ApoA-I, ApoA-II, ApoC-I, ApoC-II, Apo-E, apolipophorin III, myoglobin, or hemoglobin.
  • the additional amphipathic helix sequence is not derived from ApoA-I, ApoA-II, ApoC-I, ApoC-II, ny-2692819 Attorney Docket No.: 283912000140 Apo-E, apolipophorin III, myoglobin, or hemoglobin.
  • the additional amphipathic helix sequence is synthetic or not naturally occurring.
  • the additional amphipathic helix sequence comprises at least one (e.g., 1, 2, 3, 4, 5, or more) ⁇ - helixes or fragments thereof.
  • the MSP comprises an additional helix sequence, such as one, two, or three of any of first, second, or third helix sequences.
  • the additional amphipathic helix sequence is added to the N-terminus of an apolipoprotein (e.g., ApoA-I).
  • the additional amphipathic helix sequence is added to the C-terminus of an apolipoprotein (e.g., ApoA-I).
  • the additional amphipathic helix sequence is inserted between the original helixes of an apolipoprotein (e.g., ApoA-I).
  • the additional amphipathic helix sequences is added after the last Q residue of H3 into the original MSP1 sequence (e.g., SEQ ID NO: 2).
  • each additional amphipathic helix sequence has about 10 to about 30 (including for example any of about 15 to about 25, about 20, or about 22) amino acids.
  • the total additional amphipathic helix sequence is about any of 20-25, 40-45, 60- 65 amino acids long.
  • the MSP does not comprise the GLOB domain of SEQ ID NO: 19.
  • an amphipathic helix sequence of helix 4 can be inserted to MSP1D1 to generate MSP1E1D1, as defined by SEQ ID NO: 16.
  • 2 amphiphilic helices of Helix 4 (H4) and Helix 5 (H5) can be inserted into MSP1D1, generating MSP1E2D1 (SEQ ID NO: 17).
  • three amphiphilic helices, H4, H5 and H6, can be inserted into MSP1D1, generating MSP1E3D1 defined by SEQ ID NO: 8.
  • an additional 9 amphipathic helices is added to MSP1D1 (SEQ ID NO: 3) with a GT peptide linker to generate an extended MSP—MSP2N2 (SEQ ID NO: 18).
  • the MSP comprises an additional helix sequence, such as one, two, or three of any of first, second, or third helix sequences.
  • the additional amphipathic helix sequence is added to the N-terminus of an apolipoprotein (e.g., ApoA-I).
  • the additional amphipathic helix sequence is added to the C- terminus of an apolipoprotein (e.g., ApoA-I).
  • the additional amphipathic helix sequence is inserted between the helixes of an apolipoprotein (e.g., ApoA-I).
  • each additional amphipathic helix sequence has about 10 to about 30 (including, for example, any of about 15 to about 25, about 24, about 20, about 22, about 11) amino acids.
  • ny-2692819 Attorney Docket No.: 283912000140
  • the total additional amphipathic helix sequence is about any of about 20- 25, about 40-46, about 60-68, or about 100-200 amino acids long.
  • the MSP is MSP1E1, MSP1E2, or MSP1E3.
  • MSP1E1, MSP1E2, and MSP1E3 are all extended membrane scaffold proteins, and are obtained via insertion of one, two, or three extra 22-mer amphipathic helices after the last Q residue of H3 into the original MSP1 sequence.
  • MSP1E1 comprises the amino acid sequence of SEQ ID NO: 5.
  • MSP1E2 comprises the amino acid sequence of SEQ ID NO: 6.
  • MSP1E3 comprises the amino acid sequence of SEQ ID NO: 7.
  • the MSP has at least about 70% (e.g., at least about any of 80%, 85%, 90%, 95%, 99%, or 100%) sequence identity to any of SEQ ID NOs: 5-7.
  • the MSP comprises both an additional amphipathic helix sequence and a truncation relative to ApoA-I.
  • the MSP is MSP1E3D1.
  • MSP1E3D1 comprises the amino acid sequence of SEQ ID NO: 8.
  • the MSP has at least about 70% (e.g., at least about any of 80%, 85%, 90%, 95%, 99%, or 100%) sequence identity to SEQ ID NO: 8.
  • the MSP is a variant of MSP1E3D1 that does not comprise the N-terminal His-tag portion (his-tag removed variant) (hereinafter referred to as MSP1E3D1(-)), which comprises the amino acid sequence of SEQ ID NO: 15.
  • MSP1E3D1(-) comprises the amino acid sequence of SEQ ID NO: 15.
  • MSP2N2 is generated by extending the His-tagged MSP1D1, adding a GT spacer and an additional Helix2 through Helix10. Detailed composition and preparation procedures for MSP2N2 are described in Grinkova et al. Protein Engineering Design and Selection 201023, 843-848, the content of which is incorporated herein by reference in its entirety.
  • the histidine tag is removed from MSP1D1, MSPE1D1, MSP1E2D1, MSPE3D1 or MSP2N2 to generate MSP1D1(-), MSPE1D1(-), MSP1E2D1(-), MSPE3D1(-) or MSP2N2(-), respectively, by cleaving with TEV protease.
  • TEV protease cleavage at the boxed area shown in Table 1 can result in a residual glycine (G) ny-2692819 Attorney Docket No.: 283912000140 amino acid residue on the N-terminal of the resulting MSP protein.
  • the MSP is a variant of Apo-C.
  • the Apo-C family comprises three closely related proteins: ApoC-I, ApoC-II, and ApoC-III, that are mostly made by the liver and, to a lesser degree, in the intestine.
  • ApoC-I, ApoC-II, and ApoC-III comprises the amino acid sequence of SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11, respectively.
  • the MSP has at least about 70% (e.g., at least about any of 80%, 85%, 90%, 95%, 99%, or 100%) sequence identity to any of SEQ ID NOs: 9-11.
  • the MSP is a variant of Apo-E.
  • ApoE is responsible for the uptake and transport of cholesterol in the blood.
  • Apo-E comprises the amino acid sequence of SEQ ID NO: 12.
  • the MSP has at least about 70% (e.g., at least about any of 80%, 85%, 90%, 95%, 99%, or 100%) sequence identity to SEQ ID NO: 12.
  • the MSP is a variant of Apo-M.
  • Apo-M is found associated with high density lipoproteins and to a lesser extent with low density lipoproteins and triglyceride-rich lipoproteins.
  • Apo-M comprises the amino acid sequence of SEQ ID NO: 13.
  • the MSP has at least about 70% (e.g., at least about any of 80%, 85%, 90%, 95%, 99%, or 100%) sequence identity to SEQ ID NO: 13.
  • Phospholipids generally refer to a class of lipid with a hydrophilic head of phosphate moiety and two hydrophobic tails derived from fatty acids, joined by an alcohol or glycerol backbone.
  • the phospholipid is a sphingolipid.
  • Sphingolipids typically refers to a class of lipids containing a backbone of sphingoid bases, which are a set of aliphatic amino alcohols that includes sphingosine.
  • the sphingolipid is sphingosine-1- phosphate.
  • the phospholipid comprises one or more acyl chains. In some embodiments, each acyl chain independently has a length of about 5 to about 30 carbon units, such as about 10 to about 22 carbon units or about 10 to about 16 carbon units.
  • the phospholipid has a glycerol backbone.
  • the phospholipid is phosphatidylcholine (PC).
  • PC generally refers to the class of phospholipids that comprise choline as a head group, glycerophosphoric unit, and a variety of fatty acids.
  • the phospholipid comprises a PC selected from the group consisting of 1-oleoyl-2- palmitoyl-sn-glycero-3-phosphocholine (OPPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 2-dierucoyl-sn-glycero-3- phosphocholine (DEPC), 1-palmitoyl-2-oleoyl-sn-glycero-3- phosphorylcholine (POPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2- dilauroyl-sn-glycero-3-phosphocholine (DLPC), dimyristoylphosphatidylcholine (DMPC), 1,2- distearoyl-sn-glycero-3-phosphocholine (DSPC), and any combination thereof.
  • PC 1-oleoyl-2- palmitoyl-sn-glycero-3-phosphocholine
  • the phospholipid is selected from the group consisting of DMPC, POPC, DPPC, DSPC, and any combination thereof.
  • the phospholipid is DMPC, DPPC, or POPC.
  • the phospholipid is a single type of PC.
  • the phospholipid comprises a mixture of at least two types of PC.
  • the phospholipid is selected from the group consisting of DMPC, DMPS, POPC, DPPC, DSPC, POPS, PiP2, and any combination thereof.
  • the phospholipid is DMPC, DPPC, or POPC.
  • the phospholipid is a single type of PC.
  • the phospholipid comprises a mixture of at least two types of PC. [0115] In some embodiments, the one or more acyl chains of the phospholipid are fully saturated. In some embodiments, the phospholipid is selected from the group consisting of DMPC, DSPC, DPPC, and DMPS. In some embodiments, the phospholipid is DMPC. DMPC is a synthetic phospholipid that comprises two myristoyl fatty acids attached in ester linkage to the first and second carbon of glycerol, and choline attached through a phosphodiester linkage to the third carbon of the glycerol. In some embodiments, the phospholipid is DPPC.
  • DPPC is a synthetic phospholipid that comprises two palmitoyl fatty acids attached in ester linkage to the first and second carbon of glycerol, and choline attached through a phosphodiester linkage to the third carbon of the glycerol.
  • the phospholipid is 1,2-dimyristoyl-sn- glycero-3-phospho-L-serine (DMPS), such as a 14:0 DMPS.
  • DMPS 1,2-dimyristoyl-sn- glycero-3-phospho-L-serine
  • the phospholipid is DSPC.
  • DSPC is a synthetic phospholipid that comprises two stearoyl fatty acids attached in ester linkage to the first and second carbon of glycerol, and choline attached through a phosphodiester linkage to the third carbon of the glycerol.
  • the one or more acyl chains of the phospholipid are unsaturated.
  • the phospholipid is POPC, phosphatidylinositol 4,5-bisphosphate (PiP2), or palmitoyl-oleoyl phosphatidylserine (POPS).
  • the phospholipid is POPC.
  • POPC is a synthetic phospholipid that comprises two fatty acids, palmitic acid and oleic acid, attached in ester linkage to the first and second carbon of glycerol, respectively, and choline attached through a phosphodiester linkage to the third carbon of the glycerol.
  • the phospholipid is PiP2, which could also be referred to as PI(4,5)P2.
  • the PiP2 is 18:1 PI(4,5)P2 or 18:0-20:4 PI(4,5)P2.
  • the PiP2 is 18:1 PI(4,5)P2.
  • the PiP2 is 18:0-20:4 PI(4,5)P2.
  • the phospholipid is DOPC.
  • DOPC is a synthetic phospholipid that comprises two oleoyl fatty acids attached in ester linkage to the first and second carbon of glycerol, and choline attached through a phosphodiester linkage to the third carbon of the glycerol.
  • the phospholipid is DEPC.
  • DEPC is a synthetic phospholipid that comprises two erucoyl fatty acids attached in ester linkage to the first and second carbon of glycerol, and choline attached through a phosphodiester linkage to the third carbon of the glycerol.
  • the phospholipid is 1,2-dimyristoyl-sn-glycero-3-phospho-L- serine (DMPS), such as a 14:0 DMPS.
  • DMPS 1,2-dimyristoyl-sn-glycero-3-phospho-L- serine
  • the phospholipid is DSPC.
  • DSPC is a synthetic phospholipid that comprises two stearoyl fatty acids attached in ester linkage to the first and second carbon of glycerol, and choline attached through a phosphodiester linkage to the third carbon of the glycerol.
  • the phospholipid is PiP 2 , which could also be referred to as PI(4,5)P 2 .
  • the PiP 2 is 18:1 PI(4,5)P 2 or 18:0-20:4 PI(4,5)P 2 .
  • the PiP2 is 18:1 PI(4,5)P2.
  • the PiP2 is 18:0-20:4 PI(4,5)P2.
  • the phospholipid is cationic, anionic, or zwitterionic, or any combination thereof.
  • MSP to Phospholipid ratio [0120] The stoichiometry of MPS and phospholipid is crucial for the formulation, functions, shape homogeneity and size distribution of the nanodiscs used herein.
  • the molar ratio of phospholipid to MSP referred herein is the molar ratio (e.g., average or median molar ratio) of phospholipid to MSP in the pharmaceutical composition. In some embodiments, the molar ratio of phospholipid to MSP referred herein is the molar ratio (e.g., average or median molar ratio) of phospholipid to MSP in the nanodisc.
  • the amount of phospholipid needed to saturate a nanodisc may depend on the identity of the MSP and the phospholipid.
  • phospholipids may comprise a phosphatidyl choline (PC) head group and more than 12 hydrocarbon tails.
  • phospholipids may have the tendency to condense into a bilayer structure.
  • the MSP may contact with a bilayer formed by the phospholipids, wherein the hydrophobic part of the MSP stays in proximity to the hydrophobic tails of the phospholipids and the hydrophilic part of the MSP stays in proximity to a solvent in the pharmaceutical composition.
  • the MSP is a variant of ApoA-I, and the amino acids forming the globular domain and those of the first 11 amino acids of Helix 1 (H1, Table 1) are not in contact with the lipids (Denisov et. Al, J Am Chem Soc. 2004 Mar 24. 126(11):3477-87).
  • Variants of the MSPs without the globular domain, and/or without the H1 domain or portion thereof, and/or without any amino or carboxy terminal tag and protease cleavage sites such as the MSP1D1(-), MSPE1D1(-), MSPE2D1(-), MSPE3D1(-) and MSP2N2(-) provided herein, may have the same molar ratio of the phospholipid to the MSP in the pharmaceutical composition needed to saturate the nanodisc, compared to their parental protein, e.g., MSP1D1, MSPE1D1, MSPE2D1, MSPE3D1, and MSP2N2, respectively.
  • At least about 85% (e.g., at least about any of 90%, 95%, 96%, 97%, 98%, 99%, or 100%) of the plurality of nanodiscs in the pharmaceutical composition are fully-lipidated.
  • “Fully-lipidated”, as used herein, refers to the nanodisc wherein the molar ratio of the phospholipid to the MSP in the pharmaceutical composition is at or slightly above the phospholipid-MSP saturation ratio NS, such as equal to or 0-10% above the phospholipid-MSP saturation ratio N S .
  • the N S for exemplary lipid and MSP combinations are shown in Table 2.
  • the nanodisc comprises a phospholipid and a MSP, wherein at least about 85% (e.g., at least about any of 90%, 95%, 96%, 97%, 98%, 99%, or 100%) of the plurality of nanodiscs are under-lipidated.
  • Under-lipidated refers to the nanodisc wherein the molar ratio of the phospholipid to the MSP in the pharmaceutical composition is less than the phospholipid-MSP saturation ratio N S , such as less than about 95% (e.g., less than about any of 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, or 40%) of the phospholipid-MSP saturation ratio NS.
  • the NS for exemplary lipid and MSP combinations are shown in Table 2.
  • the molar ratio of the phospholipid to the MSP in the pharmaceutical composition is less than a phospholipid-MSP saturation ratio NS.
  • the phospholipid- MSP saturation ratio NS may be calculated according to Denisov et al. (J Am Chem Soc.2004 Mar 24. 126(11):3477-87) and Denisov and Sligar (Chem Rev.2017 Mar 22; 117(6): 4669– 4713), the contents of each of which are incorporated herein by reference in their entirety. Denisov et al.
  • phospholipid-MSP saturation ratio NS is a function of the length M as well as the mean area occupied by a single phospholipid type.
  • AL of Equation 1.1 is a constant depending on the phospholipid, e.g., may be the area occupied by a single phospholipid in a bilayer (in nm 2 ).
  • the area AL may depend on the identity of the lipid used as well as the temperature of assembly relative to the bulk bilayer phase transition temperature.
  • M can be calculated according to Equation 1.2: (Equation 1.2) ny-2692819 Attorney Docket No.: 283912000140 wherein r is the mean radius of the MSP ⁇ -helix (e.g., about 0.55 nm or about 5.5 ⁇ ), L is the helical pitch per MSP residue (e.g., about 0.15 nm or about 1.5 ⁇ ), AL is the mean surface area per phospholipid, N is the number of phospholipid per one bilayer of the phospholipids.
  • M can be referred to from Table 2.
  • the phospholipid-MSP saturation ratio N S may be calculated by Equation 1.1, wherein A L is about 0.52 for DPPC and DMPC, and A L is about 0.69 for POPC.
  • the length of the helical belt that contacts the lipids (M) is the same for the MSP variants of with the H(0.5) helical sequence, compared to their parental MSP protein containing H1 (see Table 1). In some embodiments, this helical belt length (M) may also be the same with or without an added histidine or other tag.
  • the helical belt length M of MSP1D1, MSPE1D1, MSPE2D1, MSPE3D1, and MSP2N2 for calculating NS is the same as the helical belt length M of MSP1D1(-), MSPE1D1(-), MSPE2D1(-), MSPE3D1(-) and MSP2N2(-) for calculating N S , respectively, as the tag and protease cleavage site is not involved in stabilizing the resulting nanodisc.
  • the number of lipids (DMPC, DPPC and POPC) needed to saturate the MSP1D1, MSPE1D1, MSPE2D1, MSPE3D1 nanodiscs may be calculated by Equation 1.1 and the calculation results are summarized in Table 2. Table 2.
  • the molar ratio of the phospholipid to MSP in the pharmaceutical composition is less than the phospholipid-MSP saturation ratio N S , such as less than about 95% (e.g., less than about any of 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, or 40%) of the phospholipid-MSP saturation ratio NS.
  • the NS for exemplary lipid and MSP are shown in Table 2.
  • the MSP is MSP1D1 or MSP1
  • the molar ratio of phospholipid to the MSP is from about 5:1 to about 120:1 (including for example any of about 10:1 to about 100:1, about 10:1 to about 90:1, about 20:1 to about 90:1, about 10:1 to about 85:1, about 30:1 to about 85:1, about 30:1 to about 85:1, about 30:1 to about 65:1, about 32.5:1 to about 65:1, about 10:1 to about 65:1, about 10:1 to about 50:1, about 20:1 to about 65:1, and about 25:1 to about 50:1).
  • the molar ratio of phospholipid to the MSP (e.g., MSP1D1 or MSP1) (e.g., in the nanodisc, or in the pharmaceutical composition) is about any of 10:1, 15:1, 20:1, 25:1, 30:1, 32.5:1, 35:1, 40:1, 45:1, 50:1, 55:1, 60:1, 65:1, 70:1, 75:1, 80:1, 85:1, 90:1, and 95:1.
  • the MSP is MSP1D1 or MSP1
  • the molar ratio of phospholipid to MSP (e.g., in the nanodisc, or in the pharmaceutical composition) is from about 10:1 to about 90:1.
  • the MSP is MSP1D1 or MSP1
  • the phospholipid is POPC
  • the molar ratio of POPC to the MSP is any of about 10:1 to about 90:1, about 32.5:1 to about 85:1, about 50:1 to about 65:1, about 10:1 to about 65:1, about 10:1 to about 85:1, about 32.5:1, about 50:1, or about 65:1.
  • the MSP is MSP1
  • the phospholipid is POPC
  • the molar ratio of POPC to MSP1D1 is about 32.5:1.
  • the MSP is MSP1D1
  • the phospholipid is POPC
  • the molar ratio of POPC to MSP1 (e.g., in the nanodisc, or in the pharmaceutical composition) is about 50:1.
  • the MSP is MSP1D1 or MSP1
  • the phospholipid is POPC
  • the molar ratio of POPC to MSP1D1 or MSP1 (e.g., in the nanodisc, or in the pharmaceutical composition) is about 65:1.
  • the MSP is MSP1D1, MSP1, or His-tag removed variant thereof
  • the phospholipid is POPC
  • the molar ratio of POPC to the MSP is from about 10:1 to about 65:1.
  • the MSP is MSP1D1 (e.g., SEQ ID NO: 3) or His-tag removed variant thereof (e.g., SEQ ID NO: 14)
  • the phospholipid is POPC, and at least about 85% (e.g., at least about any of 90%, 95%, 96%, 97%, 98%, 99%, or 100%) of the plurality of nanodiscs are fully-lipidated.
  • the N S is about 62.
  • the MSP is MSP1D1 or His-tag removed variant thereof
  • the phospholipid is POPC, wherein the molar ratio of POPC to the MSP (e.g., in the nanodisc, or in the pharmaceutical composition) is about 65:1.
  • the MSP is MSP1D1 (e.g., SEQ ID NO: 3) or His-tag removed variant thereof (e.g., SEQ ID NO: 14), the phospholipid is POPC, and at least about 85% (e.g., at least about any of 90%, 95%, 96%, 97%, 98%, 99%, or 100%) of the plurality of nanodiscs in the pharmaceutical composition are under-lipidated.
  • the molar ratio of POPC to MSP1D1 or His-tag removed variant thereof in the pharmaceutical composition is less than a phospholipid-MSP saturation ratio N S of about 62:1, such as less than about 95% (e.g., less than about any of 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, or 40%) of the phospholipid-MSP saturation ratio NS of about 62:1.
  • the molar ratio of POPC to MSP1D1 or His-tag removed variant thereof in the pharmaceutical composition is less than or equal to 60:1, such as less than or equal to about any of 58:1, 55:1, 50:1, 45:1, 40:1, 35:1, 32.5:1, 30:1, or less.
  • the molar ratio of POPC to MSP1D1 in the pharmaceutical composition is about 10:1 to about 60:1, such as any of about 20:1 to about 60:1, about 30:1 to about 55:1, about 40:1 to about 55:1, about 58:1, about 55:1, about 50:1, about 45:1, about 40:1, about 35:1, about 32.5:1, or about 30:1.
  • the MSP is MSP1D1 or His-tag removed variant thereof, the phospholipid is POPC, and the molar ratio of POPC to the MSP in the pharmaceutical composition is less than about 58:1, such as about 50:1 or about 32.5:1.
  • the MSP is MSP1D1 comprising the amino acid sequence of SEQ ID NO: 3.
  • the MSP is an MSP1D1 His-tag removed variant comprising the amino acid sequence of SEQ ID NO: 14.
  • the MSP is MSP1D1 or MSP1
  • the phospholipid is DMPC
  • the molar ratio of DMPC to MSP is any of about 10:1 to about 85:1, about 10:1 to about 120:1, about 50:1 to about 100:1, about 50:1 to about 90:1, about 70:1 to about 90:1, about 80:1, or about 85:1.
  • the MSP is MSP1
  • the phospholipid is DMPC
  • the molar ratio of DMPC to MSP1 is about 80:1.
  • the MSP is MSP1 or His-tag removed variant thereof
  • the phospholipid is DMPC, wherein the molar ratio of DMPC to the MSP1 or His-tag removed variant thereof e.g., in the nanodisc, or in the pharmaceutical composition) is about 80:1.
  • the MSP is MSP1D1
  • the phospholipid is DMPC
  • the molar ratio of DMPC to MSP1D1 is about 85:1.
  • the MSP is MSP1D1, MSP1, ny-2692819 Attorney Docket No.: 283912000140 or His-tag removed variant thereof
  • the phospholipid is DMPC, wherein the molar ratio of DMPC to the MSP in the pharmaceutical composition is from about 10:1 to about 85:1.
  • the MSP is MSP1D1 (e.g., SEQ ID NO: 3) or His-tag removed variant thereof (e.g., SEQ ID NO: 14), the phospholipid is DMPC, and at least about 85% (e.g., at least about any of 90%, 95%, 96%, 97%, 98%, 99%, or 100%) of the plurality of nanodiscs are fully-lipidated.
  • the N S is about 81.
  • the MSP is MSP1D1 or His-tag removed variant thereof
  • the phospholipid is DMPC, wherein the molar ratio of DMPC to the MSP in the pharmaceutical composition is about 85:1.
  • the MSP is MSP1D1 (e.g., SEQ ID NO: 3) or His-tag removed variant thereof (e.g., SEQ ID NO: 14), the phospholipid is DMPC, wherein at least about 85% (e.g., at least about any of 90%, 95%, 96%, 97%, 98%, 99%, or 100%) of the plurality of nanodiscs in the pharmaceutical composition are under-lipidated.
  • MSP1D1 e.g., SEQ ID NO: 3
  • His-tag removed variant thereof e.g., SEQ ID NO: 14
  • the phospholipid is DMPC, wherein at least about 85% (e.g., at least about any of 90%, 95%, 96%, 97%, 98%, 99%, or 100%) of the plurality of nanodiscs in the pharmaceutical composition are under-lipidated.
  • the molar ratio of DMPC to MSP1D1 or His-tag removed variant thereof in the pharmaceutical composition is less than a phospholipid-MSP saturation ratio N S of about 81:1, such as less than about 95% (e.g., less than about any of 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, or 40%) of the phospholipid-MSP saturation ratio NS of about 81:1.
  • the molar ratio of DMPC to MSP1D1 or His-tag removed variant thereof in the pharmaceutical composition is less than or equal to about 78:1, such as less than or equal to about any of 77:1, 76:1, 75:1, 74:1, 72:1, 70:1, 65:1, 60:1, 55:1, 51:1, 50:1, 45:1, 40:1, or less.
  • the molar ratio of DMPC to MSP1D1 or His-tag removed variant thereof in the pharmaceutical composition is about 10:1 to about 80:1, such as any of about 10:1 to about 78:1, about 20:1 to about 78:1, about 20:1 to about 78:1, about 30:1 to about 78:1, about 70:1 to about 80:1, about 74:1 to about 77:1, about 40:1 to about 60:1, about 65:1 to about 75:1, about 70:1 to about 75:1, about 72:1 to about 78:1, about 46:1 to about 55:1, about 75:1, about 54:1 to about 56:1, about 65:1, about 60:1, about 55:1, about 51:1, about 50:1, about 45:1, about 40:1, about 35:1, or about 30:1.
  • the molar ratio of DMPC to MSP1D1 or His-tag removed variant thereof in the pharmaceutical composition is about 65:1 to about 75:1.
  • the MSP is MSP1D1 (e.g., SEQ ID NO: 3) or His-tag removed variant thereof (e.g., SEQ ID NO: 14)
  • the phospholipid is DMPC, wherein the molar ratio of the ny-2692819 Attorney Docket No.: 283912000140 phospholipid to the MSP in the pharmaceutical composition is about 72:1 to about 78:1, such as about 75:1.
  • the MSP is MSP1D1 (e.g., SEQ ID NO: 3) or His-tag removed variant thereof (e.g., SEQ ID NO: 14),
  • the phospholipid is DMPC, wherein the molar ratio of the phospholipid to the MSP in the pharmaceutical composition is from about 40:1 to about 60:1, such as about 46:1 to about 55:1, or about 51:1.
  • the MSP is MSP1D1 or MSP1
  • the phospholipid is DPPC
  • the molar ratio of DPPC to the MSP is any of about 10:1 to about 120:1, about 10:1 to about 100:1, about 50:1 to about 100:1, about 70:1 to about 100:1, about 80:1 to about 100:1, about 80:1, about 85:1, or about 90:1.
  • the MSP is MSP1
  • the phospholipid is DPPC
  • the molar ratio of DPPC to MSP1 is about 90:1.
  • the MSP is MSP1D1, MSP1, or His-tag removed variant thereof
  • the phospholipid is DPPC, wherein the molar ratio of DPPC to the MSP (e.g., in the nanodisc, or in the pharmaceutical composition) is from about 10:1 to about 90:1.
  • the MSP is MSP1D1 (e.g., SEQ ID NO: 3) or His-tag removed variant thereof (e.g., SEQ ID NO: 14), the phospholipid is DPPC, wherein at least about 85% (e.g., at least about any of 90%, 95%, 96%, 97%, 98%, 99%, or 100%) of the plurality of nanodiscs in the pharmaceutical composition are fully-lipidated.
  • the NS is about 81.
  • the MSP is MSP1 or His-tag removed variant thereof
  • the phospholipid is DPPC, wherein the molar ratio of DPPC to the MSP in the pharmaceutical composition is about 90:1.
  • the MSP is MSP1D1 (e.g., SEQ ID NO: 3) or His-tag removed variant thereof (e.g., SEQ ID NO: 14), the phospholipid is DPPC, wherein at least about 85% (e.g., at least about any of 90%, 95%, 96%, 97%, 98%, 99%, or 100%) of the plurality of nanodiscs in the pharmaceutical composition are under-lipidated.
  • MSP1D1 e.g., SEQ ID NO: 3
  • His-tag removed variant thereof e.g., SEQ ID NO: 14
  • the phospholipid is DPPC, wherein at least about 85% (e.g., at least about any of 90%, 95%, 96%, 97%, 98%, 99%, or 100%) of the plurality of nanodiscs in the pharmaceutical composition are under-lipidated.
  • the molar ratio of DPPC to MSP1D1 or His-tag removed variant thereof in the pharmaceutical composition is less than a phospholipid-MSP saturation ratio NS of about 81:1, such as less than about 95% (e.g., less than about any of 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, or 40%) of the phospholipid-MSP saturation ratio N S of about 81:1.
  • the ny-2692819 Attorney Docket No.: 283912000140 molar ratio of DPPC to MSP1D1 or His-tag removed variant thereof in the pharmaceutical composition is less than or equal to about 78:1, such as less than or equal to about any of 77:1, 76:1, 75:1, 74:1, 72:1, 70:1, 65:1, 60:1, 55:1, 51:1, 50:1, 45:1, 40:1, or less.
  • the molar ratio of DPPC to MSP1D1 or His-tag removed variant thereof in the pharmaceutical composition is about 10:1 to about 80:1, such as any of about 10:1 to about 78:1, about 20:1 to about 78:1, about 20:1 to about 78:1, about 30:1 to about 78:1, about 70:1 to about 80:1, about 74:1 to about 77:1, about 40:1 to about 60:1, about 65:1 to about 75:1, about 70:1 to about 75:1, about 72:1 to about 78:1, about 46:1 to about 55:1, about 75:1, about 54:1 to about 56:1, about 65:1, about 60:1, about 55:1, about 51:1, about 50:1, about 45:1, about 40:1, about 35:1, or about 30:1.
  • the MSP is MSP1D1 or His-tag removed variant thereof, the phospholipid is DPPC, the molar ratio of DPPC to the MSP in the pharmaceutical composition is less than about 75:1.
  • the MSP is MSP1E3, and the molar ratio of phospholipid to MSP1E3 (e.g., the molar ratio of phospholipid to MSP1E3 in the nanodisc, or in the pharmaceutical composition) is from about 50:1 to about 250:1 (including for example any of about 50:1 to about 200:1, about 100:1 to about 200:1, about 100:1 to about 180:1, about 100:1 to about 170:1, about 130:1 to about 180:1, or about 130:1 to about 170:1).
  • the molar ratio of phospholipid to MSP1E3 (e.g., in the nanodisc, or in the pharmaceutical composition) is about any of 50:1, 100:1, 110:1, 120:1, 130:1, 140:1, 150:1, 160:1, 170:1, 180:1, 190:1, 200:1, 210:1, 220:1, 230:1, 240:1, or 250:1.
  • at least about 85% e.g., at least about any of 90%, 95%, 96%, 97%, 98%, 99%, or 100%
  • the plurality of nanodiscs in the pharmaceutical composition are fully-lipidated.
  • the MSP is MSP1E3, MSP1E3D1, or His-tag removed variant thereof, and the molar ratio of the phospholipid to the MSP (e.g., in the nanodisc, or in the pharmaceutical composition) is from about 50:1 to about 200:1, such as any of about 50:1 to about 100:1, about 100:1 to about 200:1, about 100:1 to about 180:1, about 100:1 to about 170:1, about 130:1 to about 180:1, or about 130:1 to about 170:1.
  • the MSP is MSP1E3
  • the phospholipid is POPC
  • the molar ratio of POPC to MSP1E3 is about 100:1 to about 250:1 (including for example any of about 100:1 to about 200:1, about 120:1 to about 190:1, about 120:1 to about 180:1, and about 130:1 to about 170:1).
  • the MSP is MSP1E3
  • the phospholipid is POPC
  • the molar ratio of POPC to MSP1E3 is about any of 100:1, 110:1, 120:1, 130:1, 140:1, 150:1, 160:1, and 170:1.
  • the molar ratio of POPC to MSP1E3 is about 130:1.
  • the MSP is MSP1E3 or His-tag removed variant thereof
  • the phospholipid is POPC, wherein the molar ratio of POPC to the MSP (e.g., in the nanodisc, or in the pharmaceutical composition) is about 130:1.
  • the MSP is MSP1E3
  • the phospholipid is DMPC
  • the molar ratio of DMPC to MSP1E3 is about 100:1 to about 200:1 (including for example about any of about 130:1 to about 190:1, or about 120:1 to about 170:1).
  • the molar ratio of DMPC to MSP1E3 is about any of 100:1, 110:1, 120:1, 130:1, 140:1, 150:1, 160:1, and 170:1.
  • the molar ratio of DMPC to MSP1E3 (e.g., in the nanodisc, or in the pharmaceutical composition) is about 150:1.
  • the MSP is MSP1E3 or His-tag removed variant thereof
  • the phospholipid is DMPC, wherein the molar ratio of DMPC to the MSP (e.g., in the nanodisc, or in the pharmaceutical composition) is about 150:1.
  • the MSP is MSP1E3
  • the phospholipid is DPPC
  • the molar ratio of DPPC to MSP1E3 is about 120:1 to about 220:1 (including for example about any of about 140:1 to about 200:1, or about 150:1 to about 190:1).
  • the molar ratio of DPPC to MSP1E3 is about any of 130:1, 140:1, 150:1, 160:1, 170:1, 180:1, 190:1, 200:1, 210:1, 220:1, and 230:1.
  • the molar ratio of DPPC to MSP1E3 (e.g., in the nanodisc, or in the pharmaceutical composition) is about 170:1.
  • the MSP is MSP1E3 or His-tag removed variant thereof
  • the phospholipid is DPPC, wherein the molar ratio of DPPC to the MSP (e.g., in the nanodisc, or in the pharmaceutical composition) is about 170:1.
  • the MSP is MSP1E3D1 (e.g., SEQ ID NO: 8) or His-tag removed variant thereof (e.g., SEQ ID NO: 15)
  • the phospholipid is DMPC, wherein at least about 85% (e.g., at least about any of 90%, 95%, 96%, 97%, 98%, 99%, or 100%) of the plurality of nanodiscs in the pharmaceutical composition are fully-lipidated.
  • the N S is about 167.
  • the MSP is MSP1E3D1 (e.g., SEQ ID NO: 8) or His-tag removed variant thereof (e.g., SEQ ID NO: 15), the phospholipid is DMPC, wherein at least about 85% (e.g., at least about any of 90%, 95%, 96%, 97%, 98%, 99%, or 100%) of the plurality of nanodiscs in the pharmaceutical composition are under-lipidated.
  • MSP1E3D1 e.g., SEQ ID NO: 8
  • His-tag removed variant thereof e.g., SEQ ID NO: 15
  • the phospholipid is DMPC, wherein at least about 85% (e.g., at least about any of 90%, 95%, 96%, 97%, 98%, 99%, or 100%) of the plurality of nanodiscs in the pharmaceutical composition are under-lipidated.
  • the MSP is MSP1E3D1 or His-tag removed variant thereof
  • the phospholipid is DMPC, wherein the molar ratio of the phospholipid to the MSP in the pharmaceutical composition is less than a phospholipid-MSP saturation ratio NS of about 167:1, such as less than about 95% (e.g., less than about any of 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, or 40%) of the phospholipid-MSP saturation ratio N S of about 167:1.
  • the molar ratio of DMPC to MSP1E3D1 or His-tag removed variant thereof in the pharmaceutical composition is less than or equal to about 160:1, such as less than or equal to about any of 158:1, 155:1, 150:1, 145:1, 140:1, 135:1, 130:1, 125:1, 120:1, 115:1, 110:1, 105:1, 100:1, 95:1, 90:1, 85:1, 80:1, 75:1, or less.
  • the molar ratio of DMPC to MSP1E3D1 or His-tag removed variant thereof in the pharmaceutical composition is about 50:1 to about 160:1, such as any of about 50:1 to about 150:1, about 60:1 to about 140:1, about 60:1 to about 130:1, about 60:1 to about 120:1, about 60:1 to about 100:1, about 60:1 to about 70:1, about 90:1 to about 100:1, about 50:1 to about 100:1, about 87:1 to about 96:1, about 58:1 to about 68:1, about 60:1, about 62:1, about 63:1, about 65:1, about 70:1, about 80:1, about 90:1, about 93:1, about 100:1, or about 110:1.
  • the MSP is MSP1E3 or His-tag removed variant thereof
  • the phospholipid is DMPC
  • the molar ratio of DMPC to the MSP in the pharmaceutical composition is about 150:1.
  • the MSP is MSP1E3D1 (e.g., SEQ ID NO: 8) or His-tag removed variant thereof (e.g., SEQ ID NO: 15)
  • the phospholipid is DMPC, wherein the molar ratio of DMPC to the MSP in the pharmaceutical composition is about 87:1 to about 96:1, such as about 93:1, or about 58:1 to about 68:1, such as about 63:1, or about 62:1.
  • the MSP is MSP1E3D1
  • the phospholipid is DMPC
  • the molar ratio of the phospholipid to MSP in the pharmaceutical composition is about 58:1 to about 68:1, such as about 62:1.
  • the MSP is MSP1E3D1.
  • the MSP is an MSP1E3D1 His-tag removed variant comprising the amino acid sequence of SEQ ID NO: 15.
  • the MSP is MSP1E3D1 (e.g., SEQ ID NO: 8) or His-tag removed variant thereof (e.g., SEQ ID NO: 15), the phospholipid is DPPC, wherein at least about 85% (e.g., at least about any of 90%, 95%, 96%, 97%, 98%, 99%, or 100%) of the plurality of nanodiscs in the pharmaceutical composition are fully-lipidated.
  • the NS is about 167.
  • the MSP is MSP1E3 or His-tag removed variant thereof
  • the phospholipid is DPPC, wherein the molar ratio of DPPC to the MSP in the pharmaceutical composition is about 170:1.
  • the MSP is MSP1E3D1 (e.g., SEQ ID NO: 8) or His-tag removed variant thereof (e.g., SEQ ID NO: 15), the phospholipid is DPPC, wherein at least about 85% (e.g., at least about any of 90%, 95%, 96%, 97%, 98%, 99%, or 100%) of the plurality of nanodiscs in the pharmaceutical composition are under-lipidated.
  • MSP1E3D1 e.g., SEQ ID NO: 8
  • His-tag removed variant thereof e.g., SEQ ID NO: 15
  • the phospholipid is DPPC, wherein at least about 85% (e.g., at least about any of 90%, 95%, 96%, 97%, 98%, 99%, or 100%) of the plurality of nanodiscs in the pharmaceutical composition are under-lipidated.
  • the MSP is MSP1E3D1 or His-tag removed variant thereof
  • the phospholipid is DPPC
  • the molar ratio of DPPC to the MSP in the pharmaceutical composition is less than a phospholipid- MSP saturation ratio NS of about 167:1, such as less than about 95% (e.g., less than about any of 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, or 40%) of the phospholipid-MSP saturation ratio N S of about167:1.
  • the molar ratio of DPPC to MSP1E3D1 or His-tag removed variant thereof in the pharmaceutical composition is less than or equal to about 160:1, such as less than or equal to about any of 158:1, 155:1, 150:1, 145:1, 140:1, 135:1, 130:1, 125:1, 120:1, 115:1, 110:1, 105:1, 100:1, 95:1, 90:1, 85:1, 80:1, 75:1, or less.
  • the molar ratio of DPPC to MSP1E3D1 or His-tag removed variant thereof in the pharmaceutical composition is about 70:1 to about 160:1, such as any of about 80:1 to about 150:1, about 70:1 to about 140:1, about 70:1 to about 130:1, about 80:1 to about 120:1, about 80:1 to about 100:1, about 80:1, about 90:1, about 100:1 about 87:1 to about 96:1, about 58:1 to about 68:1, about 60:1, about 62:1, about 63:1, about 65:1, about 70:1, about 80:1, about 90:1, about 93:1, about 100:1, or about 110:1.
  • the MSP is MSP1E3 or His-tag removed variant thereof
  • the phospholipid is DPPC, wherein the molar ratio of DPPC to ny-2692819 Attorney Docket No.: 283912000140 the MSP in the pharmaceutical composition is less than about 158:1.
  • the MSP is MSP1E3D1.
  • the MSP is an MSP1E3D1 His-tag removed variant comprising the amino acid sequence of SEQ ID NO: 15.
  • the MSP is MSP1E3D1 (e.g., SEQ ID NO: 8) or His-tag removed variant thereof (e.g., SEQ ID NO: 15), the phospholipid is POPC, wherein at least about 85% (e.g., at least about any of 90%, 95%, 96%, 97%, 98%, 99%, or 100%) of the plurality of nanodiscs are fully-lipidated.
  • the N S is about 126.
  • the MSP is MSP1E3D1 (e.g., SEQ ID NO: 8) or His-tag removed variant thereof (e.g., SEQ ID NO: 15), the phospholipid is POPC, wherein at least about 85% (e.g., at least about any of 90%, 95%, 96%, 97%, 98%, 99%, or 100%) of the plurality of nanodiscs are under-lipidated.
  • MSP1E3D1 e.g., SEQ ID NO: 8
  • His-tag removed variant thereof e.g., SEQ ID NO: 15
  • the phospholipid is POPC, wherein at least about 85% (e.g., at least about any of 90%, 95%, 96%, 97%, 98%, 99%, or 100%) of the plurality of nanodiscs are under-lipidated.
  • the MSP is MSP1E3D1 or His-tag removed variant thereof
  • the phospholipid is POPC
  • the molar ratio of POPC to the MSP in the pharmaceutical composition is less than a phospholipid-MSP saturation ratio N S of about 126: 1, such as less than about 95% (e.g., less than about any of 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, or 40%) of the phospholipid-MSP saturation ratio N S of about 126:1.
  • the molar ratio of POPC to MSP1E3D1 or His-tag removed variant thereof in the pharmaceutical composition is less than or equal to about 120:1, such as less than or equal to about any of 118:1, 115:1, 110:1, 105:1, 100:1, 95:1, 90:1, 85:1, 80:1, 75:1, 70:1, 65:1, or less.
  • the molar ratio of POPC to MSP1E3D1 or His-tag removed variant thereof in the pharmaceutical composition is about 60:1 to about 120:1, such as any of about 70:1 to about 120:1, about 70:1 to about 110:1, about 70:1 to about 100:1, about 80:1 to about 110:1, about 80:1 to about 100:1, about 80:1, about 90:1, about 100:1, or about 110:1.
  • the MSP is MSP1E3D1 or His-tag removed variant thereof
  • the phospholipid is POPC, wherein the molar ratio of POPC to the MSP in the pharmaceutical composition is less than about 118:1.
  • the MSP is MSP1E3D1.
  • the MSP is an MSP1E3D1 His-tag removed variant comprising the amino acid sequence of SEQ ID NO: 15.
  • the MSP is MSP1E1D1 (e.g., SEQ ID NO: 16) or His-tag removed variant thereof
  • the phospholipid is DMPC, wherein at least about 85% (e.g., at least ny-2692819 Attorney Docket No.: 283912000140 about any of 90%, 95%, 96%, 97%, 98%, 99%, or 100%) of the plurality of nanodiscs in the pharmaceutical composition are fully-lipidated.
  • the NS is about 107.
  • the MSP is MSP1E1D1 (e.g., SEQ ID NO: 16) or His-tag removed variant thereof
  • the phospholipid is DMPC, wherein at least about 85% (e.g., at least about any of 90%, 95%, 96%, 97%, 98%, 99%, or 100%) of the plurality of nanodiscs in the pharmaceutical composition are under-lipidated.
  • the MSP is MSP1E1D1 or His-tag removed variant thereof
  • the phospholipid is DMPC, wherein the molar ratio of the phospholipid to MSP in the pharmaceutical composition is less than a phospholipid-MSP saturation ratio NS of about 107:1, such as less than about 95% (e.g., less than about any of 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, or 40%) of the phospholipid-MSP saturation ratio NS of about 107:1.
  • the molar ratio of DMPC to MSP1D1E1 or His- tag removed variant thereof in the pharmaceutical composition is less than or equal to about 105:1, such as less than or equal to about any of 100:1, 95:1, 90:1, 85:1, 80:1, 75:1, 70:1, 65:1, 60:1, 55:1, 50:1, 45:1, 40:1, or less.
  • the molar ratio of DMPC to MSP1E1D1 or His-tag removed variant thereof in the pharmaceutical composition is about 30:1 to about 100:1, such as any of about 40:1 to about 90:1, about 50:1 to about 80:1, about 50:1 to about 70:1, about 60:1 to about 70:1, about 60:1 to about 65:1, about 70:1, about 65:1, about 60:1, or about 55:1.
  • the molar ratio of DMPC to MSP1E1D1 or His-tag removed variant thereof in the pharmaceutical composition is about 60:1, about 62:1, about 64:1 to about 66:1, or about 68:1.
  • the MSP is MSP1E1D1.
  • the MSP is a His-tag removed variant of MSP1E1D1.
  • the MSP is MSP1E1D1 (e.g., SEQ ID NO: 16) or His-tag removed variant thereof
  • the phospholipid is DPPC, wherein at least about 85% (e.g., at least about any of 90%, 95%, 96%, 97%, 98%, 99%, or 100%) of the plurality of nanodiscs in the pharmaceutical composition are fully-lipidated.
  • the NS is about 107.
  • the MSP is MSP1E1D1 (e.g., SEQ ID NO: 16) or His-tag removed variant thereof
  • the phospholipid is DPPC, wherein at least about 85% (e.g., at least about any of 90%, 95%, 96%, 97%, 98%, 99%, or 100%) of the plurality of nanodiscs in the pharmaceutical composition are under-lipidated.
  • the MSP is MSP1E1D1 or His-tag removed variant thereof
  • the phospholipid is DPPC, wherein the molar ratio of the ny-2692819 Attorney Docket No.: 283912000140 phospholipid to MSP in the pharmaceutical composition is less than a phospholipid-MSP saturation ratio NS of about 107:1, such as less than about 95% (e.g., less than about any of 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, or 40%) of the phospholipid-MSP saturation ratio N S of about 107:1.
  • the molar ratio of DPPC to MSP1D1E1 or His- tag removed variant thereof in the pharmaceutical composition is less than or equal to about 105:1, such as less than or equal to about any of 100:1, 95:1, 90:1, 85:1, 80:1, 75:1, 70:1, 65:1, 60:1, 55:1, 50:1, 45:1, 40:1, or less.
  • the molar ratio of DPPC to MSP1E1D1 or His-tag removed variant thereof is about 30:1 to about 100:1, such as any of about 40:1 to about 90:1, about 50:1 to about 80:1, about 50:1 to about 70:1, about 60:1 to about 70:1, about 60:1 to about 65:1, about 70:1, about 65:1, about 60:1, or about 55:1.
  • the molar ratio of DPPC to MSP1E1D1 or His-tag removed variant thereof in the pharmaceutical composition is about 60:1, 62:1, about 64:1 to about 66:1, or about 68:1.
  • the MSP is MSP1E1D1.
  • the MSP is a His-tag removed variant of MSP1E1D1.
  • the MSP is MSP1E1D1 (e.g., SEQ ID NO: 16) or His-tag removed variant thereof
  • the phospholipid is POPC, wherein at least about 85% (e.g., at least about any of 90%, 95%, 96%, 97%, 98%, 99%, or 100%) of the plurality of nanodiscs in the pharmaceutical composition are fully-lipidated.
  • the NS is about 80.
  • the MSP is MSP1E1D1 (e.g., SEQ ID NO: 16) or His-tag removed variant thereof
  • the phospholipid is POPC, and at least about 85% (e.g., at least about any of 90%, 95%, 96%, 97%, 98%, 99%, or 100%) of the plurality of nanodiscs in the pharmaceutical composition are under-lipidated.
  • the MSP is MSP1E1D1 (or His-tag removed variant thereof, the phospholipid is POPC, wherein the molar ratio of the phospholipid to the MSP in the pharmaceutical composition is less than a phospholipid-MSP saturation ratio NS of about 80:1, such as less than about 95% (e.g., less than about any of 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, or 40%) of the phospholipid-MSP saturation ratio N S of about 80:1.
  • a phospholipid-MSP saturation ratio NS such as less than about 95% (e.g., less than about any of 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, or 40%) of the phospholipid-MSP saturation ratio N S of about 80:1.
  • the molar ratio of POPC to MSP1E1D1 or His-tag removed variant thereof in the pharmaceutical composition is less than or equal to about 76:1, such as less than or equal to about any of 76:1, 70:1, 65:1, 60:1, 55:1, 50:1, 45:1, 40:1, or less.
  • the molar ratio of POPC to MSP1D1E1 or His-tag removed variant thereof in the pharmaceutical composition is about 10:1 to about 75:1, such as any of about 20:1 ny-2692819 Attorney Docket No.: 283912000140 to about 75:1, about 20:1 to about 60:1, about 30:1 to about 60:1, about 40:1 to about 60:1, about 65:1, about 60:1, about 55:1, about 50:1, about 45:1, about 40:1, about 35:1, or about 30:1.
  • the molar ratio of POPC to MSP1E1D1 or His-tag removed variant thereof in the pharmaceutical composition is about 50:1, about 54:1 to about 56:1, or about 55:1.
  • the MSP is MSP1E1D1. In some embodiments, the MSP is a His-tag removed variant of MSP1E1D1. [0162] In some embodiments, the MSP is MSP1E2D1 (e.g., SEQ ID NO: 17) or His-tag removed variant thereof, the phospholipid is DMPC, wherein at least about 85% (e.g., at least about any of 90%, 95%, 96%, 97%, 98%, 99%, or 100%) of the plurality of nanodiscs in the pharmaceutical composition are fully-lipidated. In some embodiments, the NS is about 135.
  • the MSP is MSP1E2D1 (e.g., SEQ ID NO: 17) or His-tag removed variant thereof
  • the phospholipid is DMPC, wherein at least about 85% (e.g., at least about any of 90%, 95%, 96%, 97%, 98%, 99%, or 100%) of the plurality of nanodiscs in the pharmaceutical composition are under-lipidated.
  • the MSP is MSP1E2D1 or His-tag removed variant thereof
  • the phospholipid is DMPC, wherein the molar ratio of the phospholipid to the MSP in the pharmaceutical composition is less than a phospholipid-MSP saturation ratio NS of about 135:1, such as less than about 95% (e.g., less than about any of 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, or 40%) of the phospholipid-MSP saturation ratio NS of about 135:1.
  • the molar ratio of DMPC to MSP1E2D1 or His- tag removed variant thereof in the pharmaceutical composition is less than or equal to about 128:1, such as less than or equal to about any of 125:1, 120:1, 115:1, 110:1, 105:1, 100:1, 95:1, 90:1, 85:1, 80:1, 75:1, 70:1, 65:1, 60:1, 55:1, or less.
  • the molar ratio of DMPC to MSP1E2D1 or His-tag removed variant thereof in the pharmaceutical composition is about 50:1 to about 130:1, such as any of about 50:1 to about 120:1, about 60:1 to about 120:1, about 70:1 to about 110:1, about 70:1 to about 100:1, about 70:1, about 75:1, about 80:1, or about 85:1.
  • the MSP is MSP1E2D1.
  • the MSP is a His-tag removed variant of MSP1E2D1.
  • the MSP is MSP1E2D1 (e.g., SEQ ID NO: 17) or His-tag removed variant thereof
  • the phospholipid is DPPC, wherein at least about 85% (e.g., at least ny-2692819 Attorney Docket No.: 283912000140 about any of 90%, 95%, 96%, 97%, 98%, 99%, or 100%) of the plurality of nanodiscs in the pharmaceutical composition are fully-lipidated.
  • the NS is about 135.
  • the MSP is MSP1E2D1 (e.g., SEQ ID NO: 17) or His-tag removed variant thereof
  • the phospholipid is DPPC, wherein at least about 85% (e.g., at least about any of 90%, 95%, 96%, 97%, 98%, 99%, or 100%) of the plurality of nanodiscs in the pharmaceutical composition are under-lipidated.
  • the MSP is MSP1E2D1 or His-tag removed variant thereof
  • the phospholipid is DPPC
  • the molar ratio of the phospholipid to the MSP in the pharmaceutical composition is less than a phospholipid-MSP saturation ratio NS of about 135:1, such as less than about 95% (e.g., less than about any of 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, or 40%) of the phospholipid-MSP saturation ratio NS of about 135:1.
  • the molar ratio of DPPC to MSP1E2D1 or His- tag removed variant thereof in the pharmaceutical composition is less than or equal to about 128:1, such as less than or equal to about any of 125:1, 120:1, 115:1, 110:1, 105:1, 100:1, 95:1, 90:1, 85:1, 80:1, 75:1, 70:1, 65:1, 60:1, 55:1, or less.
  • the molar ratio of DPPC to MSP1E2D1 or His-tag removed variant thereof in the pharmaceutical composition is about 50:1 to about 130:1, such as any of about 50:1 to about 120:1, about 60:1 to about 120:1, about 70:1 to about 110:1, about 70:1 to about 100:1, about 70:1, about 75:1, about 80:1, or about 85:1.
  • the MSP is MSP1E2D1.
  • the MSP is a His-tag removed variant of MSP1E2D1.
  • the MSP is MSP1E2D1 (e.g., SEQ ID NO: 17) or His-tag removed variant thereof
  • the phospholipid is POPC, wherein at least about 85% (e.g., at least about any of 90%, 95%, 96%, 97%, 98%, 99%, or 100%) of the plurality of nanodiscs in the pharmaceutical composition are fully-lipidated.
  • the NS is about 102.
  • the MSP is MSP1E2D1 (e.g., SEQ ID NO: 17) or His-tag removed variant thereof
  • the phospholipid is POPC, and at least about 85% (e.g., at least about any of 90%, 95%, 96%, 97%, 98%, 99%, or 100%) of the plurality of nanodiscs in the pharmaceutical composition are under-lipidated.
  • the MSP is MSP1E2D1 or His-tag removed variant thereof
  • the phospholipid is POPC
  • the molar ratio of the phospholipid to the MSP in the pharmaceutical composition is less than a phospholipid-MSP saturation ratio N S of about 102:1, such as less than about 95% (e.g., less than about any of 90%, ny-2692819 Attorney Docket No.: 283912000140 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, or 40%) of the phospholipid-MSP saturation ratio NS of about 102:1.
  • the molar ratio of POPC to MSP1E2D1 or His- tag removed variant thereof in the pharmaceutical composition is less than about 96:1, such as less than about any of 95:1, 90:1, 85:1, 80:1, 75:1, 70:1, 65:1, 60:1, 55:1, 50:1, or less.
  • the molar ratio of POPC to MSP1E2D1 or His-tag removed variant thereof in the pharmaceutical composition is about 40:1 to about 95:1, such as any of about 50:1 to about 90:1, about 60:1 to about 90:1, about 70:1 to about 90:1, about 70:1 to about 80:1, about 70:1, about 75:1, about 80:1, or about 85:1.
  • the MSP is MSP1E2D1. In some embodiments, the MSP is a His-tag removed variant of MSP1E2D1. [0168] In some embodiments, the MSP is MSP2N2 (e.g., SEQ ID NO: 18) or His-tag removed variant thereof, the phospholipid is DMPC, wherein at least about 85% (e.g., at least about any of 90%, 95%, 96%, 97%, 98%, 99%, or 100%) of the plurality of nanodiscs in the pharmaceutical composition are fully-lipidated. In some embodiments, the N S is about 387.
  • the MSP is MSP2N2 (e.g., SEQ ID NO: 18) or His-tag removed variant thereof, the phospholipid is DMPC, and at least about 85% (e.g., at least about any of 90%, 95%, 96%, 97%, 98%, 99%, or 100%) of the plurality of nanodiscs in the pharmaceutical composition are under-lipidated.
  • the MSP is MSP2N2 or His-tag removed variant thereof, the phospholipid is DMPC, wherein the molar ratio of the phospholipid to the MSP in the pharmaceutical composition is less than a phospholipid-MSP saturation ratio NS of about 387:1, such as less than about 95% (e.g., less than about any of 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, or 40%) of the phospholipid-MSP saturation ratio N S of about 387:1.
  • the molar ratio of DMPC to MSP2N2 or His-tag removed variant thereof in the pharmaceutical composition is less than or equal to about 370:1, such as less than or equal to about any of 367:1, 360:1, 350:1, 340:1, 330:1, 320:1, 310:1, 300:1, 290:1, 280:1, 270:1, 260:1, 250:1, 240:1, 230:1, 220:1, 210:1, 200:1, 190:1, 180:1, 170:1, 160:1, 150:1, 140:1, 130:1, 120:1, 110:1, 100:1, or less.
  • the molar ratio of DMPC to MSP2N2 or His-tag removed variant thereof in the pharmaceutical composition is about 150:1 to about 350:1, such as any of about 200:1 to about 350:1, about 250:1 to about 350:1, about 250:1 to about 330:1, about 280:1 to about 320:1, about 280:1 to about 300:1, about 280:1, about 290:1, about 300:1, or about 310:1.
  • the MSP is MSP2N2. In some embodiments, the MSP is a His-tag removed variant of MSP2N2.
  • the MSP is MSP2N2 (e.g., SEQ ID NO: 18) or His-tag removed variant thereof
  • the phospholipid is DPPC, and at least about 85% (e.g., at least about any of 90%, 95%, 96%, 97%, 98%, 99%, or 100%) of the plurality of nanodiscs in the pharmaceutical composition are fully-lipidated.
  • the N S is about 387.
  • the MSP is MSP2N2 (e.g., SEQ ID NO: 18) or His-tag removed variant thereof
  • the phospholipid is DPPC, wherein at least about 85% (e.g., at least about any of 90%, 95%, 96%, 97%, 98%, 99%, or 100%) of the plurality of nanodiscs in the pharmaceutical composition are under-lipidated.
  • the MSP is MSP2N2 or His-tag removed variant thereof
  • the phospholipid is DPPC
  • the molar ratio of the phospholipid to the MSP in the pharmaceutical composition is less than a phospholipid-MSP saturation ratio NS of about 387: 1, such as less than about 95% (e.g., less than about any of 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, or 40%) of the phospholipid-MSP saturation ratio NS of about 387:1.
  • the molar ratio of DPPC to MSP2N2 or His-tag removed variant thereof in the pharmaceutical composition is less than or equal to about 370:1, such as less than or equal to about any of 367:1, 360:1, 350:1, 340:1, 330:1, 320:1, 310:1, 300:1, 290:1, 280:1, 270:1, 260:1, 250:1, 240:1, 230:1, 220:1, 210:1, 200:1, 190:1, 180:1, 170:1, 160:1, 150:1, 140:1, 130:1, 120:1, 110:1, 100:1, or less.
  • the molar ratio of DPPC to MSP2N2 or His-tag removed variant thereof in the pharmaceutical composition is about 150:1 to about 350:1, such as any of about 200:1 to about 350:1, about 250:1 to about 350:1, about 250:1 to about 330:1, about 280:1 to about 320:1, about 280:1 to about 300:1, about 280:1, about 290:1, about 300:1, or about 310:1.
  • the MSP is MSP2N2. In some embodiments, the MSP is a His-tag removed variant of MSP2N2.
  • the MSP is MSP2N2 (e.g., SEQ ID NO: 18) or His-tag removed variant thereof
  • the phospholipid is POPC, and at least about 85% (e.g., at least about any of 90%, 95%, 96%, 97%, 98%, 99%, or 100%) of the plurality of nanodiscs in the pharmaceutical composition are fully-lipidated.
  • the NS is about 291.
  • the MSP is MSP2N2 (e.g., SEQ ID NO: 18) or His-tag removed variant thereof
  • the phospholipid is POPC, wherein at least about 85% (e.g., at least about any of 90%, 95%, 96%, 97%, 98%, 99%, or 100%) of the plurality of nanodiscs in the pharmaceutical composition are under-lipidated.
  • the MSP is MSP2N2 or His-tag removed ny-2692819 Attorney Docket No.: 283912000140 variant thereof
  • the phospholipid is POPC, wherein the molar ratio of the phospholipid to the MSP in the pharmaceutical composition is less than a phospholipid-MSP saturation ratio NS of about 291:1, such as less than about 95% (e.g., less than about any of 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, or 40%) of the phospholipid-MSP saturation ratio N S , which is 291:1.
  • the molar ratio of POPC to MSP2N2 or His-tag removed variant thereof in pharmaceutical the composition is less than or equal to about 280:1, such as less than or equal to about any of 276:1, 270:1, 260:1, 250:1, 240:1, 230:1, 220:1, 210:1, 200:1, 190:1, 180:1, 170:1, 160:1, 150:1, 140:1, 130:1, 120:1, 110:1, 100:1, or less.
  • the molar ratio of POPC to MSP2N2 or His-tag removed variant thereof in the pharmaceutical composition is about 100:1 to about 280:1, such as any of about 100:1 to about 260:1, about 120:1 to about 260:1, about 140:1 to about 260:1, about 160:1 to about 240:1, about 180:1 to about 220:1, about 180:1, about 190:1, about 200:1, or about 210:1.
  • the MSP is MSP2N2.
  • the MSP is a His-tag removed variant of MSP2N2.
  • the pharmaceutical composition comprises nanodiscs purified by chromatography, such as size exclusion chromatography (SEC).
  • the nanodisc increases the cholesterol efflux rate by at least about 2 folds (e.g., at least about any of 2.5, 3, 4, 5, 10, 15-fold, or more) compared to an untreated state (e.g., before administering the nanodisc) or the cholesterol efflux rate by ApoA-I or the MSP (e.g., MSP1D1).
  • the nanodisc increases (e.g., increasing at least about any of 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 10-fold, or more) the cholesterol efflux rate in retinal pigment epithelium cells, or macrophages.
  • the nanodisc reduces lipid or prevents lipid accumulation in the eye of the individual by at least about 5% (e.g., at least about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%).
  • the nanodisc pharmaceutical compositions described herein increase the cholesterol efflux rate by at least about 20% (e.g., at least about any of 30%, 40%, ny-2692819 Attorney Docket No.: 283912000140 50%, 60%, 70%, 80%, 90%, 100%, 2-fold, 2.5-fold, 3-fold, 4-fold, 5-fold, 10-fold, 15-fold, or more) compared to an untreated state (e.g., before administering the nanodiscs) or the cholesterol efflux rate by ApoA-I or a high density lipoprotein (HDL) alone.
  • an untreated state e.g., before administering the nanodiscs
  • HDL high density lipoprotein
  • the under-lipidated nanodisc pharmaceutical composition described herein increases the cholesterol efflux rate by at least about 20% (e.g., at least about any of 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 2-fold, 2.5-fold, 3-fold, 4-fold, 5-fold, 10-fold, 15-fold, or more) compared to a fully-lipidated nanodisc pharmaceutical composition containing the same MSP and phospholipid (the molar ratio of the phospholipid to the MSP in the pharmaceutical composition is about the phospholipid-MSP saturation ratio NS).
  • the nanodisc does not comprise a cell-penetrating peptide (CPP).
  • the CPP is selected from the group consisting of penetratin, polyarginine (R8), LL-37, transportan, Pep-1, and membrane translocating sequence (MTS).
  • the nanodisc does not comprise a target protein. In some embodiments, the nanodisc does not comprise a prophylactic or therapeutic agent.
  • the diameter of the nanodisc is any of about 2 nm to about 100 nm, about 5 nm to about 100 nm, about 5 nm to about 75 nm, about 5 nm to about 50 nm, about 5 nm to about 40 nm, about 5 nm to about 30 nm, about 5 nm to about 20 nm, or about 10 nm to about 20 nm.
  • the diameter of the nanodisc is about 10 nm to about 20 nm.
  • the average size of the nanodiscs can be tuned by the length of the MSP.
  • the diameter of the nanodisc allows for the penetration of the nanodisc into or through ocular muscles.
  • the diameter of the nanodisc allows for the reachability of the nanodisc to the posterior of the eye without CPP. In some embodiments, the diameter of the nanodisc can be tuned by the length of the MSP belt. [0180] In some embodiments, the average diameter of the nanodiscs is any of about 2 nm to about 100 nm, about 5 nm to about 75 nm, about 5 nm to about 50 nm, about 5 nm to about 40 nm, about 5 nm to about 30 nm, about 5 nm to about 20 nm, or about 10 nm to about 20 nm.
  • the average diameter of the nanodiscs is about 2 nm to about 20 nm, such as about 10 nm to about 20 nm. In some embodiments, the average diameter of the nanodiscs can be tuned by the length of the MSP. ny-2692819 Attorney Docket No.: 283912000140 [0181] In some embodiments, the thickness of the nanodisc is any of about 2 nm to about 20 nm, about 3 nm to about 15 nm, about 3 nm to about 10 nm, about 4 nm to about 7 nm, about 4 nm, about 5 nm, or about 6 nm. In some embodiments, the thickness of the nanodisc can be tuned by the type of phospholipid.
  • each nanodisc comprises any of about 50 to about 300, about 100 to about 300, about 100 to about 250, or about 100 to about 200 phospholipid molecules. In some embodiments, each nanodisc comprises any of about 2, 3, 4, or more MSPs.
  • the molecular weight of the nanodisc is any of about 100,000 g/mol to about 400,000 g/mol, about 120,000 g/mol to about 400,000 g/mol, about 150,000 g/mol to about 350,000 g/mol, about 150,000 g/mol to about 300,000 g/mol, about 150,000 g/mol to about 250,000 g/mol, about 150,000 g/mol, about 200,000 g/mol, about 250,000 g/mol, about 300,000 g/mol, or about 320,000 g/mol.
  • the nanodisc comprises MSP in a weight percentage of any of about 15% to about 50%, about 20% to about 40%, about 22% to about 38%, about 24%, about 25%, about 27%, about 29%, about 30%, about 33%, or about 35%.
  • the nanodisc comprises a phospholipid in a weight percentage of any of about 50% to about 90%, about 60% to about 80%, about 63% to about 77%, about 64%, about 65%, about 66%, about 67%, about 70%, about 73%, or about 75%.
  • the nanodisc comprises MSP and phospholipid in a weight ratio of about any of 1.5:8.5 to 1:1, 2:8 to 1:1, 2.5:7.5:1.1, 3:7 to 1:1, 1.5:7.5, 2:8, 3:7, 3.5:6.5, 4:6, or 4.5:5.5. In some embodiment, about any of 80% (w/w), 85% (w/w), 90% (w/w), 92% (w/w), 94% (w/w), 96% (w/w), 99% (w/w), or 99.5% (w/w) of the total amount of protein in the naodisc is MSP.
  • the polydispersity index (a measure of the heterogeneity) of the size of the nanodiscs in the pharmaceutical composition is less than about 0.5, such as less than about any of 0.4, 0.3, 0.2, 0.1, 0.09, 0.08, 0.07, 0.06, 0.05, 0.04, 0.03, 0.02, or 0.01.
  • the size and size distribution of the nanodiscs in the pharmaceutical composition may be determined by combines multi-angle light scattering with size-exclusion chromatography (SEC-MALS).
  • At least about 60%, such as at least about any of 70%, 75%, 80%, 85%, 90%, 95%, or 99% of the nanodiscs in the pharmaceutical composition have a size of about 2 nm to about 20 nm. In some embodiments, at least about 60%, such as at least about any of 70%, 75%, 80%, 85%, 90%, 95%, or 99% of the nanodiscs remain to have sizes of ny-2692819 Attorney Docket No.: 283912000140 about 2 nm to about 20 nm after being frozen and thawed for one or more times, such as being frozen at -80°C and thawed with or without a cryoprotectant.
  • At least about 60%, such as at least about any of 70%, 75%, 80%, 85%, 90%, 95%, or 99% of the nanodiscs in the pharmaceutical composition have sizes substantially unchanged (e.g., a size change of the nanodiscs in the pharmaceutical composition of less than about any of 20%, 15%, 10%, 5%, 2%, or 1%) after being frozen and thawed for one or more times, such as being frozen at -80 °C and thawed with or without a cryoprotectant.
  • the polydispersity index (a measure of the heterogeneity) of the lipid content of the nanodiscs in the pharmaceutical composition is less than about 0.5, such as less than about any of 0.4, 0.3, 0.2, 0.1, 0.09, 0.08, 0.07, 0.06, 0.05, 0.04, 0.03, 0.02, or 0.01.
  • At least about 60%, such as at least about any of 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% of the nanodiscs have molar ratio between phospholipid and MSP of less than about 95% (e.g., less than about any of 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, or 40%) of the phospholipid- MSP saturation ratio N S .
  • At least about 60%, such as at least about any of 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% of the nanodiscs have molar ratio between phospholipid and MSP of less than about 95% (e.g., less than about any of 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, or 40%) of the phospholipid- MSP saturation ratio NS after being frozen and thawed for one or more times, such as being frozen at -80°C and thawed, with or without a cryoprotectant.
  • At least about 60%, such as at least about any of 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% of the nanodiscs have molar ratio between phospholipid and MSP remains substantially unchanged (e.g., a size change of the nanodiscs in the pharmaceutical composition of less than about any of 20%, 15%, 10%, 5%, 2%, or 1%) after being frozen and thawed for one or more times, such as being frozen at -80°C and thawed with or without a cryoprotectant.
  • the molar ratio between phospholipid and MSP in the pharmaceutical composition remains substantially unchanged (e.g., a size change of the nanodiscs in the pharmaceutical composition of less than about any of 20%, 15%, 10%, 5%, 2%, or 1%) after being storied under room temperature for at least about 1 hour, such as at least about any of 2, 5, 10, 12, 15, 20, or 24 hours, or 2, 3, 4, 5, 6, 7, 8, 15, 30, 40, 50, 100 days.
  • the molar ratio between phospholipid and MSP remains substantially unchanged (e.g., a size change of the nanodiscs in the pharmaceutical composition of less than about any of ny-2692819 Attorney Docket No.: 283912000140 20%, 15%, 10%, 5%, 2%, or 1%) after being storied under -80°C for at least about 1 month, such as at least about any of 2, 3, 4, 5, 6, 7, 8, 15, 30, 40, 50, 100 months.
  • Method of preparation [0187] Nanodiscs can be formed by the interaction between the MSP and the phospholipid. Method of preparing nanodiscs is known in the art, and description of the exemplary methods can be found in Timothy H. Bayburt, Yelena V.
  • a method of preparing a pharmaceutical composition comprising a plurality of nanodiscs (e.g., any of the nanodisc pharmaceutical compositions described herein, e.g., fully-lipidated or under-lipidated), the method comprising: (1) incubating the MSP and the phospholipid to obtain a preparation mixture, wherein the phospholipid is solubilized by a detergent or organic compound prior to the incubation; and (2) removing the detergent or organic compound from the preparation mixture, thereby obtaining the nanoparticles.
  • the molar ratio of the phospholipid to the MSP in the preparation mixture is less than (e.g., less than about any of 95%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, or lower) or equal to the molar ratio of the phospholipid to the MSP in the pharmaceutical composition.
  • the method further comprises purifying the obtained nanodiscs, e.g., by SEC (one or more times).
  • the method further comprises verifying the obtained nanodisc pharmaceutical composition, e.g., after the final purification step.
  • the MSP is any of the MSPs described herein.
  • the phospholipid is any of the phospholipids described herein.
  • the nanodiscs can be synthesized by incubating solubilized phospholipid with MSP.
  • the phospholipid is solubilized by a detergent or an organic compound.
  • the solubilized phospholipid is prepared by adding solution of detergent or an organic compound to a thin film of phospholipid.
  • Exemplary detergents or organic compounds include, but are not limited to, alkyglucosides such as n- dodecyl- ⁇ -D-maltoside (DDM), octyl- ⁇ -glucoside (OG), Triton X-100, 3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate (CHAPS), 2,2-didecylpropane-1,3-bis- ⁇ -D- ny-2692819 Attorney Docket No.: 283912000140 maltopyranoside (LMNG), cholate, and any combination thereof.
  • the phospholipid is solubilized by a cholate salt, such as sodium cholate.
  • the MSP is provided in a solution comprising a buffer.
  • the buffer has a pH of about 5 to about 9, such as about 6 to about 8, about 7, or about 7.5.
  • the MSP in the solution is at least about 0.1 mg/mL, such as at least about any of 0.2 mg/mL, 0.3 mg/mL, 0.4 mg/mL, 0.5 mg/mL, 1 mg/mL, 1.5 mg/mL, 2 mg/mL, or 2.5 mg/mL.
  • the MSP in the solution is about 0.5 mg/mL to about 10 mg/mL, such as any of about 1 mg/mL to about 8 mg/mL, about 1 mg/mL to about 5 mg/mL, about 1.5 mg/mL to about 6 mg/mL, about 2 mg/mL to about 5 mg/mL, or about 2.5 mg/ml to about 4 mg/mL.
  • the method comprises removing the detergent or organic compound from the preparation mixture, thereby obtaining the nanodiscs.
  • the detergent or organic compound e.g., cholate
  • absorbent polymer beads e.g., cholate
  • the absorbent polymer beads comprise styrene-divinylbenzene (macroreticular) beads (e.g., Amberlite® XAD-2® Beads).
  • the formation and size of nanodiscs can be determined by tools known in the art, such as size-exclusion chromatography (SEC) and electron microscopy.
  • the method further comprises isolating the nanodiscs.
  • the nanodiscs are isolated by chromatography.
  • the method further comprises subjecting the nanodiscs to one or more freeze thaw cycles with or without cryoprotectant.
  • the method further comprises purifying the prepared nanodiscs, such as separating nanodiscs with different phospholipid to MSP ratios.
  • the purification comprises the use of SEC.
  • the method comprises more than one purification cycles until a desired distribution of phospholipid to MSP ratio (e.g., fully- saturated, or less than phospholipid-polymer saturation ratio NS) is reached.
  • the method further comprises measuring the actual phospholipid to MSP ratio in the nanodisc pharmaceutical compositions.
  • the actual phospholipid to MSP ratio in the nanodisc pharmaceutical compositions can be determined by measuring the fatty acid content using gas chromatography (GC) analysis.
  • GC gas chromatography
  • the molar ratio of phospholipid to the MSP in the preparation mixture is the same as that in the obtained nanodisc pharmaceutical composition. In some embodiments, the molar ratio of phospholipid to the MSP in the preparation mixture (e.g., mixture of method of preparation step (1) described above) is less than (e.g., less than about any of 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, or 40% of) that in the obtained nanodisc pharmaceutical composition.
  • the molar ratio of the phospholipid to the MSP in the preparation mixture is determined such that at least about 60%, such as at least about any of 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100%, of the as-prepared nanodics are under- lipidated, such as having a molar ratio of the phospholipid to the MSP less than about 95% (e.g., less than about any of 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, or 40%) of the phospholipid-MSP saturation ratio N S .
  • the molar ratio of the phospholipid to the MSP in the preparation mixture is determined such that at least about 60%, such as at least about any of 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100%, of the as-prepared nanodics are fully-lipidated.
  • the pharmaceutical composition further comprises a prophylactic or therapeutic agent.
  • the pharmaceutical composition does not comprise a prophylactic or therapeutic agent. Routes of administration and Dose [0197] Nanodics or pharmaceutical compositions thereof described herein can be administered by any suitable administration routes.
  • the nanodics or pharmaceutical compositions thereof are administered via local ocular administration.
  • the suitable administration routes include, but are not limited to, intravitreal, topical, periocular injections, intra- or periocular implants, intravitreal implants, and suprachoroidal implants or particles or polymeric composition, or any releasing systems such as emulsions, solid non-biodegradable or degradable implants or tablets, or mini pumps.
  • the nanodiscs or pharmaceutical compositions thereof are directly administered to ny-2692819 Attorney Docket No.: 283912000140 the eye by ocular tissue injection such as periocular, conjunctival, subtenon, intracameral, intravitreal, intraocular, subretinal, subconjunctival, retrobulbar, suprachoroidal or intracanalicular injections.
  • the nanodiscs or pharmaceutical compositions thereof are administered by direct application to the eye using a catheter or other placement device such as a retinal pellet, intraocular insert, suppository or an implant comprising a porous, non-porous, or gelatinous material.
  • the nanodiscs or pharmaceutical compositions thereof are administered by topical ocular drops or ointments.
  • the nanodiscs or pharmaceutical compositions thereof are administered by a slow release device in the cul-de-sac or implanted adjacent to the sclera (transscleral) or in the sclera (intrascleral) or suprachoroidal or within the eye.
  • Intracameral injection may be through the cornea into the anterior chamber to allow the agent to reach the trabecular meshwork.
  • Intracanalicular injection may be into the venous collector channels draining Schlemm's canal or into Schlemm's canal.
  • the nanodiscs or pharmaceutical compositions thereof are administered intravitreally.
  • the pharmaceutical composition comprising a nanodisc may be in the form of isotonic, sterile, saline solutions (monosodium or disodium phosphate, sodium, potassium, calcium or magnesium chloride and the like or mixtures of such salts), sterile aqueous dispersions (e.g., water-oil emulsion), or dry, especially freeze- dried compositions which upon addition, depending on the case, of sterilized water or physiological saline, permit the constitution of injectable solutions.
  • saline solutions monosodium or disodium phosphate, sodium, potassium, calcium or magnesium chloride and the like or mixtures of such salts
  • sterile aqueous dispersions e.g., water-oil emulsion
  • dry especially freeze- dried compositions which upon addition, depending on the case, of sterilized water or physiological saline, permit the constitution of injectable solutions.
  • the pharmaceutical composition comprising a nanodisc (e.g., a plurality of nanodiscs) and a pharmaceutically acceptable excipient is administered as an ophthalmic eye drop.
  • Suitable pharmaceutically acceptable excipient include, but are not limited to, ophthalmologically acceptable preservatives, solvents, surfactants, viscosity enhancers, penetration enhancers, buffers, isotonic agents, stabilizer, pH regulators, or water to form an aqueous, sterile ophthalmic suspension or solution.
  • the retention of the pharmaceutical composition can be further improved by viscosity building agents, such as hydroxymethyl cellulose, hydroxyethyl cellulose, hydroxypropyl methylcellulose, methylcellulose, carboxymethylcellulose, polyvinylpyrrolidone, hyaluronic acid, or the like.
  • viscosity building agents such as hydroxymethyl cellulose, hydroxyethyl cellulose, hydroxypropyl methylcellulose, methylcellulose, carboxymethylcellulose, polyvinylpyrrolidone, hyaluronic acid, or the like.
  • ny-2692819 Attorney Docket No.: 283912000140
  • the pharmaceutical composition comprising a nanodisc (e.g., a plurality of nanodiscs) and a pharmaceutically acceptable excipient is administered as an ophthalmic ointment composition.
  • the pharmaceutically acceptable excipient comprises a preservative in an appropriate vehicle, such as mineral oil, liquid lanolin, or white petrolatum.
  • the pharmaceutical composition is an anhydrous dosage form comprising a nanodisc. Such an anhydrous dosage form can be prepared using anhydrous or low moisture containing ingredients and low moisture or low humidity conditions.
  • the pharmaceutical composition further comprises a buffer.
  • the buffer is an isotonic buffer.
  • the isotonic buffer is phosphate-buffer saline (PBS).
  • the isotonic buffer does not comprise tromethamine (tris).
  • the pharmaceutical composition further comprises a preservative.
  • Suitable preservatives include, but are not limited to, sodium bisulfite, sodium bisulfate, sodium thiosulfate, benzalkonium chloride, chlorobutanol, thimerosal, phenylmercuric acetate, phenylmercuric nitrate, methylparaben, polyvinyl alcohol and phenylethyl alcohol.
  • the excipient can be selected by one of ordinary skill in the art. Exemplary excipients include, for example, those described in the Handbook of Pharmaceutical Excipients, Rowe et al. (Eds.) 6th Ed.
  • a doctor can determine the dosage which they consider most appropriate according to a preventive or curative treatment and according to the age, weight, condition, and other factors specific to the individual to be treated.
  • the frequency and dosage may also vary according to factors specific for each individual depending on the specific therapy (e.g., therapeutic or prophylactic), the route of administration, as well as age, body, weight, response, and the past medical history of the individual.
  • effective doses may be extrapolated from dose- response curves derived from in vitro or animal model test systems.
  • dose of the nanodiscs or pharmaceutical compositions thereof, when administered to remove lipid or prevent lipid (e.g., cholesterol) accumulation in the eye, or to prevent or treat an eye disease characterized by lipid (e.g., cholesterol) accumulation in an individual is about 50 ⁇ g/kg to about 50 mg/kg (including for example any of about 50 ⁇ g/kg to about 30 mg/kg, about 70 ⁇ g/kg to about 20 mg/kg, about 100 ⁇ g/kg to about 10 mg/kg, about 100 ⁇ g/kg to about 500 ⁇ g /kg, or about 200 ⁇ g/kg to about 500 ⁇ g /kg) of the individual weight.
  • dose of the nanodiscs or pharmaceutical compositions thereof, when administered to remove lipid or prevent lipid (e.g., cholesterol) accumulation in the eye, or to prevent or treat an eye disease characterized by lipid (e.g., cholesterol) accumulation in an individual is about 0.1 ⁇ g/kg to about 20 ⁇ g/kg (including for example any of about 0.5 ⁇ g/kg to about 18 mg/kg, about 1 ⁇ g/kg to about 15 mg/kg, about 1 ⁇ g/kg to about 10 mg/kg, about 1.5 ⁇ g/kg to about 9 ⁇ g /kg, or about 2 ⁇ g/kg to about 9 ⁇ g /kg) of the individual weight.
  • dose of the nanodiscs or pharmaceutical compositions thereof for methods described herein is about 1 ⁇ g to about 50 mg (including for example any of about 1 ⁇ g to about 30 mg, about 1 ⁇ g to about 20 mg, about 1 ⁇ g to about 10 mg, about 10 ⁇ g to about 20 mg, about 10 ⁇ g, or about 15 mg). In some embodiments, dose of the nanodiscs or pharmaceutical compositions thereof for methods described herein is about 1 ⁇ g to about 1000 ⁇ g (including for example any of about 5 ⁇ g to about 800 mg, about 5 ⁇ g to about 600 mg, about 8 ⁇ g to about 600 mg, or about 10 ⁇ g to about 600 mg).
  • the dose of the nanodiscs or pharmaceutical compositions thereof can be administered according to a suitable schedule, for example, any of about every day, about every two days, about every three days, about every four days, about every five days, about every six days, about once a week, about once every two weeks, about once every three weeks, and about once a month.
  • the administration of the nanodiscs or pharmaceutical compositions thereof can be repeated about every three days.
  • the administration of the nanodiscs or pharmaceutical compositions thereof can be repeated about every 2 to 4 weeks.
  • the administration of the nanodiscs or pharmaceutical compositions thereof can be repeated about every 3 weeks.
  • administration of the nanodiscs or pharmaceutical compositions thereof may be repeated, such as separated by at least 1 day, 2 days, 3 days, 5 days, 10 days, 15 days, 30 days, 45 days, 2 months, 75 days, 3 months, or 6 months.
  • ny-2692819 Attorney Docket No.: 283912000140 [0210] It may be necessary to use dosages of the nanodiscs or pharmaceutical compositions thereof outside the ranges disclosed herein in some cases, as will be apparent to those of ordinary skill in the art.
  • the clinician or treating physician will know how and when to interrupt, adjust, or terminate therapy in conjunction with the individual’s response.
  • a method of removing lipid or preventing lipid accumulation in the eye of an individual comprising administering to the eye of the individual an effective amount of a pharmaceutical composition comprising a nanodisc, wherein the nanodisc comprises a membrane scaffold protein (MSP) and a phospholipid.
  • MSP membrane scaffold protein
  • Embodiment 2. A method of preventing or treating an eye disease characterized by lipid accumulation in an individual, comprising administering to the eye of the individual an effective amount of a pharmaceutical composition comprising a nanodisc, wherein the nanodisc comprises an MSP and a phospholipid.
  • the MSP comprises a variant of an apolipoprotein selected from the group consisting of apolipoprotein A-I (ApoA-I), ApoA-II, ApoC, ApoE, and ApoM.
  • Embodiment 4 The method of any one of embodiments 1-3, wherein the MSP is a variant of ApoA-I.
  • Embodiment 5. The method of embodiment 4, wherein the MSP comprises one or more amphipathic helix sequences relative to ApoA-I.
  • Embodiment 6. The method of embodiment 5, wherein the one or more amphipathic helix sequences are derived from ApoA-I.
  • Embodiment 8 The method of any one of embodiments 4-7, wherein the MSP comprises an N- terminus truncation relative to ApoA-I. ny-2692819 Attorney Docket No.: 283912000140 Embodiment 9. The method of embodiment 8, wherein the MSP is MSP1, MSP1D1, MSP1D2, or MSPE3D1. Embodiment 10. The method of any one of embodiments 1-9, wherein the phospholipid is a phosphatidylcholine (PC). Embodiment 11.
  • PC phosphatidylcholine
  • the phospholipid is selected from the group consisting of dimyristoylphosphatidylcholine (DMPC), palmitoyloleoylphosphatidylcholine (POPC), dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylcholine (DSPC), and a combination thereof.
  • DMPC dimyristoylphosphatidylcholine
  • POPC palmitoyloleoylphosphatidylcholine
  • DPPC dipalmitoylphosphatidylcholine
  • DSPC distearoylphosphatidylcholine
  • Embodiment 12 The method of any one of embodiment 10 or 11, wherein the phospholipid is DMPC, DPPC, or POPC.
  • Embodiment 13 The method of any one of embodiments 1-12, wherein the MSP is MSP1D1 or MSP1, and wherein the molar ratio of phospholipid to MSP is from about 10:1 to about 90
  • Embodiment 15 The method of embodiment 14, wherein the MSP is MSP1D1, and wherein the molar ratio of POPC to MSP1D1 is about 50:1 or about 32.5:1.
  • Embodiment 16 The method of embodiment 14, wherein the MSP is MSP1D1 or MSP1, and wherein the molar ratio of POPC to MSP1D1 or MSP1 is about 65:1.
  • Embodiment 17 The method of embodiment 13, wherein the phospholipid is DMPC, and wherein the molar ratio of DMPC to MSP is from about 10:1 to about 85:1.
  • ny-2692819 Attorney Docket No.: 283912000140 Embodiment 22.
  • Embodiment 23. The method of embodiment 21, wherein the phospholipid is DMPC, and wherein the molar ratio of DMPC to MSP1E3 is about 150:1.
  • the method of embodiment 21, wherein the phospholipid is DPPC, and wherein the molar ratio of DPPC to MSP1E3 is about 170:1.
  • Embodiment 25 The method of any one of embodiments 1-24, wherein the nanodisc is about 5 nm to about 20 nm in diameter.
  • Embodiment 26 The method of any one of embodiments 1-25, wherein the lipid for removal or prevention from accumulation in the eye is cholesterol.
  • Embodiment 27 The method of any one of embodiments 1-26, wherein the nanodisc does not comprise a cell-penetrating peptide (CPP).
  • Embodiment 28 The method of embodiment 27, wherein the CPP is selected from the group consisting of penetratin, polyarginine (R8), LL-37, transportan, Pep-1, and membrane translocating sequence (MTS).
  • Embodiment 29 The method of any one of embodiments 1-28, wherein the nanodisc does not comprise a target protein.
  • Embodiment 30 The method of any one of embodiments 1-25, wherein the lipid for removal or prevention from accumulation in the eye is cholesterol.
  • Embodiment 28 The method of any one of embodiments 1-26, wherein the nanodisc does not comprise a cell-penetrating peptide (CPP).
  • CPP cell-penetrating peptide
  • Embodiment 31 The method of any one of embodiments 26-30, wherein the nanodisc increases cholesterol efflux rate by at least about 2-fold compared to an untreated state, or the cholesterol efflux rate by ApoA-I or the MSP.
  • Embodiment 32 The method of any one of embodiments 1-31, wherein the pharmaceutical composition is administered via intravitreal injection.
  • Embodiment 33 The method of any one of embodiments 1-31, wherein the pharmaceutical composition is administered via intravitreal injection.
  • any one of embodiments 2-32, wherein the eye disease characterized by lipid accumulation is selected from the group consisting of age-related macular degeneration (AMD), photoreceptor neurodegeneration, optic nerve atrophy, loss of acuity, ny-2692819 Attorney Docket No.: 283912000140 hemianopia, visual agnosia, strabismus, retinal neurovascular disorder, lipid keratopathy, corneal lipidosis, and a combination thereof.
  • AMD age-related macular degeneration
  • Embodiment 34 The method of embodiment 33, wherein the eye disease characterized by lipid accumulation is AMD.
  • the method of embodiment 34, wherein the AMD is dry AMD.
  • Embodiment 36 Embodiment 36.
  • Nanodiscs were assembled from a phospholipid, a MSP, and a detergent.
  • Phospholipid stocks were prepared in chloroform at 50-100 mM for long-term storage at –20oC in 4 ml glass vials with Teflon-lined screw caps. Concentration of the stock solution was determined by phosphate analysis, the desired amount of chloroform lipid stock was dispensed into a disposable glass culture tube, and the solvent was dried up using a gentle stream of nitrogen gas in a fume hood. The tube was rotated at an angle to obtain a thin film on the lower walls. Residual solvent was removed by placing the tube in a vacuum desiccator under high vacuum for at least 4 hours.
  • ny-2692819 Attorney Docket No.: 283912000140
  • Buffer comprising a detergent and sodium cholate was added to the tube so that the ratio of cholate to lipid in the mixture was 2:1.
  • the tube was vortexed, heated under hot tap water, and sonicated in an ultrasonic bath until the solution was clear and no lipid remained on the walls of the tube.
  • MSP was added to the solution and incubated for at least 15 minutes.
  • 0.5-0.8 g of Amberlite XAD 2 beads (Sigma) were added per every ml of the reconstitution mixture. The suspension was placed on the orbital shaker and incubated overnight.
  • Example 2A Nanodisc preparation at different lipid to MSP ratios
  • Example 2A Nanodisc preparation at different lipid to MSP ratios
  • MSP1D1 was added to the sodium cholate lipid mixture at a lipid to MSP ratio of 10:1, 32.5:1, 50:1, and 65:1 for POPC lipids, and 20:1, 30:1, 40:1, 50:1, 60:1, and 85:1 for DMPC lipids. No lipid addition served as control (lipid free).
  • a POPC to MSP1D1 ratio of 65:1 resulted in a fully lipidated (10 nm diameter) nanodisc.
  • FIG. 2B at a molar ratio of 85:1 DMPC to MSP1D1, a fully lipidated nanodisc was formed.
  • Nanodiscs containing DMPC (Lipid A) or POPC (Lipid B) at different lipid concentrations were formed with MSP1D1 using the methods of Examples 1 and 2.
  • J774 cells derived from a murine macrophage cell line were plated and radiolabeled with 74 kBq of 3 H- cholesterol per millimeter.
  • ABCA1 was up-regulated by means of a 6-hour incubation with 0.3 mM 8-(4-chlorophenylthio)-cAMP.
  • the POPC (Lipid B) containing nanodiscs with lipid to MSP1D1 ratios of 50:1 and 32.5:1 had higher cholesterol efflux percentage than fully lipidated POPC (Lipid B) nanodiscs with a lipid to MSP1D1 ratio of 65:1.
  • Nanodiscs containing POPC at lipid to MSP1D1 ratios from 32.5:1 to 65:1 all demonstrated better cholesterol efflux properties compared to nanodiscs containing DMPC at lipid to MSP1D1 ratio of 85:1. Further, all tested nanodiscs demonstrated at least about 2-fold cholesterol efflux of that achieved by MSP1D1 protein per se.
  • Example 4 Exemplary nanodisc removes cholesterol from J774 murine macrophages in vitro
  • a nanodisc with a DMPC to MSP1D1 ratio of 85:1 was formed using the methods of Examples 1 and 2.
  • the methods of Example 3 were repeated to measure cholesterol efflux by such exemplary nanodisc in comparison to naturally occurring ApoA-I. As shown in FIG.4, the percent of cholesterol efflux with the exemplary nanodisc was more than two times that of naturally occurring ApoA-I.
  • Example 5 Exemplary nanodisc removes cholesterol from human retinal pigment epithelial cells in vitro
  • Nanodiscs with a POPC to MSP1D1 ratio of 65:1 (MSP1D1 Lipid B) and nanodiscs with a DMPC to MSP1D1 ratio of 85:1 (MSP1D1 Lipid A) were formed using the methods of Examples 1 and 2.
  • the methods of measuring cholesterol efflux in Example 3 were conducted on human retinal pigment epithelial (RPE) cells (Neuromics) to measure cholesterol efflux at 2, 4 and 6 hours of incubation. Passive efflux (no protein or nanodisc added) and MSP1D1 protein alone were used for comparison.
  • RPE retinal pigment epithelial
  • FIGs.5A-5C show percent cholesterol efflux from RPE cells after incubation for 2 hours (FIG.5A), 4 hours (FIG.5B), and 6 hours (FIG.5C). These results collectively demonstrate that nanodiscs with POPC to MSP1D1 ratio of 65:1 and nanodiscs with DMPC to MSP1D1 ratio of 85:1 are both capable of continuously promoting cholesterol efflux from RPE cells for at least 6 hours, which were significantly more than that achieved by MSP1D1 protein alone.
  • nanodiscs with DMPC to MSP1D1 ratio of 85:1 seemed to efflux cholesterol faster than nanodiscs with POPC to MSP1D1 ratio of 65:1 during 2-4 hour incubation window, but nanodiscs with POPC to MSP1D1 ratio of 65:1 caught up speed by 6 hour.
  • Example 6 In vivo delivery of exemplary nanodisc in murine eyes [0226] To determine the penetrability and persistence of the exemplary nanodiscs in the eye in vivo, nanodiscs comprising MSP1D1 and DMPC in a molar ratio of 1:85 was prepared, with the MSP1D1 linked to a green fluorescent label via a cysteine residue engineered into the MSP1D1 protein.
  • ny-2692819 Attorney Docket No.: 283912000140 [0227]
  • the nanodiscs were prepared the day before the procedure and stored at 4°C.
  • 6-week old C57Bl/6J mice were anesthetized with 87.6mg/kg Ketamine plus 10mg/kg Xylazine cocktail via IP injection and administered 0.5% proparacaine hydrochloride ophthalmic drops for pain management.
  • the prepared nanodiscs were administered at a dose of 7 ⁇ g/eye in a volume of 1.14 ⁇ l via intravitreal injection.
  • mice had the left eye dilated with 1% Tropicamide ophthalmic solution, followed by application of 2.5% Gonak hypromellose demulcent solution and noninvasive fundus imaging using Phoenix Micron III imaging system at various time points.
  • mice were sacrificed and eyes were enucleated. Eyes were fixed in 10% Neutral buffered formalin for 2 hrs at room temperature (RT) before being transferred to graded sucrose (10% followed by 30%) overnight. The fixed, cryopreserved eyes were mounted in optimal cutting temperature (O.C.T.) and slides were prepared by cutting sections (20 ⁇ M) on a Lieca 1850 cryostat.
  • O.C.T. optimal cutting temperature
  • Coverslips were applied using Vectasheild antifade mounting medium with Dapi to counterstain nuclei and to avoid additional processing steps associated with other counterstaining methods.
  • the prepared slides were imaged on an Olympus FV1000 confocal microscope using system-optimized z step distances for each respective objective. This allowed for direct visualization of fluorescently labeled nanodiscs following the successful removal of cholesterol from the retina.
  • the nanodisc was successfully delivered to the murine eye via intravitreal injection, and the nanodisc was able to enter from the vitreous, got to the retinal space, into the RPE, and into the ocular muscle by 6 hours. By 24 hours, the disc appeared to have left the eye.
  • Example 7 In vivo safety and efficacy of exemplary nanodisc in murine eyes [0230] To determine in vivo safety and efficacy of the nanodiscs in the eye, an exemplary nanodisc comprising MSP1D1 and DMPC at a molar ratio of 1:85 was prepared, with the MSP1D1 linked to a green fluorescent label via a cysteine residue engineered into the MSP1D1 protein.
  • the fluorescence-labelled exemplary nanodisc was intravitreally delivered into the eyes of anesthetized mice, and its path and rate of diffusion within the eye was visualized by imaging ny-2692819 Attorney Docket No.: 283912000140 post-mortem retinal tissue along a 24-hour post-injection time course (FIG. 7A). As shown in FIG.7B, the fluorescence signal was detectable within 15 minutes of injection and diffused rapidly across the retina in a vitreous-to-RPE (retinal pigment epithelium) gradient, indicating that the nanodisc can be delivered into the eye and traffic through the retina effectively to target tissues.
  • RPE retina pigment epithelium
  • a model of ‘wet’ AMD mice was used, which had a laser-induced injury of the Bruch’s membrane to cause choroidal neovascularization (CNV).
  • CNV choroidal neovascularization
  • the exemplary nanodisc was intravitreally injected in tandem with, or 3 days after (‘+d3’), laser injury to examine the resulting CNV area size (FIG.8A).
  • the mean CNV area was similar across control and exemplary nanodisc -treated mice, suggesting that the exemplary nanodisc does not exacerbate prominent features of AMD in the mouse (FIG. 8B and FIG. 8C).
  • FIG.9A for the first cohort of mice (Study 1), baseline (T0) optical coherence tomography (OCT) fundus images of the mouse retina were collected prior to high-fat diet induction and intravitreal delivery of the exemplary nanodisc (‘Disc’ in FIG.9B and FIG.9C) or a blank injection (‘Vehicle’ in FIG.9B and FIG. 9C). A second intravitreal injection was administered 3 weeks later. After 6 weeks of high-fat diet exposure, follow-up OCT fundus imaging (T1) and blood sample collection were performed followed by electroretinography (ERG) visual function testing and tissue harvest.
  • OCT optical coherence tomography
  • RPE disruption and hypertransmission, ellipsoid zone abnormalities, inner retinal subsidence, and hyperreflective foci are primary anatomical changes linked to AMD progression are identified with noninvasive imaging.
  • T1 optical coherence tomography (OCT) images were examined for potential structural changes resulting from the genetic mutation and high-fat diet.
  • IS/OS inner segment/outer segment
  • RPE disruptions including pigment epithelial detachments (PED) were found in images of vehicle-injected mice following 6 weeks of high-fat diet absent in baseline T0 images (FIG.9F).
  • these abnormalities were rarely detected or absent in exemplary nanodisc-treated mouse eyes, indicating ameliorating features of AMD on OCT imaging.
  • mice In analyzing follow-up OCT imaging, several more lesions in the IS/OS junction and RPE were observed in vehicle-injected mice, but far fewer lesions were observed in mice injected with the exemplary nanodisc. Furthermore, greater numbers of lesions in both groups were observed compared to the first study, suggesting that exposing Abca1/g1-rod/-rod mice to a high-fat diet for 2 weeks prior to initial intravitreal injection worsened the disease (Table B-2 below, which summarizes cumulative incidence of retinal lesions in vehicle and exemplary nanodisc-treated mice via OCT imaging examination across two studies). Regardless, treatment of mice with the exemplary nanodisc resulted in reduced retinal lesions in both studies by nearly four times compared to vehicle.
  • Example 8 Preparation of Exemplary Under-lipidated Nanodiscs
  • MSP1D1(-) refers to MSP1D1 with the N-terminal Histidine affinity tag removed, which was used for the preparation herein.
  • MSP1D1(-) comprises the sequence of SEQ ID NO: 14.
  • Phospholipid e.g., DMPC, DPPC
  • concentration was measured by determination of total phosphorous (see, e.g., Chen, Toribara, and Warner (1956) Anal. Chem.28:1756-1758, the content of which is incorporated herein by reference in its entirety).
  • Other reagents used for the preparation included Phosphate Buffered Saline (PBS), 200 mM Sodium Cholate detergent, Superdex S-200 Size Exclusion Column, methanol, ethyl acetate, ethanol, hexane, concentrated hydrochloric acid, and water.
  • DMPC/MSP1D1(-) Nanodisc ny-2692819 Attorney Docket No.: 283912000140 [0240] All manipulations were performed at room temperature (20 – 22°C). [0241] DMPC/chloroform stock was added to a glass tube so that the final ratio of DMPC to MSP1D1(-) in the preparation mixture was 30:1. DMPC was dried under a stream of nitrogen while rotating the glass tube, forming a thin film on the sides of the tube. The tube containing DMPC was then placed in a desiccator and vacuum was applied for a minimum of 4 hours, for instance, overnight. [0242] DMPC was then hydrated in water.
  • the desired final sodium cholate concentration was first determined.
  • the final cholate to lipid ratio was determined to be greater than 2.
  • One exemplary concentration was 17 mM cholate.
  • the amount of water and 200 mM sodium cholate were determined such that the final concentration of Sodium Cholate was 17 mM Sodium Cholate.
  • the amount of protein was then determined such that the final protein concentration was about 3.5 mg/ml. Only water was added to the dried DMPC at this time. [0243] The water/DMPC mixture was vortexed until the solution was milky white.200 mM sodium cholate was then added to the hydrated DMPC to give a final cholate concentration of 17 mM in the assembly mixture.
  • the sodium cholate/DMPC mixture was fully solubilized by placing the tube in a sonicating water bath for 5 min, followed by gentle heating at ⁇ 40°C, repeated for 2 times. MSP1D1(-) was then added to the sodium cholate/DMPC mixture and mixed well, arriving at a preparation mixture with a protein concentration of about 3.5 mg/ml. The preparation mixture was incubated for 10 – 20 minutes at room temperature. [0244] The assembly of the nanodisc was initiated by addition of Amberlite XAD-2 resin, where Amberlite absorbed the cholate detergent.
  • the amount of packed Amberlite XAD-2 resin gave at least 50% of the volume of the assembly mixture, for example, for a 5 ml preparation mixture, Amberlite resin was added to the preparation mixture until the volume was 7.5 ml.
  • the assembly mixture containing Amberlite resin was gently agitated by placing on a platform shaker or a tube rotator for at least four hours at room temperature.
  • the supernatant containing assembled nanodiscs was recovered from the Amberlite XAD resin using a pipette and placed in a new tube.
  • the Amberlite XAD resin was washed 2 times with PBS, and the washing solution containing residual assembled nanodiscs was added to a sample tube. Large nanodiscs were removed from the sample by passing through a 0.2 micron filter.
  • Under-lipidated nanodisc comprising DMPC and MSP1E3D1(-) was similarly prepared with a different lipid to MSP molar ratio in the preparation mixture.
  • the lipid to MSP molar ratios used for various DMPC/MSP1E3D1(-) compositions were also detailed in Example 12.
  • MSP1E3D1(-) refers to MSP1E3D1 with the N-terminal Histidine affinity tag removed, which was used for the preparation herein.
  • MSP1E3D1(-) comprises the sequence of SEQ ID NO: 15.
  • MSP1E3D1 refers to the N-terminal Histidine affinity tag not-removed format, comprising SEQ ID NO: 8.
  • under-lipidated nanodisc To make under-lipidated nanodisc, the final ratio of DMPC to MSP1E3D1(-) in the preparation mixture was about 50:1.
  • Another under-lipidated nanodisc comprising DMPC and MSP1E3D1(-) was similarly made with molar ratio of DMPC to MSP1E3D1(-) in the preparation mixture of about 90:1.
  • Nanodiscs comprising other MSPs (or other phospholipid/MSP ratios were prepared following similar protocols detailed herein.
  • Example 9 Purification of Exemplary Under-lipidated Nanodiscs
  • the filtered samples containing DMPC/MSP1D1(-) nanodiscs or DMPC/MSP1E3D1(-) nanodiscs from Example 8 were further purified by size exclusion chromatography (SEC). Control nanodiscs were similarly purified.
  • SEC size exclusion chromatography
  • the chromatogram of the preparative SEC of DMPC/MSP1D1(-) nanodisc preparation mixture ratio of about 30:1) are shown in FIG.11A.
  • FIG.11A two peaks were isolated.
  • the area on the right (dark grey) corresponding to a more under-lipidated (less lipid to protein ratio) nanodisc containing approximately 51 lipids per MSP1D1(-).
  • the peak positioned at 20 minutes is the residual lipid-free protein.
  • ny-2692819 Attorney Docket No.: 283912000140 [0252]
  • the chromatograms of the preparative SEC of DMPC/MSP1E3D1(-) nanodisc (preparation mixture ratio of about 50:1) are shown in FIG. 11B. As shown in FIG.
  • Example 10 Measurement of the Phospholipid to MSP Ratio
  • This assay measured the phospholipid to MSP ratio by first extracting the phospholipid, followed by saponification and methylation of the fatty acids for rapid gas chromatography (GC) analysis. The measured quantity of fatty acids/ MSP was twice of that of the starting phospholipid. Since phospholipid DPPC was used as standard, 2 fatty acids per phospholipid was already taken into account. [0254] The phospholipid to MSP ratio in the nanodisc was determined following the procedures detailed below.
  • the MSP concentration of the nanodisc described herein was measured by UV-Vis spectroscopy.
  • Nanodisc sample was added to a 2 ml GC vial so that there was about 1 ⁇ g phospholipid. Each nanodisc sample was run in triplicate.
  • the nanodisc sample was diluted to 100 ⁇ L with PBS. 10 ⁇ L of standard (DPPC) was added to each nanodisc sample at a known concentration.
  • the total phospholipid was extracted following the modified Bligh and Dyer technique described in conditions E and E’ in the phase diagrams of FIG.7 in Int. J. Mol. Sci.2017, 18(4), 708. [0256]
  • the extracted phospholipids were dried under a stream of nitrogen.
  • the fatty acids were saponified and methylated simultaneously by dissolving the nanodisc sample in 100 ⁇ L of toluene.0.75 ml of methanol and 0.15 ml of 8% Hydrochloric Acid/Methanol solution were added to the mixture. (See J Lipid Res.2010; 51(3):635-40)). The mixture was incubated at 100°C for 1 hour. In other batches, the mixture was incubated at 45°C for overnight. [0257] 0.5 ml water and 0.5 hexane were then added to the mixture. The mixture was vortexed, top hexane layer was recovered for GC analysis.
  • FIG. 12 An exemplary result generated by the GC method was shown in FIG. 12 using the DMPC/MSP1D1(-) nanodisc (preparation mixture ratio of about 30:1) from Example 8.
  • DMPCfame is methyl myristate
  • DPPCfame is methly palmitate, i.e., the methylated fatty acids derived from DMPC and DPPC, respectively.
  • compositions comprising a plurality of DMPC/MSP1D1(-) nanodiscs were synthesized and purified following the procedures described in Examples 8 and 9, and measured for ratio as in Example 10.
  • DMPC/MSP1D1(-) preparation mixture ratio 80:1, 55:1, and 40:1
  • the collected fraction from the first SEC was repurified with a second SEC (repurification).
  • DMPC/MSP1D1(-) preparation mixture ratio of 40:1 a third SEC purification (repurification 2) was further conducted.
  • DMPC/MSP1D1(-) preparation mixture ratio of 30:1 only one step of SEC was conducted.
  • the lipid to protein ratio was measured in the final nanodisc after the final purification step.
  • the results are shown in FIGs.13A-13D.
  • a starting DMPC/MSP1D1(-) ratio of 80:1 in the preparation mixture yielded nanodisc wherein the DMPC/MSP1D1(-) ratio was about 81:1.
  • a starting DMPC/MSP1D1(-) ratio of 55:1 in the preparation mixture yielded nanodisc wherein the DMPC/MSP1D1(-) ratio was about 75:1.
  • a starting DMPC/MSP1D1(-) ratio of 40:1 in the preparation mixture yielded nanodisc wherein the ny-2692819 Attorney Docket No.: 283912000140 DMPC/MSP1D1(-) ratio was about 51:1.
  • a starting DMPC/MSP1D1(-) ratio of 30:1 in the preparation mixture yielded nanodisc wherein the DMPC/MSP1D1(-) ratio was about 51:1.
  • the 30:1 (prep. ratio) DMPC to MSP1D1(-) was adequately isolated in a single purification step.
  • a low phospholipid/MSP ratio e.g., a starting DMPC/MSP1D1(-) ratio of less than 81:1 (phospholipid- MSP saturation ratio N S of DMPC and MSP1D1) in the preparation mixture
  • a low phospholipid/MSP ratio e.g., a starting DMPC/MSP1D1(-) ratio of less than 81:1 (phospholipid- MSP saturation ratio N S of DMPC and MSP1D1) in the preparation mixture
  • Example 12 Correspondence between phospholipid/MSP1E3D1(-) ratio in the preparation mixture and the final nanodisc composition [0270] The correspondence between the phospholipid/MSP1E3D1(-) ratio in the preparation mixture and the final nanodisc composition was examined.
  • Exemplified nanodiscs comprising a plurality of DMPC/MSP1E3D1(-) nanodiscs were synthesized and purified following the procedures described in Examples 8 and 9, and measured for ratio as in Example 10. Only one step of SEC was conducted. The lipid to protein ratio was measured in the final nanodisc after the final purification step. The results are shown in FIGs. 14A-14C. As shown in FIG.14A, a starting DMPC/MSP1E3D1(-) ratio of 90:1 in the preparation mixture yielded nanodisc wherein the DMPC/MSP1D1(-) ratio was about 93, such as 93 ⁇ 3.
  • a starting DMPC/MSP1E3D1(-) ratio of 60:1 in the preparation mixture yielded nanodisc wherein the DMPC/MSP1D1(-) ratio was about 63:1, such as 63 ⁇ 4 :1.
  • a starting DMPC/MSP1D1(-) ratio of 50:1 in the preparation mixture yielded nanodisc wherein the DMPC/MSP1D1(-) ratio was about 62:1, such as 62 ⁇ 0.5 :1.
  • Example 13 Cholesterol Efflux Assay
  • the protocol for this assay was performed as described in the product information of Cholesterol Efflux Assay Kit (Cell-based) (ab196985).
  • J774 mouse macrophage cells were cultured and placed in a 96 well tissue culture plate. Cells were labeled with fluorescent cholesterol, rinsed, and incubated overnight.
  • FIG. 15A and FIG. 15B show efflux assays performed on purified exemplary nanodiscs as described in Examples 8-10. The phospholipid to MSP ratios were ratios measured from the final nanodiscs.
  • MSP1D1(-) protein only, lipid free
  • Apo-AI protein only, lipid free
  • HDL high-density lipoprotein
  • the phospholipid-MSP saturation ratio N S of a nanodisc comprising DMPC and MSP1E3D1 (or MSP1E3D1(-)) is 167 (see Table 2).
  • FIG. 1 shows that removing N- terminal his-tag portion of the MSP protein did not affect cholesterol efflux
  • Example 14 Stability of Under-Lipidated Nanodisc Compositions to Freeze thaw Cycles
  • the stability of the prepared nanodiscs from Examples 8-10 was tested.
  • the sample was frozen at -80°C with or without 10% glycerol as a cryoprotectant. Then the frozen samples were removed from -80°C storage and thawed rapidly. Samples were subjected to SEC and compared to the chromatograms taken prior to freezing.

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Abstract

The present disclosure relates to methods of removing lipid (e.g., cholesterol) or preventing lipid accumulation in the eye of an individual (e.g., human) and methods of preventing or treating an eye disease characterized by lipid accumulation (e.g., age-related macular degeneration) in an individual, comprising administering to the eye of the individual an effective amount of a pharmaceutical composition comprising a plurality of nanodiscs, wherein the nanodisc comprises a membrane scaffold protein (MSP) and a phospholipid.

Description

Attorney Docket No.: 283912000140 METHODS FOR TREATING LIPID-RELATED EYE DISEASES CROSS REFERENCE TO RELATED APPLICATIONS [0001] This application claims priority benefit of United States Provisional Patent Application No.63/450,945, filed March 8, 2023, and United States Provisional Patent Application No. 63/610,922, filed December 15, 2023, the contents of each of which are incorporated herein by reference in their entirety. REFERENCE TO AN ELECTRONIC SEQUENCE LISTING [0002] The content of the electronic sequence listing (283912000140SEQLIST.xml; Size: 24,766 bytes; and Date of Creation: March 7, 2024) is incorporated herein by reference in its entirety. FIELD [0003] The present disclosure generally relates to methods of removing lipid (e.g., cholesterol) or preventing lipid accumulation in the eye of an individual (e.g., human) and methods of preventing or treating an eye disease characterized by lipid accumulation in an individual. The present disclosure also provides pharmaceutical compositions comprising a plurality of nanodiscs, and methods of using and making thereof. BACKGROUND [0004] Nanodiscs, comprising a membrane scaffold protein (MSP) and a phospholipid, provide a native-like lipid environment for the incorporation of membrane proteins, and they have become a valuable platform for the study of membrane biophysics and for therapeutical applications. [0005] Accumulation of lipid (e.g., cholesterol), has been among the leading causes of numerous eye diseases, including neurodegeneration, entropy, macular degeneration, and inflammation in the eye. Among these diseases, age-related macular degeneration (AMD) affects about 14-24% of the people aged 65 to 74 and about 35% of the people over 75, and about 200 million people around the world, which has been the leading cause of legal blindness in ny-2692819 Attorney Docket No.: 283912000140 developed countries. Despite the progress in mechanistic study on the roles of lipid in these eye diseases, developing efficient methods of removing lipid or preventing lipid accumulation in the eye remains an unmet need. This application addresses this and other needs. BRIEF SUMMARY OF THE INVENTION [0006] In one aspect, provided herein is a method of removing lipid or preventing lipid accumulation in the eye of an individual, comprising administering to the eye of the individual an effective amount of a pharmaceutical composition comprising a plurality of nanodiscs, wherein the nanodisc comprises a membrane scaffold protein (MSP) and a phospholipid. [0007] In one aspect, provided herein is a method of preventing or treating an eye disease characterized by lipid accumulation in an individual, comprising administering to the eye of the individual an effective amount of a pharmaceutical composition comprising a plurality of nanodiscs, wherein the nanodisc comprises an MSP and a phospholipid. [0008] In some embodiments, the MSP is selected from the group consisting of apolipoprotein A-I (ApoA-I), ApoA-II, ApoC, ApoE, ApoM, and a variant thereof. In some embodiments, the MSP is a variant of ApoA-I. In some embodiments, the MSP comprises one or more amphipathic helix sequences derived from ApoA-I. In some embodiments, the MSP comprises an N-terminus truncation relative to ApoA-I. In some embodiments, the MSP is selected from the group consisting of MSP1E1, MSP1E2, MSP1E3, MSP1, MSP1D1, MSP1E1D1, MSP1E2D1, MSP1E3D1, MSP2N2, and N-terminal His-tag portion removed variant thereof (His-tag removed variant). In some embodiments, the MSP is: (i) an MSP1D1 His-tag removed variant comprising the amino acid sequence of SEQ ID NO: 14; or (ii) an MSP1E3D1 His-tag removed variant comprising the amino acid sequence of SEQ ID NO: 15. [0009] In some embodiments, the phospholipid comprises DMPC, DPPC, DMPS, DSPC, POPC, POPS, PiP2, or a combination thereof. In some embodiments, the phospholipid is a phosphatidylcholine (PC). In some embodiments, the phospholipid is selected from the group consisting of DMPC, POPC, DPPC, DSPC, and a combination thereof. [0010] In some embodiments, at least about 85% of the plurality of nanodiscs in the pharmaceutical composition are fully-lipidated. In some embodiments, at least about 85% of the plurality of nanodiscs in the pharmaceutical composition are under-lipidated. In some ny-2692819 Attorney Docket No.: 283912000140 embodiments, the molar ratio of the phospholipid to the MSP in the pharmaceutical composition is less than a phospholipid-MSP saturation ratio NS. In some embodiments, the molar ratio of the phospholipid to the MSP in the pharmaceutical composition is less than about 95% of the phospholipid-MSP saturation ratio NS. [0011] In some embodiments, the MSP is MSP1D1, MSP1, or His-tag removed variant thereof, and the molar ratio of the phospholipid to the MSP in the pharmaceutical composition is from about 10:1 to about 90:1. In some embodiments, the MSP is MSP1D1, MSP1, or His-tag removed variant thereof, wherein the phospholipid is POPC, and the molar ratio of POPC to the MSP in the pharmaceutical composition is from about 10:1 to about 65:1. In some embodiments, the MSP is MSP1D1 or His-tag removed variant thereof, and the molar ratio of POPC to the MSP in the pharmaceutical composition is about 65:1. In some embodiments, the MSP is MSP1D1 or His-tag removed variant thereof, the phospholipid is POPC, and the NS is about 62. In some embodiments, the molar ratio of POPC to the MSP in the pharmaceutical composition is less than about 58:1. In some embodiments, the molar ratio of POPC to the MSP in the pharmaceutical composition is about 50:1 or about 32.5:1. [0012] In some embodiments, the MSP is MSP1D1, MSP1, or His-tag removed variant thereof, wherein the phospholipid is DMPC, and the molar ratio of DMPC to the MSP in the pharmaceutical composition is from about 10:1 to about 85:1. In some embodiments, the MSP is MSP1D1 or His-tag removed variant thereof, the phospholipid is DMPC, and the molar ratio of DMPC to the MSP in the pharmaceutical composition is about 85:1. In some embodiments, the MSP is MSP1D1 or His-tag removed variant thereof, the phospholipid is DMPC, and the NS is about 81. In some embodiments, the molar ratio of DMPC to the MSP in the pharmaceutical composition is less than about 78:1. In some embodiments, the molar ratio of DMPC to the MSP in the pharmaceutical composition is about 65:1 to about 75:1. In some embodiments, the molar ratio of DMPC to the MSP in the pharmaceutical composition is from about 40:1 to about 60:1. In some embodiments, the molar ratio of DMPC to the MSP in the pharmaceutical composition is about 46:1 to about 55:1. In some embodiments, the MSP is MSP1 or His-tag removed variant thereof, and the molar ratio of DMPC to the MSP is about 80:1. [0013] In some embodiments, the MSP is MSP1D1, MSP1, or His-tag removed variant thereof, the phospholipid is DPPC, and the molar ratio of DPPC to the MSP in the ny-2692819 Attorney Docket No.: 283912000140 pharmaceutical composition is from about 10:1 to about 90:1. In some embodiments, the MSP is MSP1 or His-tag removed variant thereof, the phospholipid is DPPC, and the molar ratio of DPPC to the MSP in the pharmaceutical composition is about 90:1. In some embodiments, the MSP is MSP1D1 or His-tag removed variant thereof, the phospholipid is DPPC, and the NS is about 81. In some embodiments, the molar ratio of DPPC to the MSP in the pharmaceutical composition is less than about 75:1. [0014] In some embodiments, the MSP is MSP1E3D1, MSP1E3, or His-tag removed variant thereof, and the molar ratio of the phospholipid to the MSP in the pharmaceutical composition is from about 50:1 to about 200:1. In some embodiments, the MSP is MSP1E3 or His-tag removed variant thereof, wherein the phospholipid is POPC, and the molar ratio of POPC to the MSP in the pharmaceutical composition is about 130:1. In some embodiments, the MSP is MSP1E3D1 or His-tag removed variant thereof, the phospholipid is POPC, and the NS is about 126. In some embodiments, the molar ratio of POPC to the MSP in the pharmaceutical composition is less than about 118:1. In some embodiments, the MSP is MSP1E3D1 or His-tag removed variant thereof, the phospholipid is DMPC, and the NS is about 167. In some embodiments, the molar ratio of DMPC to the MSP in the pharmaceutical composition is less than about 158:1. In some embodiments, the molar ratio of DMPC to the MSP in the pharmaceutical composition is from about 50:1 to about 160:1. In some embodiments, the molar ratio of DMPC to the MSP in the pharmaceutical composition is from about 50:1 to about 100:1. In some embodiments, the molar ratio of DMPC to the MSP in the pharmaceutical composition is about 87:1 to about 96:1, about 58:1 to about 68:1, about 63:1, or about 62:1. In some embodiments, the MSP is MSP1E3 or His-tag removed variant thereof, the phospholipid is DMPC, and the molar ratio of DMPC to the MSP in the pharmaceutical composition is about 150:1. In some embodiments, the MSP is MSP1E3 or His-tag removed variant thereof, the phospholipid is DPPC, and the molar ratio of DPPC to the MSP in the pharmaceutical composition is about 170:1. In some embodiments, the MSP is MSP1E3D1 or His-tag removed variant thereof, the phospholipid is DPPC, and the NS is about 167. In some embodiments, the molar ratio of DPPC to the MSP in the pharmaceutical composition is less than about 158:1. [0015] In some embodiments, the MSP is MSP1E1D1 or His-tag removed variant thereof, the phospholipid is DMPC, and the NS is about 107. In some embodiments, the molar ratio of DMPC to the MSP in the pharmaceutical composition is less than about 100:1. In some ny-2692819 Attorney Docket No.: 283912000140 embodiments, the molar ratio of DMPC to the MSP in the pharmaceutical composition is from about 60:1 to about 65:1. [0016] In some embodiments, the nanodisc in the pharmaceutical composition is about 2 nm to about 20 nm in diameter. In some embodiments, the lipid for removal or prevention from accumulation in the eye is cholesterol. In some embodiments, the nanodisc does not comprise: (i) a cell-penetrating peptide (CPP); (ii) a target protein; and/or (iii) a prophylactic or therapeutic agent. [0017] In some embodiments, the nanodisc increases cholesterol efflux rate by at least about 2- fold compared to an untreated state, or the cholesterol efflux rate by ApoA-I or the MSP. In some embodiments, the pharmaceutical composition is administered via intravitreal injection. In some embodiments, the eye disease characterized by lipid accumulation is selected from the group consisting of age-related macular degeneration (AMD), photoreceptor neurodegeneration, optic nerve atrophy, loss of acuity, hemianopia, visual agnosia, strabismus, retinal neurovascular disorder, lipid keratopathy, corneal lipidosis, and a combination thereof. In some embodiments, the eye disease characterized by lipid accumulation is AMD. In some embodiments, the AMD is dry AMD. In some embodiments, the dry AMD is geographic atrophy (GA). In some embodiments, the nanodisc reduces lipid or prevents lipid accumulation in the eye of the individual by at least about 5%. BRIEF DESCRIPTION OF THE DRAWINGS [0018] The drawings illustrate certain embodiments of the features and advantages of this disclosure. These embodiments are not intended to limit the scope of the appended claims in any manner. [0019] FIG. 1 displays an exemplary chromatogram of nanodisc preparation. [0020] FIG. 2A displays a chromatogram of the formation of POPC MSP1D1 nanodiscs at different POPC to MSP1D1 ratios. The results indicate that a ratio of 65:1 forms a fully lipidated nanodisc. FIG. 2B displays a chromatogram of the formation of DMPC MSP1D1 nanodiscs at different DMPC to MSP1D1 ratios. The results indicate that fully lipidated nanodics form at 85:1 DMPC to MSP1D1 ratio. ny-2692819 Attorney Docket No.: 283912000140 [0021] FIG. 3 shows the percent of cholesterol efflux from macrophages in vitro using various nanodiscs with different lipid to MSP ratios. Lipid A refers to DMPC, and Lipid B refers to POPC. [0022] FIG. 4 shows that a DMPC MSP1D1 nanodisc with a lipid to MSP ratio of 85:1 has the ability to remove almost two times more cholesterol from macrophages than naturally occurring ApoA-I after 4 hours of incubation. [0023] FIGs. 5A-5C shows the percent of cholesterol efflux from human retinal pigment epithelial (RPE) cells in vitro using a nanodisc with POPC to MSP1D1 ratio of 65:1 (MSP1D1 Lipid B), a nanodisc DMPC to MSP1D1 ratio of 85:1 (MSP1D1 Lipid A), and a MSP1D1 protein alone after 2 hours (FIG. 5A), 4 hours (FIG. 5B), and 6 hours (FIG.5C) of incubation. [0024] FIG. 6A and FIG.6B show fluorescence images following intravitreal delivery of a GFP-labeled exemplary nanodisc into the murine eye in vivo. From left to right, FIG.6A shows fluorescence images at 15 minutes, 30 minutes, 60 minutes, 6 hours, and 24 hours after delivery of a GFP-labeled exemplary nanodisc into the murine eye in vivo. The first row in FIG.6B, from left to right, shows fluorescence images at 7 minutes, 7 minutes, 10 minutes, 15 minutes, and 20 minutes after delivery of a GFP-labeled exemplary nanodisc in to the murine eye. The second row in FIG. 6B, from left to right, shows fluorescence images at 30 minutes, 30 minutes, 33 minutes, and 47 minutes after delivery of a GFP-labeled exemplary nanodisc in to the murine eye. The third row in FIG.6B, from left to right, shows fluorescence images at 6 hours, 6 hours, and 24 hours after delivery of a GFP-labeled exemplary nanodisc in to the murine eye. [0025] FIG. 7A is a schematic depicting schedule for intravitreal injection of fluorescent labeled exemplary nanodisc and tissue harvest. FIG.7B shows representative images of immunostained retina sections taken from injected mice depicting retinal neurons (solid arrows)and fluorescent labeled exemplary nanodisc (blank arrows). FIG.7B, from left to right, shows fluorescence images at 0 minutes, 15 minutes, 30 minutes, 1 hour, 6 hours and 24 hours after delivery of a fluorescent-labelled exemplary nanodisc in to the murine eye. In FIG.7B, from top to bottom, the images show the fluorescence signals at retinal pigment epithelium (RPE), outer nuclear layer (ONL), and inner nuclear layer (INL). [0026] FIG.8A is a schematic depicting schedule for laser injury-induced choroidal neovascularization (CNV), intravitreal injection of the exemplary nanodisc, intravenous injection ny-2692819 Attorney Docket No.: 283912000140 of FITC Dextran, and tissue harvest. FIG.8B shows representative confocal image of CNV area labeled by FITC Dextran and outlined in a circle following laser injury in the mouse. FIG. 8C shows dot plots showing CNV area in mice that received 0 (‘Cntl’), 1 (‘Nanodisc’), or 2 (‘Nanodisc +d3) intravitreal injections of the exemplary nanodisc. Data (n=8-10 mice per group) are presented as a mean (±SEM). [0027] FIG.9A shows a schematic depicting schedule for high-fat diet administration, intravitreal injections, baseline (T0) and follow-up (T1) imaging and tissue harvest for Abca1/g1-rod/-rod mice. FIG. 9B and FIG.9C show line graphs showing full-field scotopic (FIG. 9B) and light bleach recovery (FIG.9C) response amplitudes and ratios in vehicle (‘Vehicle’) and exemplary nanodisc-treated (‘Disc’) mouse groups. Data (Vehicle, n=3; nanodisc, n=4) are presented as mean (±SEM). FIG.9D shows representative images of hematoxylin and eosin-stained (H&E) sections of whole eyes (left) and retinas (right). FIG.9E shows representative images of immunostained retina sections showing retinal neuron (solid arrows) and complement-3 (blank arrows) expression in the central (left) and mid-peripheral (right) retina. FIG.9F shows representative baseline (top) and follow-up (bottom) optical coherence tomography (OCT) images of living mouse retina cross section showing incidence of (Vehicle) and absence of (Disc) inner/outer segment abnormalities (arrowhead in the bottom left image) and retinal pigment epithelium detachements (arrowhead in the bottom right image) in reference to baseline (asterisk and arrowhead in the top images). [0028] FIG.10A shows a schematic depicting schedule for high-fat diet administration, intravitreal injections, baseline (T0) and follow-up (T1) imaging and blood collection, and tissue harvest for Abca1/g1-rod/-rod mice. FIG.10B and FIG.10C show line graphs showing full- field scotopic (FIG.10B) and light bleach recovery (FIG.10C) response amplitudes and ratios in vehicle (‘Vehicle’) and the exemplary nanodisc-treated (‘Disc’) mouse groups. The y-axis shows Amplitude (µV). Data (Vehicle, n=3; Disc, n=4) are presented as mean (±SEM). FIG. 10D shows box plots showing cholesteryl ester (CE) proportions of retinal lipids measured in Vehicle- and nanodisc- treated mice. Data (Vehicle, n=3; Disc, n=4) is presented as a median with interquartile range, minimum, and maximum values indicated. [0029] FIG.11A shows one chromatogram of preparative size exclusion chromatography (SEC) of the exemplary DMPC/MSP1D1(-) nanodisc prepared at 30:1 ratio. (-) denotes the removal of ny-2692819 Attorney Docket No.: 283912000140 the His tag from the MSP1D1. The dark-grey shaded area of the peak (upper panel) corresponding to an under-lipidated nanodisc containing approximately 51 lipids per MSP1D1(-) (lower panel). FIG.11B shows one chromatogram of preparative SEC of an exemplary DMPC/MSP1E3D1(-) nanodisc prepared at 50:1 ratio. A single highlighted peak (upper panel) was collected, which corresponds to an under-lipidated nanodisc with about 62 lipids per MSP1E3D1(-). [0030] FIG.12 shows gas chromatography (GC) result for determining phospholipid to MSP ratio of the exemplary DMPC/MSP1D1(-) nanodisc prepared at 30:1 ratio. [0031] FIGs. 13A-13D show the correspondence between the phospholipid/MSP1D1(-) ratio in the preparation mixture and the final nanodisc for DMPC/MSP1D1(-) about 81:1 nanodisc (preparation mixture 80:1; final nanodisc 81±3 : 1) (FIG.13A), DMPC/MSP1D1(-) nanodisc about 75:1 (preparation mixture 55:1; final nanodisc 75±1.5 :1) (FIG. 13B), DMPC/MSP1D1(-) about 51:1 nanodisc (preparation mixture 40:1; final nanodisc 51±1 :1) (FIG.13C), and DMPC/MSP1D1(-) about 51:1 nanodisc (preparation mixture 30:1; final nanodisc 51±1 :1) (FIG. 13D). [0032] FIGs. 14A-14C show the correspondence between the phospholipid/MSP1E3D1(-) ratio in the preparation mixture and the final nanodisc for DMPC/MSP1E3D1(-) about 93:1 nanodisc (preparation mixture 90:1; final nanodisc 93±3 :1) (FIG.14A), DMPC/MSP1E3D1(-) about 63:1 nanodisc (preparation mixture 60:1; final nanodisc 63±4 :1) (FIG.14B), and DMPC/MSP1E3D1(-) about 62:1 nanodisc (preparation mixture 50:1; final composition 62±0.5 :1) (FIG.14C). [0033] FIG. 15A shows the cholesterol efflux assay results using various under-lipidated nanodiscs. The phospholipid to MSP ratios were measured from the final nanodiscs (not preparation mixture).2.5 μg of samples were used. Controls include a fully-lipidated nanodiscs with DMPC:MSP1D1(-)=81:1, a fully-lipidated nanodiscs with DMPC:MSP1D1=83:1, lipid-free MSP1D1(-), lipid-free Apo-AI , lipid-free HDL, positive control (from the assay kit), and a PBS buffer blank (not shown). FIG.15 shows cholesterol efflux dose dependence of fully-lipidated nanodiscs DMPC:MSP1D1(-)=81:1 and an exemplary under-lipidated nanodisc composition DMPC:MSP1D1(-)=51:1 at 2.5 μg and 10 µg doses. (-) denotes the removal of the N-terminal His tag. ny-2692819 Attorney Docket No.: 283912000140 [0034] FIG. 16 shows the SEC chromatogram of exemplary under-lipidated DMPC/MSP1D1(-) nanodiscs prepared at 30:1 ratio (final composition ratio was about 51:1) after freezing and thawing without cryoprotectant, compared to before-freezing. [0035] FIG. 17 shows the cholesterol efflux results of exemplary under-lipidated DMPC:MSP1D1(-) nanodiscs prepared at 30:1 ratio (final ratio in the nanodiscs was about 51:1) before and after freezing and thawing, in comparison with controls including fully-lipidated DMPC:MSP1D1 nanodiscs prepared at 85:1 (final composition ratio was 83:1), HDL, and PBS solution. DETAILED DESCRIPTION [0036] The present application provides a method of removing lipid (e.g., cholesterol) or preventing lipid accumulation in the eye of an individual (e.g., human), and/or preventing or treating an eye disease characterized by lipid accumulation in an individual, comprising administering to the eye of the individual an effective amount of a pharmaceutical composition comprising a nanodisc (e.g., a plurality of nanodiscs), wherein the nanodisc comprises a membrane scaffold protein (MSP) and a phospholipid. The present invention is based, at least in part, on the surprising discovery that nanodiscs described herein i) are able to reach the posterior segment of the eye with excellent penetrance, ii) have good PK properties and do not dwell in the eye for an extended period of time (e.g., >24 hours); iii) can effectively remove lipids; iv) do not require the assistance of any targeting moiety or cell penetrating peptide (CPP); and v) is both effective and safe in treating eye disease characterized by lipid accumulation (e.g., age-related macular degeneration (AMD)) in vivo. The nanodiscs and methods described herein are therefore particularly helpful in treating a broad range of eye diseases characterized by lipid accumulation, including macular degeneration and atrophy in the eye. I. Definitions [0037] As used herein, the singular forms “a,” “an,” and “the” include the plural referents unless the context clearly indicates otherwise. [0038] The term “about” indicates and encompasses an indicated value and a range above and below that value. In certain embodiments, the term “about” indicates the designated ny-2692819 Attorney Docket No.: 283912000140 value ± 10%, ± 5%, or ± 1%. In certain embodiments, the term “about” indicates the designated value ± one standard deviation of that value. [0039] The terms “effective amount” and “pharmaceutically effective amount” refer to a sufficient amount of an agent to provide the desired biological result. That result can be reduction (e.g., reducing at least about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or 100%) and/or alleviation of the signs, symptoms, or causes of a disease or disorder, or any other desired alteration of a biological system. An appropriate effective amount in any individual case may be determined by one of ordinary skill in the art using routine experimentation. [0040] The terms “patient,” “subject,” “individual,” and the like are used interchangeably herein, and refer to any animal, in some embodiments a mammal, and in some embodiments a human, having a complement system, including a human in need of therapy for, or susceptible to, a condition or its sequelae. The individual may include, for example, dogs, cats, pigs, cows, sheep, goats, horses, rats, rabbits, hamsters, guinea pigs, monkeys, mice, and humans. In some embodiments, the individual is a human. [0041] The term “treatment” typically refers to clinical intervention designed to alter the natural course of the individual or cell being treated during the course of clinical pathology. Desirable effects of treatment include decreasing the rate of disease progression, ameliorating or palliating the disease state, and remission or improved prognosis. For example, an individual is successfully “treated” if one or more symptoms associated with disease or disorder are mitigated or eliminated, including, but not limited to, decreasing the frequency and/or severity of a sign and/or symptoms resulting from the disease, increasing the quality of life of those suffering from the disease, decreasing the dose of other medications required to treat the disease, and/or prolonging survival of individuals. Treatment may be prophylactic (to prevent or delay the onset of the disease, or to prevent the manifestation of clinical or subclinical symptoms thereof) or therapeutic suppression or alleviation of symptoms after the manifestation of the disease. [0042] As used herein, “percent (%) amino acid sequence identity” and “homology” with respect to a peptide, polypeptide or antibody sequence are defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the specific peptide or polypeptide sequence, after aligning the sequences and introducing gaps, if ny-2692819 Attorney Docket No.: 283912000140 necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN or MEGALIGNTM (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared. [0043] As used herein, “amphipathic” molecules refer to molecules that have both hydrophilic and hydrophobic properties. Examples include phospholipids, cholesterol, glycolipids and fatty acids. [0044] It is understood that embodiments of the invention described herein include “consisting of” and/or “consisting essentially of” embodiments. II. Methods of Removing Lipid or Preventing Lipid Accumulation, and Methods of Treating Eye Diseases [0045] In one aspect, provided herein is a method of removing lipid (e.g., cholesterol) or preventing lipid accumulation in the eye of an individual (e.g., human), comprising administering to the eye of the individual an effective amount of a pharmaceutical composition comprising a nanodisc, wherein the nanodisc comprises an MSP and a phospholipid. In one aspect, provided herein is a method of removing lipid or preventing lipid accumulation in the eye of an individual, comprising administering to the eye of the individual an effective amount of a pharmaceutical composition comprising a plurality of nanodiscs, wherein the nanodisc comprises a membrane scaffold protein (MSP) and a phospholipid. In some embodiments, the MSP is a variant of ApoA-I. In some embodiments, the MSP is selected from the group consisting of MSP1E1, MSP1E2, MSP1E3, MSP1, MSP1D1, MSP1E1D1, MSP1E2D1, MSP1E3D1, MSP2N2, and N-terminal His-tag portion removed variant thereof (His-tag removed variant). In some embodiments, the MSP is selected from the group consisting of MSP1E1, MSP1E2, MSP1E3, MSP1, MSP1D1, MSP1D2, and MSPE3D1. In some embodiments, the MSP is (i) an MSP1D1 His-tag removed variant comprising the amino acid sequence of SEQ ID NO: 14; or (ii) an MSP1E3D1 His-tag removed variant comprising the amino acid sequence of SEQ ID NO: ny-2692819 Attorney Docket No.: 283912000140 15. In some embodiments, the phospholipid comprises DMPC, DPPC, DMPS, DSPC, POPC, POPS, PiP2, or a combination thereof. In some embodiments, the phospholipid is a PC, such as selected from the group consisting of DMPC, POPC, DPPC, DSPC, and a combination thereof. [0046] In some embodiments, provided herein is a method of removing lipid or preventing lipid accumulation in the eye of an individual, comprising administering to the eye of the individual an effective amount of a pharmaceutical composition comprising a plurality of nanodiscs, wherein at least about 85% of the plurality of nanodiscs in the pharmaceutical composition are fully-lipidated. In some embodiments, provided herein is a method of removing lipid or preventing lipid accumulation in the eye of an individual, comprising administering to the eye of the individual an effective amount of a pharmaceutical composition comprising a plurality of nanodiscs, wherein at least about 85% of the plurality of nanodiscs in the pharmaceutical composition are under-lipidated. In some embodiments, the molar ratio of the phospholipid to the MSP in the pharmaceutical composition is less than a phospholipid-MSP saturation ratio NS. In some embodiments, the molar ratio of the phospholipid to the MSP in the pharmaceutical composition is less than about 95% of the phospholipid-MSP saturation ratio NS. [0047] In some embodiments, the MSP is MSP1D1, MSP1, or His-tag removed variant thereof, and the molar ratio of the phospholipid to the MSP in the pharmaceutical composition is from about 10:1 to about 90:1. In some embodiments, the MSP is MSP1D1 or MSP1, and the molar ratio of phospholipid to MSP is from about 10:1 to about 90:1. In some embodiments, the MSP is MSP1D1, MSP1, or His-tag removed variant thereof, the phospholipid is POPC, and the molar ratio of POPC to the MSP in the pharmaceutical composition is from about 10:1 to about 65:1. In some embodiments, the MSP is MSP1D1 or MSP1, the phospholipid is POPC, and the molar ratio of POPC to MSP1D1 or MSP1 is from about 10:1 to about 65:1. In some embodiments, the MSP is MSP1D1, the phospholipid is POPC, and the molar ratio of POPC to MSP1D1 is about 50:1 or about 32.5:1. In some embodiments, the MSP is MSP1 or MSP1D1, the phospholipid is POPC, and the molar ratio of POPC to MSP1 or MSP1D1 is about 65:1. In some embodiments, the MSP is MSP1D1 or His-tag removed variant thereof, the phospholipid is POPC, and the molar ratio of POPC to the MSP in the pharmaceutical composition is about 65:1. In some embodiments, the MSP is MSP1D1 or His-tag removed variant thereof, the phospholipid is POPC, and the NS is about 62. In some embodiments, the MSP is MSP1D1 or His-tag removed variant thereof, the phospholipid is POPC, the molar ratio of POPC to MSP1D1 ny-2692819 Attorney Docket No.: 283912000140 or His-tag removed variant thereof in the pharmaceutical composition is less than about 58:1. In some embodiments, the MSP is MSP1D1 or His-tag removed variant thereof, the phospholipid is POPC, the molar ratio of POPC to MSP1D1 or His-tag removed variant thereof in the pharmaceutical composition is about 50:1 or about 32.5:1. [0048] In some embodiments, the MSP is MSP1D1, MSP1, or His-tag removed variant thereof, the phospholipid is DMPC, wherein the molar ratio of DMPC to the MSP in the pharmaceutical composition is from about 10:1 to about 85:1. In some embodiments, the MSP is MSP1D1 or MSP1, the phospholipid is DMPC, and the molar ratio of DMPC to MSP1D1 or MSP1 is from about 10:1 to about 85:1. In some embodiments, the MSP is MSP1D1 or His-tag removed variant thereof, the phospholipid is DMPC, and the molar ratio of DMPC to the MSP in the pharmaceutical composition is about 85:1. In some embodiments, the MSP is MSP1D1, the phospholipid is DMPC, and the molar ratio of DMPC to MSP1D1 is about 85:1. In some embodiments, the molar ratio of DMPC to MSP1D1 in the pharmaceutical composition is about 30:1 to about 80:1, such as any of about 40:1 to about 60:1, about 46:1 to about 55:1, about 51:1, about 55:1, or about 50:1. In some embodiments, the MSP is MSP1D1 or His-tag removed variant thereof, the phospholipid is DMPC, and the NS is about 81. In some embodiments, the MSP is MSP1, the phospholipid is DMPC, and the molar ratio of DMPC to MSP1 is about 80:1. In some embodiments, the MSP is MSP1D1 or His-tag removed variant thereof, the phospholipid is DMPC, and the molar ratio of DMPC to the MSP in the pharmaceutical composition is from about 40:1 to about 60:1, such as about 46:1 to about 55:1, about 51:1, about 55:1, or about 50:1. [0049] In some embodiments, the MSP is MSP1D1, MSP1, or His-tag removed variant thereof, wherein the phospholipid is DPPC, and the molar ratio of DPPC to the MSP1D1, MSP1, or His-tag removed variant thereof in the pharmaceutical composition is from about 10:1 to about 90:1. In some embodiments, the MSP is MSP1 or His-tag removed variant thereof, the phospholipid is DPPC, and the molar ratio of DPPC to the MSP1D1, MSP1, or His-tag removed variant thereof in the pharmaceutical composition is about 90:1. In some embodiments, the MSP is MSP1D1 or His-tag removed variant thereof, the phospholipid is DPPC, and the NS is about 81. In some embodiments, the molar ratio of DPPC to the MSP1D1 or His-tag removed variant thereof in the pharmaceutical composition is less than about 75:1. In some embodiments, the MSP is MSP1, the phospholipid is DPPC, and the molar ratio of DPPC to MSP1 is about 90:1. ny-2692819 Attorney Docket No.: 283912000140 [0050] In some embodiments, the MSP is MSP1E3, and the molar ratio of phospholipid to MSP1E3 (e.g., in the nanodisc, or in the pharmaceutical composition) is from about 100:1 to about 200:1. In some embodiments, the phospholipid is POPC, the MSP is MSP1E3, and the molar ratio of POPC to MSP1E3 is about 130:1. In some embodiments, the phospholipid is DMPC, the MSP is MSP1E3, and the molar ratio of DMPC to MSP1E3 (e.g., in the nanodisc, or in the pharmaceutical composition) is about 150:1. In some embodiments, the phospholipid is DPPC, the MSP is MSP1E3, and the molar ratio of DPPC to MSP1E3 (e.g., in the nanodisc, or in the pharmaceutical composition) is about 170:1. [0051] In some embodiments, the MSP is MSP1E3D1 or His-tag removed variant thereof, the phospholipid is DMPC, and the molar ratio of DMPC to the MSP in the pharmaceutical composition is about 87:1 to about 96:1, about 58:1 to about 68:1, about 63:1, or about 62:1. [0052] In some embodiments, the nanodisc comprises DMPC and MSP1D1, such as in a molar ratio of about 85:1. In some embodiments, the nanodisc comprises DMPC and MSP1D1, optionally wherein the molar ratio of DMPC to MSP1D1in the pharmaceutical composition is about 46:1 to about 55:1, about 51:1, about 55:1, or about 50:1. Hence in some embodiments, there is provided a method of removing lipid (e.g., cholesterol) or preventing lipid accumulation in the eye of an individual (e.g., human), comprising administering to the eye of the individual an effective amount of a pharmaceutical composition comprising a plurality of nanodiscs, wherein the nanodisc comprises DMPC and MSP1D1, and the molar ratio of DMPC and MSP1D1 in the pharmaceutical composition is about 46:1 to about 55:1, about 51:1, about 55:1, or about 50:1. [0053] In some embodiments, the nanodisc comprises POPC and MSP1D1, such as in a molar ratio of about 65:1, about 50:1, or about 32.5:1. Hence in some embodiments, there is provided a method of removing lipid (e.g., cholesterol) or preventing lipid accumulation in the eye of an individual (e.g., human), comprising administering to the eye of the individual an effective amount of a pharmaceutical composition comprising a nanodisc, wherein the nanodisc comprises DMPC and MSP1D1 at a molar ratio of about 85:1, about 51:1, about 55:1, or about 50:1. [0054] In some embodiments, the nanodisc comprises DMPC and MSP1E3D1 and the molar ratio of DMPC and MSP1E3D1 in the pharmaceutical composition is about 87:1 to about 96:1, about 58:1 to about 68:1, about 63:1, or about 62:1. In some embodiments, the molar ratio of DMPC to MSP1E3D1 in the pharmaceutical composition is about 60:1 to about 65:1. Hence in ny-2692819 Attorney Docket No.: 283912000140 some embodiments, there is provided a method of removing lipid (e.g., cholesterol) or preventing lipid accumulation in the eye of an individual (e.g., human), comprising administering to the eye of the individual an effective amount of a pharmaceutical composition comprising a plurality of nanodiscs, wherein the nanodisc comprises DMPC and MSP1E3D1, and the molar ratio of DMPC and MSP1D1E3 in the pharmaceutical composition is about 58:1 to about 68:1, about 63:1, or about 62:1. [0055] In some embodiments, there is provided a method of removing lipid (e.g., cholesterol) or preventing lipid accumulation in the eye of an individual (e.g., human), comprising administering to the eye of the individual an effective amount of a pharmaceutical composition comprising a plurality of nanodiscs, wherein the nanodisc comprises POPC and MSP1D1 (e.g., SEQ ID NO: 3) at a molar ratio of about 65:1, about 50:1, or about 32.5:1. [0056] In some embodiments, there is provided a method of removing lipid (e.g., cholesterol) or preventing lipid accumulation in the eye of an individual (e.g., human), comprising administering to the eye of the individual an effective amount of a pharmaceutical composition comprising a plurality of nanodiscs, wherein the nanodisc comprises DMPC and MSP1D1 (e.g., SEQ ID NO: 3) or His-tag removed variant thereof (e.g., SEQ ID NO: 14), and wherein the molar ratio of DMPC and MSP1D1 (e.g., SEQ ID NO: 3) or His-tag removed variant thereof (e.g., SEQ ID NO: 14) in the pharmaceutical composition is about 46:1 to about 55:1, about 51:1, about 55:1, or about 50:1. [0057] In some embodiments, there is provided a method of removing lipid (e.g., cholesterol) or preventing lipid accumulation in the eye of an individual (e.g., human), comprising administering to the eye of the individual an effective amount of a pharmaceutical composition comprising a plurality of nanodiscs, wherein the nanodisc comprises DMPC and MSP1E3D1 (e.g., SEQ ID NO: 8) or His-tag removed variant thereof (e.g., SEQ ID NO: 15), and the molar ratio of DMPC and MSP1E3D1 (e.g., SEQ ID NO: 8) or His-tag removed variant thereof (e.g., SEQ ID NO: 15) in the pharmaceutical composition is about 58:1 to about 68:1, about 63:1, or about 62:1. [0058] In some embodiments, the lipid for removal or prevention from accumulation in the eye is cholesterol. In some embodiments, the nanodisc is administered intravitreally. In some embodiments, the nanodisc is about 2 nm to about 20 nm, such as about 5 nm to about 20 nm or ny-2692819 Attorney Docket No.: 283912000140 about 10 nm to about 20 nm in diameter. In some embodiments, the nanodisc does not comprise a CPP. In some embodiments, the nanodisc does not comprise a target protein. In some embodiments, the nanodisc does not comprise a prophylactic or therapeutic agent. In some embodiments, the nanodisc increases cholesterol efflux rate by at least about 2-fold (e.g., at least about any of 2.5, 3, 4, 5, 10, 15-fold, or more) compared to an untreated state, or the cholesterol efflux rate by ApoA-I or the MSP. In some embodiments, the nanodisc reduces lipid or prevents lipid accumulation in the eye of the individual by at least about 5% (e.g., at least about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%). [0059] In another aspect, provided herein is a method of preventing or treating an eye disease characterized by lipid (e.g., cholesterol) accumulation in an individual (e.g., human), comprising administering to the eye of the individual an effective amount of a pharmaceutical composition comprising a nanodisc, wherein the nanodisc comprises an MSP and a phospholipid. In one aspect, provided herein is a method of preventing or treating an eye disease characterized by lipid (e.g., cholesterol) accumulation in an individual (e.g., human), comprising administering to the eye of the individual an effective amount of a pharmaceutical composition comprising a plurality of nanodiscs, wherein the nanodisc comprises an MSP and a phospholipid. In some embodiments, the MSP is a variant of ApoA-I. In some embodiments, the MSP is selected from the group consisting of MSP1E1, MSP1E2, MSP1E3, MSP1, MSP1D1, MSP1E1D1, MSP1E2D1, MSP1E3D1, MSP2N2, and N-terminal His-tag portion removed variant thereof (His-tag removed variant). In some embodiments, the MSP is selected from the group consisting of MSP1E1, MSP1E2, MSP1E3, MSP1, MSP1D1, MSP1D2, and MSPE3D1. In some embodiments, the MSP is (i) an MSP1D1 His-tag removed variant comprising the amino acid sequence of SEQ ID NO: 14; or (ii) an MSP1E3D1 His-tag removed variant comprising the amino acid sequence of SEQ ID NO: 15. In some embodiments, the phospholipid comprises DMPC, DPPC, DMPS, DSPC, POPC, POPS, PiP2, or a combination thereof. In some embodiments, the phospholipid is a PC, such as selected from the group consisting of DMPC, POPC, DPPC, DSPC, and a combination thereof. [0060] In some embodiments, provided herein is a method of preventing or treating an eye disease characterized by lipid (e.g., cholesterol) accumulation in an individual (e.g., human), comprising administering to the eye of the individual an effective amount of a pharmaceutical composition comprising a nanodisc, wherein at least about 85% of the plurality of nanodiscs in ny-2692819 Attorney Docket No.: 283912000140 the pharmaceutical composition are fully-lipidated. In some embodiments, provided herein is a method of preventing or treating an eye disease characterized by lipid (e.g., cholesterol) accumulation in an individual (e.g., human), comprising administering to the eye of the individual an effective amount of a pharmaceutical composition comprising a nanodisc, wherein at least about 85% of the plurality of nanodiscs in the pharmaceutical composition are under- lipidated. In some embodiments, the molar ratio of the phospholipid to the MSP in the pharmaceutical composition is less than a phospholipid-MSP saturation ratio NS. In some embodiments, the molar ratio of the phospholipid to the MSP in the pharmaceutical composition is less than about 95% of the phospholipid-MSP saturation ratio NS. [0061] In some embodiments, the MSP is MSP1D1, MSP1, or His-tag removed variant thereof, and the molar ratio of the phospholipid to the MSP in the pharmaceutical composition is from about 10:1 to about 90:1. In some embodiments, the MSP is MSP1D1 or MSP1, and the molar ratio of phospholipid to MSP is from about 10:1 to about 90:1. In some embodiments, the MSP is MSP1D1, MSP1, or His-tag removed variant thereof, the phospholipid is POPC, and the molar ratio of POPC to the MSP in the pharmaceutical composition is from about 10:1 to about 65:1. In some embodiments, the phospholipid is POPC, and the molar ratio of POPC to MSP is from about 10:1 to about 65:1. In some embodiments, the MSP is MSP1D1, the phospholipid is POPC, and the molar ratio of POPC to MSP1D1 is about 50:1 or about 32.5:1. In some embodiments, the MSP is MSP1 or MSP1D1, the phospholipid is POPC, and the molar ratio of POPC to MSP1 or MSP1D1 is about 65:1. In some embodiments, the MSP is MSP1D1 or His- tag removed variant thereof, the phospholipid is POPC, and the molar ratio of POPC to the MSP in the pharmaceutical composition is about 65:1. In some embodiments, the MSP is MSP1D1 or His-tag removed variant thereof, the phospholipid is POPC, and the NS is about 62. In some embodiments, the molar ratio of POPC to the MSP1D1 or His-tag removed variant thereof in the pharmaceutical composition is less than about 58:1. In some embodiments, the molar ratio of POPC to the MSP1D1 or His-tag removed variant thereof in the pharmaceutical composition is about 50:1 or about 32.5:1. [0062] In some embodiments, the MSP is MSP1D1, MSP1, or His-tag removed variant thereof, the phospholipid is DMPC, wherein the molar ratio of DMPC to the MSP in the pharmaceutical composition is from about 10:1 to about 85:1. In some embodiments, the MSP is MSP1D1, MSP1, or His-tag removed variant thereof, the phospholipid is DMPC, and the molar ny-2692819 Attorney Docket No.: 283912000140 ratio of DMPC to MSP is from about 10:1 to about 85:1.In some embodiments, the MSP is MSP1D1 or His-tag removed variant thereof, the phospholipid is DMPC, and the molar ratio of DMPC to the MSP in the pharmaceutical composition is about 85:1. In some embodiments, the MSP is MSP1D1, the phospholipid is DMPC, and the molar ratio of DMPC to MSP1D1 is about 85:1. In some embodiments, the molar ratio of DMPC to MSP1D1 in the pharmaceutical composition is about 30:1 to about 80:1, such as any of about 40:1 to about 60:1, about 46:1 to about 55:1, about 51:1, about 55:1, or about 50:1. In some embodiments, the MSP is MSP1D1 or His-tag removed variant thereof, the phospholipid is DMPC, and the NS is about 81. In some embodiments, the MSP is MSP1, and the molar ratio of DMPC to MSP1 is about 80:1. In some embodiments, the MSP is MSP1D1, the phospholipid is DMPC, and the molar ratio of DMPC to MSP1D1 in the pharmaceutical composition is about 80:1, about 51:1, about 55:1, or about 50:1. In some embodiments, the MSP is MSP1D1, MSP1, or His-tag removed variant thereof, the phospholipid is DMPC, and the molar ratio of DMPC to the MSP in the pharmaceutical composition is less than about 78:1. In some embodiments, the molar ratio of DMPC to the MSP in the pharmaceutical composition is about 65:1 to about 75:1. In some embodiments, the molar ratio of DMPC to the MSP1D1, MSP1, or His-tag removed variant thereof in the pharmaceutical composition is from about 40:1 to about 60:1. In some embodiments, the molar ratio of DMPC to the MSP in the pharmaceutical composition is about 46:1 to about 55:1, about 51:1, about 55:1, or about 50:1. [0063] In some embodiments, the MSP is MSP1D1, MSP1, or His-tag removed variant thereof, wherein the phospholipid is DPPC, and the molar ratio of DPPC to the MSP in the pharmaceutical composition is from about 10:1 to about 90:1. In some embodiments, the MSP is MSP1 or His-tag removed variant thereof, the phospholipid is DPPC, and the molar ratio of DPPC to the MSP in the pharmaceutical composition is about 90:1. In some embodiments, the MSP is MSP1D1 or His-tag removed variant thereof, the phospholipid is DPPC, and the NS is about 81. In some embodiments, the molar ratio of DPPC to the MSP in the pharmaceutical composition is less than about 75:1. In some embodiments, the MSP is MSP1, the phospholipid is DPPC, and the molar ratio of DPPC to MSP1 is about 90:1. [0064] In some embodiments, the MSP is MSP1E3D1, MSP1E3, or His-tag removed variant thereof, and the molar ratio of the phospholipid to the MSP in the pharmaceutical composition is from about 50:1 to about 200:1. In some embodiments, the MSP is MSP1E3, and the molar ratio ny-2692819 Attorney Docket No.: 283912000140 of phospholipid to MSP1E3 (e.g., in the nanodisc, or in the pharmaceutical composition) is from about 100:1 to about 200:1. In some embodiments, the phospholipid is POPC, and the molar ratio of POPC to MSP1E3 (e.g., in the nanodisc, or in the pharmaceutical composition) is about 130:1. In some embodiments, the MSP is MSP1E3 or His-tag removed variant thereof, the phospholipid is POPC, and the molar ratio of POPC to the MSP1E3D1, or His-tag removed variant thereof in the pharmaceutical composition is about 130:1. In some embodiments, the MSP is MSP1E3D1 or His-tag removed variant thereof, the phospholipid is POPC, and the NS is about 126. In some embodiments, the molar ratio of POPC to the MSP1E3D1, or His-tag removed variant thereof in the pharmaceutical composition is less than about 118:1. In some embodiments, the phospholipid is DMPC, the MSP is MSP1E3, and the molar ratio of DMPC to MSP1E3 is about 150:1. In some embodiments, the phospholipid is DPPC, the MSP is MSP1E3 or His-tag removed variant thereof, and the molar ratio of DPPC to MSP1E3 is about 170:1. [0065] In some embodiments, the MSP is MSP1E3D1 or His-tag removed variant thereof, the phospholipid is DMPC, and the molar ratio of DMPC to the MSP in the pharmaceutical composition is about 87:1 to about 96:1, about 58:1 to about 68:1, about 63:1, or about 62:1. [0066] In some embodiments, the nanodisc comprises DMPC and MSP1D1, such as in a molar ratio of about 85:1. In some embodiments, the nanodisc comprises DMPC and MSP1D1 (e.g., SEQ ID NO: 3) or His-tag removed variant thereof (e.g., SEQ ID NO: 14), optionally wherein the molar ratio of DMPC to MSP1D1 (e.g., SEQ ID NO: 3) or His-tag removed variant thereof (e.g., SEQ ID NO: 14) in the pharmaceutical composition is about 46:1 to about 55:1, about 51:1, about 55:1, or about 50:1. Hence in some embodiments, there is provided a method of preventing or treating an eye disease characterized by lipid (e.g., cholesterol) accumulation in the eye of an individual (e.g., human), comprising administering to the eye of the individual an effective amount of a pharmaceutical composition comprising a plurality of nanodiscs, wherein the nanodisc comprises DMPC and MSP1D1 (e.g., SEQ ID NO: 3) or His-tag removed variant thereof (e.g., SEQ ID NO: 14), and the molar ratio of DMPC and MSP1D1 (e.g., SEQ ID NO: 3) or His-tag removed variant thereof (e.g., SEQ ID NO: 14) in the pharmaceutical composition is about 46:1 to about 55:1, about 51:1, about 55:1, or about 50:1. [0067] In some embodiments, the nanodisc comprises POPC and MSP1D1 (e.g., SEQ ID NO: 3), such as in a molar ratio of about 65:1, about 50:1, or about 32.5:1. Hence in some ny-2692819 Attorney Docket No.: 283912000140 embodiments, there is provided a method of preventing or treating an eye disease characterized by lipid (e.g., cholesterol) accumulation in the eye of an individual (e.g., human), comprising administering to the eye of the individual an effective amount of a pharmaceutical composition comprising a nanodisc, wherein the nanodisc comprises DMPC and MSP1D1 (e.g., SEQ ID NO: 3) at a molar ratio of about 85:1, about 51:1, about 55:1, or about 50:1. [0068] In some embodiments, the nanodisc comprises DMPC and MSP1E3D1 (e.g., SEQ ID NO: 8) or His-tag removed variant thereof (e.g., SEQ ID NO: 15) and the molar ratio of DMPC and MSP1E3D1 (e.g., SEQ ID NO: 8) or His-tag removed variant thereof (e.g., SEQ ID NO: 15)in the pharmaceutical composition is about 87:1 to about 96:1, about 58:1 to about 68:1, about 63:1, or about 62:1. Hence in some embodiments, there is provided a method of preventing or treating an eye disease characterized by lipid (e.g., cholesterol) accumulation in the eye of an individual (e.g., human), comprising administering to the eye of the individual an effective amount of a pharmaceutical composition comprising a plurality of nanodiscs, wherein the nanodisc comprises DMPC and MSP1E3D1 (e.g., SEQ ID NO: 8) or His-tag removed variant thereof (e.g., SEQ ID NO: 15), and the molar ratio of DMPC and MSP1E3D1 (e.g., SEQ ID NO: 8) or His-tag removed variant thereof (e.g., SEQ ID NO: 15) in the pharmaceutical composition is about 58:1 to about 68:1, about 63:1, or about 62:1. [0069] In some embodiments, there is provided a method of preventing or treating an eye disease characterized by lipid (e.g., cholesterol) accumulation in the eye of an individual (e.g., human), comprising administering to the eye of the individual an effective amount of a pharmaceutical composition comprising a nanodisc, wherein the nanodisc comprises POPC and MSP1D1 (e.g., SEQ ID NO: 3) at a molar ratio of about 65:1, about 50:1, or about 32.5:1. [0070] In some embodiments, there is provided a method of preventing or treating an eye disease characterized by lipid (e.g., cholesterol) accumulation in the eye of an individual (e.g., human), comprising administering to the eye of the individual an effective amount of a pharmaceutical composition comprising a plurality of nanodiscs, wherein the nanodisc comprises DMPC and MSP1D1 (e.g., SEQ ID NO: 3) or His-tag removed variant thereof (e.g., SEQ ID NO: 14), and the molar ratio of DMPC and MSP1D1 (e.g., SEQ ID NO: 3) or His-tag removed variant thereof (e.g., SEQ ID NO: 14) in the pharmaceutical composition is about 46:1 to about 55:1, about 51:1, about 55:1, or about 50:1. ny-2692819 Attorney Docket No.: 283912000140 [0071] In some embodiments, there is provided a method of preventing or treating an eye disease characterized by lipid (e.g., cholesterol) accumulation in the eye of an individual (e.g., human), comprising administering to the eye of the individual an effective amount of a pharmaceutical composition comprising a plurality of nanodiscs, wherein the nanodisc comprises DMPC and MSP1E3D1 (e.g., SEQ ID NO: 8) or His-tag removed variant thereof (e.g., SEQ ID NO: 15), and the molar ratio of DMPC and MSP1E3D1 (e.g., SEQ ID NO: 8) or His-tag removed variant thereof (e.g., SEQ ID NO: 15)in the pharmaceutical composition is about 58:1 to about 68:1, about 63:1, or about 62:1. [0072] In some embodiments, the lipid for removal or prevention from accumulation in the eye is cholesterol. In some embodiments, the eye disease characterized by lipid accumulation is photoreceptor neurodegeneration, a retinal neurovascular disorder, age-related macular degeneration (AMD), or atrophy. In some embodiments, the eye disease characterized by lipid accumulation is AMD, such as dry AMD. In some embodiments, the dry AMD is geographical atrophy (GA). In some embodiments, the eye disease characterized by lipid accumulation is wet AMD. In some embodiments, the eye disease is choroidal neovascularization (CNV). In some embodiments, the nanodisc is administered intravitreally. In some embodiments, the nanodisc is about 2 nm to about 20 nm, such as any of about 5 nm to about 20 nm or about 10 nm to about 20 nm in diameter. In some embodiments, the nanodisc does not comprise a CPP. In some embodiments, the nanodisc does not comprise a target protein. In some embodiments, the nanodisc does not comprise a prophylactic or therapeutic agent. In some embodiments, the nanodisc increases cholesterol efflux rate by at least about 2-fold (e.g., at least about any of 2.5, 3, 4, 5, 10, 15-fold, or more) compared to an untreated state, or the cholesterol efflux rate by ApoA-I or the MSP. In some embodiments, the nanodisc reduces lipid or prevents lipid accumulation in the eye of the individual by at least about 5% (e.g., at least about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%). [0073] In some embodiments, there is provided a method of preventing or treating an AMD in the eye of an individual (e.g., human), comprising administering to the eye of the individual an effective amount of a pharmaceutical composition comprising a nanodisc, wherein the nanodisc comprises an MSP and a phospholipid. In some embodiments, there is provided a method of preventing or treating an AMD in the eye of an individual (e.g., human), comprising administering to the eye of the individual an effective amount of a pharmaceutical composition ny-2692819 Attorney Docket No.: 283912000140 comprising a nanodisc, wherein the nanodisc comprises DMPC and MSP1D1 (e.g., SEQ ID NO: 3) at a molar ratio of about 85:1. In some embodiments, there is provided a method of preventing or treating an AMD in the eye of an individual (e.g., human), comprising administering to the eye of the individual an effective amount of a pharmaceutical composition comprising a plurality of nanodiscs, wherein the nanodisc comprises DMPC and MSP1D1 (e.g., SEQ ID NO: 3) or His- tag removed variant thereof (e.g., SEQ ID NO: 14), and the molar ratio of DMPC and MSP1D1 (e.g., SEQ ID NO: 3) or His-tag removed variant thereof (e.g., SEQ ID NO: 14) in the pharmaceutical composition is about 46:1 to about 55:1, about 51:1, about 55:1, or about 50:1. In some embodiments, there is provided a method of preventing or treating an AMD in the eye of an individual (e.g., human), comprising administering to the eye of the individual an effective amount of a pharmaceutical composition comprising a plurality of nanodiscs, wherein the nanodisc comprises DMPC and MSP1E3D1 (e.g., SEQ ID NO: 8) or His-tag removed variant thereof (e.g., SEQ ID NO: 15), and the molar ratio of DMPC and MSP1E3D1 (e.g., SEQ ID NO: 8) or His-tag removed variant thereof (e.g., SEQ ID NO: 15) in the pharmaceutical composition is about 58:1 to about 68:1, about 63:1, or about 62:1. In some embodiments, there is provided a method of preventing or treating an AMD in the eye of an individual (e.g., human), comprising administering to the eye of the individual an effective amount of a pharmaceutical composition comprising a nanodisc, wherein the nanodisc comprises POPC and MSP1D1 (e.g., SEQ ID NO: 3) at a molar ratio of about 65:1, about 50:1, or about 32.5:1. In some embodiments, the AMD is dry AMD. In some embodiments, the dry AMD is GA. In some embodiments, the AMD is wet AMD. In some embodiments, the wet AMD comprises CNV. In some embodiments, the method removes lipid (e.g., cholesterol) or prevents lipid (e.g., cholesterol) accumulation in the eye of the individual. In some embodiments, the nanodisc is administered intravitreally. In some embodiments, the nanodisc is about 2 nm to about 20 nm, such as any of about 5 nm to about 20 nm or about 10 nm to about 20 nm in diameter. In some embodiments, the nanodisc does not comprise a CPP. In some embodiments, the nanodisc does not comprise a target protein. In some embodiments, the nanodisc does not comprise a prophylactic or therapeutic agent. In some embodiments, the nanodisc increases cholesterol efflux rate by at least about 2-fold (e.g., at least about any of 2.5, 3, 4, 5, 10, 15-fold, or more) compared to an untreated state, or the cholesterol efflux rate by ApoA-I or the MSP. In some embodiments, the nanodisc reduces lipid or prevents lipid accumulation in the eye of the individual by at least about 5% (e.g., at least about any of ny-2692819 Attorney Docket No.: 283912000140 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%). In some embodiments, the AMD is GA, and the nanodisc reduces the total area of GA lesion by at least about 5% (e.g., at least about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%). In some embodiments, the nanodisc slows the progression of the total area of GA lesions in the eye of the individual by at least about 5% (e.g., at least about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%). In some embodiments, the total area of GA lesion is determined by autofluorescence imaging. [0074] In some embodiments, the lipid for removal or prevention from accumulation in the eye is selected from the group consisting of fats, sterols, phospholipids and any combination thereof. In some embodiments, the lipid comprises cholesterol esters (ChEs), wax esters (WEs), diesters, triacylglycerol (TG), free cholesterol, free fatty acids (FAs), (O-acyl)-ω-hydroxy fatty acids (OAHFAs), or any combination thereof. In some embodiments, the lipid is a fatty acid or its derivative (including tri-, di-, mono-glycerides, and phospholipids). In some embodiments, the lipid for removal or prevention from accumulation in the eye comprises two or more of the lipids described herein. In some embodiments, the lipid for removal or prevention from accumulation in the eye is cholesterol. [0075] In some embodiments, the eye diseases characterized by lipid accumulation is selected from the group consisting of AMD, photoreceptor neurodegeneration, optic nerve atrophy, loss of acuity, hemianopia, visual agnosia, strabismus, retinal neurovascular disorder, lipid keratopathy, corneal lipidosis, and any combination thereof. In some embodiments, exemplary eye diseases characterized by lipid accumulation include, but are not limited to, AMD including dry AMD (atrophic AMD) and wet AMD (neovascular AMD), choroidal neovascularization (CNV), retinal angiomatous proliferation (RAP), retinal neovascularization (RNV), juvenile macular degeneration (e.g., Stargardt disease), macular telangiectasia, maculopathy (e.g., age- related maculopathy (ARM) and diabetic maculopathy (DMP), including partial ischemic DMP), macular edema (e.g., diabetic macular edema (DME), including clinically significant DME, focal DME and diffuse DME, Irvine-Gass syndrome (postoperative macular edema), and macular edema following RVO, including central RVO and branch RVO), retinopathy (e.g., diabetic retinopathy (DR), including in patients with DME, proliferative vitreoretinopathy (PVR), Purtscher's retinopathy, and radiation retinopathy), retinal artery occlusion (RAO, e.g., central and branch RAO), retinal vein occlusion (RVO, e.g., central RVO, including central RVO with ny-2692819 Attorney Docket No.: 283912000140 cystoid macular edema (CME), and branch RVO, including branch RVO with CME), retinitis (e.g., Coats' disease (exudative retinitis) and retinitis pigmentosa (RP)), chorioretinitis, choroiditis (e.g., serpiginous choroiditis), uveitis (including anterior uveitis, intermediate uveitis, posterior uveitis with or without CME, pan-uveitis and non-infectious uveitis), retinal detachment (e.g., in von Hippel-Lindau disease), retinal pigment epithelium (RPE) detachment, dystrophies of rods or/and cones, and any combination thereof. [0076] In some embodiments, the eye disease characterized by lipid accumulation is photoreceptor neurodegeneration or retinal neurovascular disorder. [0077] In some embodiments, the eye disease characterized by lipid accumulation is AMD. In some embodiments, the AMD is dry AMD. Dry AMD is generally characterized by a buildup of yellowish deposits called drusen beneath the retina and typically affects vision in both eyes, although vision loss often occurs in one eye before the other. A more advanced stage of dry AMD is known as geographic atrophy (GA), in which areas of the macula waste away (atrophy), resulting in severe vision loss. [0078] Dry AMD can progress into wet AMD. Wet AMD is generally characterized by the growth of leaky blood vessels underneath the macula. This growth of leaky blood vessels is called choroidal neovascularization. The vessels leak blood and fluid, which damages the macula and makes central vision appear blurry and distorted. Wet macular degeneration is associated with severe vision loss that can worsen rapidly. It has been shown that high levels of intracellular cholesterol significantly increase choroidal neovascularization. Thus in some embodiments, the eye disease characterized by lipid accumulation is wet AMD. In some embodiments, the eye disease characterized by lipid accumulation is choroidal neovascularization (CNV). In some embodiments, the wet AMD comprises CNV and/or RAP. [0079] In some embodiments, the methods for removing lipid or preventing lipid accumulation, or for treating an eye disease characterized by lipid accumulation described here can achieve one or more following effects: i) removing (e.g., at least about any of 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100%) lipid (e.g., cholesterol) in the eye; ii) increasing (e.g., at least about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 1- fold, 2-fold, 2.5-fold, 5-fold, 10-fold, or more) the efflux rate of the lipid (e.g., cholesterol) in the eye; iii) reducing (e.g., reducing at least about any of 5%, 10%, 20%, 30%, 40%, 50%, 60%, ny-2692819 Attorney Docket No.: 283912000140 70%, 80%, 90%, 95%, or 100%) the likelihood of the occurrence of lipid accumulation and/or eye diseases characterized by lipid accumulation in the eye; iv) delaying (e.g., delaying at least about any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 18, 24, 36, or more months) the onset of lipid accumulation and/or eye diseases characterized by lipid accumulation in the eye; v) slowing the progression of lipid accumulation and/or eye diseases characterized by lipid accumulation in the eye; and vi) increasing cholesterol efflux rate by at least about 2-fold (e.g., at least about any of 2.5, 3, 4, 5, 10, 15-fold, or more) compared to an untreated state, or the cholesterol efflux rate by ApoA-I or the MSP (e.g., MSP1D1) alone. [0080] In some embodiments, the eye disease characterized by lipid accumulation is GA, and the method of preventing or treating GA provided herein achieves one or more following effects: i) reducing lipid (e.g., cholesterol) or preventing lipid accumulation in the eye of the individual by at least about 5% (e.g., at least about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100%); ii) reducing the total area of GA lesions in the eye of the individual by at least about 5% (such as by at least about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100%); and iii) slows the progression of the total area of GA lesions in the eye of the individual by at least about 5% (such as at least about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100%). The total area of GA lesions can be determined by any methods known in the art, such as autofluorescence imaging. In some embodiments, the autofluorescence is fundus autofluorescence (FAF). [0081] Hence in some embodiments, there is provided a method of reducing and/or slowing the progress of total area of GA lesions in the eye of an individual (e.g., human), comprising administering to the eye of the individual an effective amount of a pharmaceutical composition comprising a nanodisc, wherein the nanodisc comprises an MSP and a phospholipid. In some embodiments, there is provided a method of reducing and/or slowing the progress of total area of GA lesions in the eye of an individual (e.g., human), comprising administering to the eye of the individual an effective amount of a pharmaceutical composition comprising a nanodisc, wherein the nanodisc comprises DMPC and MSP1D1 (e.g., SEQ ID NO: 3) at a molar ratio of about 85:1. In some embodiments, there is provided a method of reducing and/or slowing the progress of total area of GA lesions in the eye of an individual (e.g., human), comprising administering to the eye of the individual an effective amount of a pharmaceutical composition comprising a plurality of nanodiscs, wherein the nanodisc comprises DMPC and MSP1D1 (e.g., SEQ ID NO: ny-2692819 Attorney Docket No.: 283912000140 3) or His-tag removed variant thereof (e.g., SEQ ID NO: 14), and the molar ratio of DMPC and MSP1D1 (e.g., SEQ ID NO: 3) or His-tag removed variant thereof (e.g., SEQ ID NO: 14) in the pharmaceutical composition is about 46:1 to about 55:1, about 51:1, about 55:1, or about 50:1. In some embodiments, there is provided a method of reducing and/or slowing the progress of total area of GA lesions in the eye of an individual (e.g., human), comprising administering to the eye of the individual an effective amount of a pharmaceutical composition comprising a plurality of nanodiscs, wherein the nanodisc comprises DMPC and MSP1E3D1 (e.g., SEQ ID NO: 8) or His- tag removed variant thereof (e.g., SEQ ID NO: 15), and the molar ratio of DMPC and MSP1E3D1 (e.g., SEQ ID NO: 8) or His-tag removed variant thereof (e.g., SEQ ID NO: 15) in the pharmaceutical composition is about 58:1 to about 68:1, about 63:1, or about 62:1. In some embodiments, there is provided a method of reducing and/or slowing the progress of total area of GA lesions in the eye of an individual (e.g., human), comprising administering to the eye of the individual an effective amount of a pharmaceutical composition comprising a nanodisc, wherein the nanodisc comprises POPC and MSP1D1 (e.g., SEQ ID NO: 3) at a molar ratio of about 65:1, about 50:1, or about 32.5:1. In some embodiments, the method removes lipid (e.g., cholesterol) or prevents lipid (e.g., cholesterol) accumulation in the eye of the individual. In some embodiments, the nanodisc is administered intravitreally. In some embodiments, the nanodisc is about 2 nm to about 20 nm, such as any of about 5 nm to about 20 nm or about 10 nm to about 20 nm in diameter. In some embodiments, the nanodisc does not comprise a CPP. In some embodiments, the nanodisc does not comprise a target protein. In some embodiments, the nanodisc does not comprise a prophylactic or therapeutic agent. In some embodiments, the nanodisc increases cholesterol efflux rate by at least about 2-fold (e.g., at least about any of 2.5, 3, 4, 5, 10, 15-fold, or more) compared to an untreated state, or the cholesterol efflux rate by ApoA-I or the MSP. In some embodiments, the nanodisc reduces lipid or prevents lipid accumulation in the eye of the individual by at least about 5% (e.g., at least about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%). In some embodiments, the nanodisc reduces the total area of GA lesion by at least about 5% (e.g., at least about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%). In some embodiments, the nanodisc slows the progression of the total area of GA lesions in the eye of the individual by at least about 5% (e.g., at least about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%). In some embodiments, the total area of GA lesion is determined by autofluorescence imaging. ny-2692819 Attorney Docket No.: 283912000140 [0082] In some embodiments, the methods described herein inhibits (e.g., inhibiting at least about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100%) neovascularization in the eye of an individual suffering from AMD. In some embodiments, the method delays (e.g., delaying at least about any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 18, 24, 36, or more months) the development of AMD or CNV. In some embodiment, the method treats and/or reduces (e.g., reducing at least about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100%) the risk of developing retinal neovascularization (RNV). In some embodiment, the method treats and/or reduces (e.g., reducing at least about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100%) the risk of developing retinal angiomatous proliferation (RAP). Hence in some embodiments, there is provided a method of i) inhibiting neovascularization, ii) delaying the development of AMD or CNV, iii) treating and/or reducing the risk of developing RNV and/or RAP in the eye of an individual (e.g., human), and/or (iv) reducing (e.g., reducing at least about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100%) lipid accumulation in retinal pigment epithelium (RPE) and/or Bruch’s membrane, and/or (v) reducing (e.g., reducing at least about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100%) retinal neurodegeneration, and/or (vi) ameliorating inner segment/outer segment (IS/OS) junction abnormalities, and/or (vii) reducing (e.g., reducing at least about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100%) RPE disruptions including pigment epithelial detachments (PED), and/or (viii) reducing (e.g., reducing at least about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100%) cumulative incidence of retinal lesions, and/or (ix) reducing (e.g., reducing at least about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100%) cholesteryl ester, comprising administering to the eye of the individual an effective amount of a pharmaceutical composition comprising a nanodisc, wherein the nanodisc comprises an MSP and a phospholipid. In some embodiments, there is provided a method of i) inhibiting neovascularization, ii) delaying the development of AMD or CNV, iii) treating and/or reducing the risk of developing RNV and/or RAP in the eye of an individual (e.g., human), and/or (iv) reducing (e.g., reducing at least about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100%) lipid accumulation in retinal pigment epithelium (RPE) and/or Bruch’s membrane, and/or (v) reducing (e.g., reducing at least about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100%) retinal neurodegeneration, and/or (vi) ameliorating inner ny-2692819 Attorney Docket No.: 283912000140 segment/outer segment (IS/OS) junction abnormalities, and/or (vii) reducing (e.g., reducing at least about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100%) RPE disruptions including pigment epithelial detachments (PED), and/or (viii) reducing (e.g., reducing at least about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100%) cumulative incidence of retinal lesions, and/or (ix) reducing (e.g., reducing at least about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100%) cholesteryl ester, comprising administering to the eye of the individual an effective amount of a pharmaceutical composition comprising a nanodisc, wherein the nanodisc comprises DMPC and MSP1D1 (e.g., SEQ ID NO: 3) at a molar ratio of about 85:1. In some embodiments, there is provided a method of i) inhibiting neovascularization, ii) delaying the development of AMD or CNV, iii) treating and/or reducing the risk of developing RNV and/or RAP in the eye of an individual (e.g., human), and/or (iv) reducing (e.g., reducing at least about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100%) lipid accumulation in retinal pigment epithelium (RPE) and/or Bruch’s membrane, and/or (v) reducing (e.g., reducing at least about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100%) retinal neurodegeneration, and/or (vi) ameliorating inner segment/outer segment (IS/OS) junction abnormalities, and/or (vii) reducing (e.g., reducing at least about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100%) RPE disruptions including pigment epithelial detachments (PED), and/or (viii) reducing (e.g., reducing at least about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100%) cumulative incidence of retinal lesions, and/or (ix) reducing (e.g., reducing at least about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100%) cholesteryl ester, comprising administering to the eye of the individual an effective amount of a pharmaceutical composition comprising a plurality of nanodiscs, wherein the nanodisc comprises DMPC and MSP1D1 (e.g., SEQ ID NO: 3) or His-tag removed variant thereof (e.g., SEQ ID NO: 14), and the molar ratio of DMPC and MSP1D1 (e.g., SEQ ID NO: 3) or His-tag removed variant thereof (e.g., SEQ ID NO: 14) in the pharmaceutical composition is about 46:1 to about 55:1, about 51:1, about 55:1, or about 50:1. In some embodiments, there is provided a method of i) inhibiting neovascularization, ii) delaying the development of AMD or CNV, iii) treating and/or reducing the risk of developing RNV and/or RAP in the eye of an individual (e.g., human), and/or (iv) reducing (e.g., reducing at least about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100%) lipid accumulation in retinal pigment epithelium (RPE) and/or Bruch’s ny-2692819 Attorney Docket No.: 283912000140 membrane, and/or (v) reducing (e.g., reducing at least about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100%) retinal neurodegeneration, and/or (vi) ameliorating inner segment/outer segment (IS/OS) junction abnormalities, and/or (vii) reducing (e.g., reducing at least about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100%) RPE disruptions including pigment epithelial detachments (PED), and/or (viii) reducing (e.g., reducing at least about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100%) cumulative incidence of retinal lesions, and/or (ix) reducing (e.g., reducing at least about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100%) cholesteryl ester, comprising administering to the eye of the individual an effective amount of a pharmaceutical composition comprising a plurality of nanodiscs, wherein the nanodisc comprises DMPC and MSP1E3D1 (e.g., SEQ ID NO: 8) or His-tag removed variant thereof (e.g., SEQ ID NO: 15), and the molar ratio of DMPC and MSP1E3D1 (e.g., SEQ ID NO: 8) or His-tag removed variant thereof (e.g., SEQ ID NO: 15) in the pharmaceutical composition is about 58:1 to about 68:1, about 63:1, or about 62:1. In some embodiments, there is provided a method of i) inhibiting neovascularization, ii) delaying the development of AMD or CNV, iii) treating and/or reducing the risk of developing RNV and/or RAP in the eye of an individual (e.g., human), and/or (iv) reducing (e.g., reducing at least about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100%) lipid accumulation in retinal pigment epithelium (RPE) and/or Bruch’s membrane, and/or (v) reducing (e.g., reducing at least about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100%) retinal neurodegeneration, and/or (vi) ameliorating inner segment/outer segment (IS/OS) junction abnormalities, and/or (vii) reducing (e.g., reducing at least about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100%) RPE disruptions including pigment epithelial detachments (PED), and/or (viii) reducing (e.g., reducing at least about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100%) cumulative incidence of retinal lesions, and/or (ix) reducing (e.g., reducing at least about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100%) cholesteryl ester, comprising administering to the eye of the individual an effective amount of a pharmaceutical composition comprising a plurality of nanodiscs, wherein administering to the eye of the individual an effective amount of a pharmaceutical composition comprising a nanodisc, wherein the nanodisc comprises POPC and MSP1D1 (e.g., SEQ ID NO: 3) at a molar ratio of about 65:1, about 50:1, or about 32.5:1. In some embodiments, the method removes lipid (e.g., cholesterol) ny-2692819 Attorney Docket No.: 283912000140 or prevents lipid (e.g., cholesterol) accumulation in the eye of the individual. In some embodiments, the nanodisc is administered intravitreally. In some embodiments, the nanodisc is about about 2 nm to about 20 nm, such as any of about 5 nm to about 20 nm or about 10 nm to about 20 nm in diameter. In some embodiments, the nanodisc does not comprise a CPP. In some embodiments, the nanodisc does not comprise a target protein. In some embodiments, the nanodisc does not comprise a prophylactic or therapeutic agent. In some embodiments, the nanodisc increases cholesterol efflux rate by at least about 2-fold (e.g., at least about any of 2.5, 3, 4, 5, 10, 15-fold, or more) compared to an untreated state, or the cholesterol efflux rate by ApoA-I or the MSP. In some embodiments, the nanodisc reduces lipid or prevents lipid accumulation in the eye of the individual by at least about 5% (e.g., at least about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%). [0083] In some embodiments, the individual is suffering from AMD and is at risk of or suffering from RAP. In some embodiments, the individual is suffering from AMD and is at risk of or suffering from CNV. In some embodiments, the individual is suffering from AMD and is at risk of or suffering from RNV. In some embodiments, the individual has been identified as having one or more genetic polymorphisms that increases the risk of AMD. In some embodiments, the method of preventing or treating an eye disease characterized by lipid accumulation (e.g., AMD) described herein further comprises determining whether the individual has a genetic polymorphism that increases the risk of AMD. In some embodiments, the individual has injury at the Bruch’s membrane. In some embodiments, the individual has deficient Abca1 and/or Abcg1 cholesterol efflux transporters in rod photoreceptors. In some embodiments, the individual has at least one of RPE disruption and hypertransmission, ellipsoid zone abnormalities, inner retinal subsidence, and hyperreflective foci. In some embodiments, the individual has at least one of inner segment/outer segment (IS/OS) junction abnormalities and RPE disruptions including pigment epithelial detachments (PED). In some embodiments, the individual has been on a high-fat diet. [0084] In some embodiments, there is provided a method of improving vision impairment and/or reducing the likelihood of vision loss associated with an eye disease characterized by lipid accumulation in the eye in an individual (e.g., human), comprising administering to the eye of the individual an effective amount of a pharmaceutical composition comprising a nanodisc, wherein the nanodisc comprises an MSP and a phospholipid. In some embodiments, the nanodisc ny-2692819 Attorney Docket No.: 283912000140 comprises DMPC and MSP1D1 (e.g., SEQ ID NO: 3), such as in a molar ratio of about 85:1. In some embodiments, there is provided a method of improving vision impairment and/or reducing the likelihood of vision loss associated with an eye disease characterized by lipid accumulation in the eye in an individual (e.g., human), comprising administering to the eye of the individual an effective amount of a pharmaceutical composition comprising a plurality of nanodiscs, wherein the nanodisc comprises DMPC and MSP1D1 (e.g., SEQ ID NO: 3) or His-tag removed variant thereof (e.g., SEQ ID NO: 14), and the molar ratio of DMPC and MSP1D1 (e.g., SEQ ID NO: 3) or His-tag removed variant thereof (e.g., SEQ ID NO: 14) in the pharmaceutical composition is about 46:1 to about 55:1, about 50:1, about 51:1, or about 55:1. In some embodiments, there is provided a method of improving vision impairment and/or reducing the likelihood of vision loss associated with an eye disease characterized by lipid accumulation in the eye in an individual (e.g., human), comprising administering to the eye of the individual an effective amount of a pharmaceutical composition comprising a plurality of nanodiscs, wherein the nanodisc comprises DMPC and MSP1E3D1 (e.g., SEQ ID NO: 8) or His-tag removed variant thereof (e.g., SEQ ID NO: 15), and the molar ratio of DMPC and MSP1E3D1 (e.g., SEQ ID NO: 8) or His-tag removed variant thereof (e.g., SEQ ID NO: 15) in the pharmaceutical composition is about 58:1 to about 68:1, about 63:1, or about 62:1. In some embodiments, the nanodisc comprises POPC and MSP1D1 (e.g., SEQ ID NO: 3), such as in a molar ratio of about 65:1, about 50:1, or about 32.5:1. In some embodiments, the method provided herein improves vision impairment and/or reduces the likelihood of vision loss associated with eye diseases characterized by lipid accumulation in the eye by at least about 5%, such as by at least about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100%. The vision impairment and/or vision loss can be associated with any of the eye diseases described herein. In some embodiments, the vision impairment or loss is associated with atrophic AMD including non-central or/and central GA or neovascular AMD including types 1, 2 or/and 3 NV, or the vision improvement can occur in a subject with atrophic AMD or neovascular AMD. [0085] In some embodiments, there is provided a method of improving the normal luminance best-corrected visual acuity (NL-BCVA) score and/or the low luminance best-corrected visual acuity (LL-BCVA) score of an individual (e.g., human), comprising administering to the eye of the individual an effective amount of a pharmaceutical composition comprising a nanodisc, wherein the nanodisc comprises an MSP and a phospholipid. In some embodiments, the nanodisc ny-2692819 Attorney Docket No.: 283912000140 comprises DMPC and MSP1D1 (e.g., SEQ ID NO: 3), such as in a molar ratio of about 85:1. In some embodiments, there is provided a method of improving the normal luminance best- corrected visual acuity (NL-BCVA) score and/or the low luminance best-corrected visual acuity (LL-BCVA) score of an individual (e.g., human), comprising administering to the eye of the individual an effective amount of a pharmaceutical composition comprising a plurality of nanodiscs, wherein the nanodisc comprises DMPC and MSP1D1 (e.g., SEQ ID NO: 3) or His- tag removed variant thereof (e.g., SEQ ID NO: 14), and the molar ratio of DMPC and MSP1D1 (e.g., SEQ ID NO: 3) or His-tag removed variant thereof (e.g., SEQ ID NO: 14) in the pharmaceutical composition is about 46:1 to about 55:1, about 51:1, about 55:1, or about 50:1. In some embodiments, there is provided a method of improving the normal luminance best- corrected visual acuity (NL-BCVA) score and/or the low luminance best-corrected visual acuity (LL-BCVA) score of an individual (e.g., human), comprising administering to the eye of the individual an effective amount of a pharmaceutical composition comprising a plurality of nanodiscs, wherein the nanodisc comprises DMPC and MSP1E3D1 (e.g., SEQ ID NO: 8) or His- tag removed variant thereof (e.g., SEQ ID NO: 15), and the molar ratio of DMPC and MSP1E3D1 (e.g., SEQ ID NO: 8) or His-tag removed variant thereof (e.g., SEQ ID NO: 15) in the pharmaceutical composition is about 58:1 to about 68:1, about 63:1, or about 62:1. In some embodiments, the nanodisc comprises POPC and MSP1D1 (e.g., SEQ ID NO: 3), such as in a molar ratio of about 65:1, about 50:1, or about 32.5:1. In some embodiments, the method provided herein improves the NL-BCVA score and/or the LL-BCVA score of the individual by at least about 5%, such as by at least about any of 10%, 20%, 30%, 40%, 50%,60%, 70%, 80%, 90%, 95%, or 100%. The NL-BCVA score and/or LL-BCVA score can be determined by any known methods in the art. [0086] In some embodiments, any of the methods of i) removing lipid or preventing lipid accumulation, ii) preventing or treating an eye disease characterized by lipid accumulation, iii) reducing and/or slowing the progress of total area of GA lesions, iv) inhibiting neovascularization, v) delaying the development of AMD or CNV, vi) treating and/or reducing the risk of developing RNV and/or RAP, vii) improving vision impairment and/or reducing the likelihood of vision loss associated with an eye disease characterized by lipid accumulation, and viii) improving the NL-BCVA score and/or the LL-BCVA score mentioned herein is compared to a baseline state before treatment (or untreated state), a natural progression without any ny-2692819 Attorney Docket No.: 283912000140 treatment, a treatment with placebo, or a treatment with ApoA-I or the MSP protein (e.g., MSP1D1) alone. In some embodiments, the efficacy of any of the methods provided herein is compared to a treatment with HDL alone. [0087] In some embodiments, the efficacy of any of the methods provided herein may be determined by any method known in the art. For example, in some embodiments, the efficacy of any of the methods provided herein may be determined by measuring photoreceptor and RPE function, optionally by full-field scotopic and light bleach recovery ERG diagnostics. In some embodiments, the efficacy of any of the methods provided herein may be determined by measuring anatomical changes linked to AMD progression such as RPE disruption and hypertransmission, ellipsoid zone abnormalities, inner retinal subsidence, and hyperreflective foci, optionally by noninvasive imaging such as optical coherence tomography (OCT). In some embodiments, the efficacy of any of the methods provided herein may be determined by measuring visual function or cumulative incidence of retinal lesions, optionally by full-field scotopic and light bleach recovery ERG diagnostics. In some embodiments, the efficacy of any of the methods provided herein may be determined by examining potential changes in individual lipid species in the eye such as cholesteryl ester (CE) composition, optionally by lipidomics analyses. [0088] In some embodiments, any of the methods provided herein is safe and leads to minimal side effects, wherein the safety of the methods can be determined by any method known in the art. In some embodiments, any of the methods provided herein does not lead to significant retinal inflammation or differences in retinal integrity. In some embodiments, the retinal inflammation or differences in retinal integrity may be measured by examining retinal histology. In some embodiments, any of the methods provided herein does not exacerbate expected complement signaling in individuals suffering dry AMD. In some embodiments, the complement signaling may be determined by retina section immunostaining. Nanodisc [0089] Nanodisc provided herein refers to a nanocomposite resembling a disc in which a lipid bilayer comprising a phospholipid is surrounded by a stabilizing belt comprising an MSP. In some embodiments, the nanodisc comprises DMPC and MSP1D1, optionally in a molar ratio ny-2692819 Attorney Docket No.: 283912000140 (e.g., the molar ratio of DMPC and MSP1D1 in the pharmaceutical composition) of about 85:1. In some embodiments, the nanodisc comprises DMPC and MSP1D1, and the molar ratio of DMPC and MSP1D1 in the pharmaceutical composition is about 46:1 to about 55:1. In some embodiments, the nanodisc comprises DMPC and MSP1E3D1, and the molar ratio of DMPC and MSP1D1E3 in the pharmaceutical composition is about 58:1 to about 68:1, about 63:1, or about 62:1. In some embodiments, the nanodisc comprises POPC and MSP1D1, optionally in a molar ratio (e.g., the molar ratio of POPC and MSP1D1 in the pharmaceutical composition) of about 65:1, or about 50:1, or about 32.5:1. In some embodiments, the nanodiscs have an average diameter of about 2 nm to about 20 nm, such as any of about 5 nm to about 20 nm, about 5 nm to about 15 nm, about 2 nm to about 15 nm, about 2 nm to about 10 nm, about 10 nm to about 20 nm, or about 10 nm to about 15 nm. In some embodiments, the polydispersity index of the size of the nanodics in the pharmaceutical composition is less than about 0.2. In some embodiments, the polydispersity index of the phospholipid content of the nanodics in the pharmaceutical composition is less than about 0.2. In some embodiments, the nanodisc does not comprise a CPP. In some embodiments, the CPP is selected from the group consisting of penetratin, polyarginine (R8), LL-37, transportan, Pep-1, and membrane translocating sequence (MTS). In some embodiments, the nanodisc does not comprise a target protein. In some embodiments, the nanodisc does not comprise a prophylactic or therapeutic agent. a. Membrane scaffold protein (MSP) [0090] In some embodiments, the MSP is selected from the group consisting of apolipoprotein A-I (ApoA-I), ApoA-II, ApoC, ApoE, ApoM, and a variant thereof. In some embodiments, the MSP is a variant of ApoA-I. [0091] Apolipoproteins play an important role in maintaining the structural integrity and solubility of lipoproteins, in lipoprotein receptor recognition, and in the regulation of certain enzymes in lipoprotein metabolism. Main types of apolipoproteins include, but are not limited to, apolipoprotein A (Apo-A) such as Apo-AI, Apo-A2, Apo-A4, and Apo-A5, apolipoprotein B (Apo-B), such as Apo-B48 and Apo B-100, apolipoprotein C (Apo-C), such as ApoC-I, apo ApoC-II, apo ApoC-III, and ApoC-IV, apolipoprotein D (Apo-D), apolipoprotein E (Apo-E), and apolipoprotein M (ApoM). Apolipoproteins can be from any organism, including but not limited to, human, cows, horses, sheep, monkeys, baboons, goats, rabbits, dogs, hedgehogs, badgers, ny-2692819 Attorney Docket No.: 283912000140 mice, rats, cats, guinea pigs, hamsters, duck, chicken, salmon, and eel. In some embodiments, the apolipoprotein is human apolipoprotein. [0092] In some embodiments, the MSP comprises a variant of an apolipoprotein selected from the group consisting of ApoA-I, ApoA-II, Apo-C, Apo-E, Apo-M, and any combination thereof. In some embodiments, the MSP further comprises a fragment derived from a natural apolipoprotein selected from the group consisting of ApoA-I, ApoA-II, Apo-C, Apo-E, and Apo- M. In some embodiments, the MSP comprises a mutation (e.g., insertion, deletion, substitution, or any combination thereof) compared to a naturally existing apolipoprotein (e.g., ApoA-I). In some embodiments, the MSP comprises one or more cysteine (Cys) substitution. In some embodiments, the mutation (e.g., Cys substitution) is at the N terminus, at the C terminus, and/or among the amphipathic helices of the MSP. In some embodiments, the Cys substitution is in one of the helixes of the MSP. In some embodiments, the cysteine may be linked to an imaging group, optionally a fluorescent group (e.g., GFP, RFP, YFP). In some embodiments, the cysteine in the MSP may be linked to an imaging group, optionally a fluorescent group (e.g., fluorescein, rhodamine, or other common luminescent small molecules). In some embodiments, the MSP is fused with a fluorescent protein such as GFP, RFP, or YFP. In some embodiments, the MSP comprises a tag, such a tag commonly used in protein expression and/or purification. In some embodiments, the tag is a polyhistidine, c-Myc, FLAG, biotin, or any combination thereof. In some embodiments, the MSP comprises an enzymatic cleavage site, e.g., Tobacco Etch Protease (TEV) recognition site (boxed in Table 1). [0093] In some embodiments, the MSP comprises a His-tag. In some embodiments, the MSP does not comprise a His-tag. The symbol “(-)” as used herein refers to an MSP with the His-tag removed. In some embodiments, the His-tag is followed by a TEV protease recognition site. Cleavage with TEV leaves an additional glycine (G) residue at the amino terminus of the MSPs. In some embodiments, the MSP is derived from a naturally occurring ApoA-I, e.g., human ApoA-I. TEV protease recognition site sequence is boxed in Table 1. [0094] ApoA-I is the major protein component of HDL particles in plasma. The protein enables efflux of fat molecules by accepting fats within cells for transport elsewhere, such as back to LDL particles or to the liver for excretion. Human ApoA-I contains an N-terminal globular domain (GLOB) made up of about 43 residues followed by 10 amphipathic helices (H1- ny-2692819 Attorney Docket No.: 283912000140 H10) made up of 11, 22, or 24 residues each. Seven of the helices are 22 amino acids in length, one helix (H10) is 24 amino acids in length, while two helices (H3, H9) are 11 amino acids in length. The helices are separated by a glycine or proline. The sequences of different domains are indicated for human ApoA-I in Table 1. A Full-length human ApoA-I comprises from N’ to C’: GLOB-H1-H2-H3-H4-H5-H6-H7-H8-H9-H10. Human ApoA-I comprises the amino acid sequence of SEQ ID NO: 1. In some embodiments, the GLOB domain comprises the amino acid sequence of DEPPQSPWDRVKDLATVYVDVLKDSGRDYVSQFEGSALGKQLN (SEQ ID NO: 19). [0095] ApoA-1 can be from any organism. In some embodiments, ApoA-1 is derived from animals selected from the group consisting of human, cows, horses, sheep, monkeys, baboons, goats, rabbits, dogs, hedgehogs, badgers, mice, rats, cats, guinea pigs, hamsters, duck, chicken, salmon, eel, and any combination thereof. In some embodiments, ApoA-1 is a human ApoA-1. In some embodiments, ApoA-1 comprises (or consists of, or consists essentially of) the amino acid sequence of SEQ ID NO: 1. [0096] In some embodiments, the MSP is a variant of ApoA-I. In some embodiments, the MSP has at least about 70% (e.g., at least about any of 80%, 85%, 90%, 95%, 99%, or higher) sequence identity to SEQ ID NO: 1. In some embodiments, MSPs containing alternate amphipathic helical polypeptide sequences can be generated by adding or removing one or more helical segments (e.g., H1, H2, etc.) or partial helical segments (e.g., removing partial H1 sequence of ApoA-I to arrive at H(0.5)), or adding, removing, or substituting amino acid residues, based off an ApoA-I (e.g., human ApoA-I) protein. In some embodiments, an MSP can be derived from ApoA-I (e.g., human ApoA-I) by removing the GLOB domain. In some embodiments, the MSP can be derived from ApoA-I (e.g., human ApoA-I) by adding one (e.g., H4), two (e.g., H4 and H5), three (e.g., H4-H6), or more helical segments. [0097] Exemplary MSPs and their contained domains and sequences are indicated in Table 1. Other MSPs with variations relative to those in Table 1 can also be used herein. For example, by adding or removing one or more helical segments or portion thereof, and/or by adding, removing, and/or substituting one or more amino acid residues (e.g., adding a peptide linker in between helical segments) at N-terminus, internally, and/or at C-terminus, and/or by adding, removing, and/or substituting a tag-sequence. ny-2692819 Attorney Docket No.: 283912000140 Table 1. Sequences
Figure imgf000039_0001
37 ny-2692819 Attorney Docket No.: 283912000140
Figure imgf000040_0001
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Figure imgf000041_0001
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Figure imgf000042_0001
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Figure imgf000043_0001
[0098] In some embodiments, the MSP comprises an N-terminus truncation relative to ApoA-I. In some embodiments, the truncation comprises deleting 1, 2, 3, 4, 5, 10, 15, 20, 30, 40, 50, 60, 100, or more consecutive amino acids from the N-terminus of ApoA-I. In some embodiments, the truncation comprises deleting about 10-70 amino acids from the N-terminus of ApoA-I. In some embodiments, the MSP is MSP1, MSP1D1, or MSP1D2. In some embodiments, the truncation comprises deleting about 10-70 amino acids from the N-terminus of ApoA-I. In some embodiments, the N-terminus GLOB domain of ApoA-I is removed in the MSP. In some embodiments, the first 11 amino acids of H1 of ApoA-I is removed in the MSP, resulting in a H(0.5) helical portion of STFSKLREQLG (SEQ ID NO: 20) (see H(0.5) in Table 1). In some embodiments, both the N-terminus GLOB domain and the first 11 amino acids of H1 of ApoA-I is removed in the MSP. [0099] In some embodiments, the MSP comprises an amino acid sequence of MGHHHHHHHDYDIPTTENLYFQG (SEQ ID NO: 21) at the N-terminus, hereinafter also referred to as “His-tag” or “His-tag portion.” The TEV protease recognition site is indicated by boxing. In some embodiments, the MSP does not comprise the N-terminal His-tag portion (his- tag removed). In some embodiments, the N-terminal His-tag (SEQ ID NO: 21) is removed from the MSP by cleaving with the TEV protease. It is understood that cleavage at the TEV protease ny-2692819 Attorney Docket No.: 283912000140 recognition site may result in a residual glycine (G) residue at the N-terminus of the resulting MSP sequence. For example, MSP1D1(-) (SEQ ID NO: 14) is obtained by cleaving MSP1D1 (SEQ ID NO: 3) with TEV protease, and MSP1E3D1(-) (SEQ ID NO: 15) is obtained by cleaving MSP1E3D1 (SEQ ID NO: 8) with TEV protease. It is also understood for MSPs wherein the indicated His-tag (SEQ ID NO: 21) is not removed, the N-terminal methionine residue will be often removed during heterologous expression, e.g., if expressed in a bacteria such as E. coli. [0100] In some embodiments, the MSP is MSP1. In some embodiments, MSP1 comprises the amino acid sequence of SEQ ID NO: 2. MSP1 is derived from the sequence of ApoA-1, but without the globular N-terminal domain of native ApoA-1. In some embodiments, the MSP has at least about 70% (e.g., at least about any of 80%, 85%, 90%, 95%, 99%, or 100%) sequence identity to MSP1 (e.g., SEQ ID NO: 2). [0101] In some embodiments, the MSP is MSP1D1. In some embodiments, MSP1D1 is derived from the sequence of ApoA-1, but without the globular N-terminal domain of native ApoA-1 and removing 11 amino acids from the first helix labeled H1 in the parent ApoA-I, generating the half helix labeled H(0.5) and MSP1D1. In some embodiments, MSP1D1 comprises the amino acid sequence of SEQ ID NO: 3. In some embodiments, the MSP has at least about 70% (e.g., at least about any of 80%, 85%, 90%, 95%, 99%, or 100%) sequence identity to SEQ ID NO: 3. In some embodiments, the MSP is a variant of MSP1D1 that does not comprise the N-terminal His- tag portion (his-tag removed variant) (hereinafter referred to as MSP1D1(-)), which comprises the amino acid sequence of SEQ ID NO: 14. [0102] In some embodiments, the MSP is MSP1D2. In some embodiments, MSP1D2 comprises the amino acid sequence of SEQ ID NO: 4. In some embodiments, the MSP has at least about 70% (e.g., at least about any of 80%, 85%, 90%, 95%, 99%, or 100%) sequence identity to SEQ ID NO: 4. [0103] In some embodiments, the MSP comprises one or more (e.g., 1, 2, 3, or more) additional amphipathic helix sequences relative to ApoA-I. In some embodiments, each additional amphipathic helix sequence is independently derived from any of ApoA-I, ApoA-II, ApoC-I, ApoC-II, Apo-E, apolipophorin III, myoglobin, or hemoglobin. In some embodiments, the additional amphipathic helix sequence is not derived from ApoA-I, ApoA-II, ApoC-I, ApoC-II, ny-2692819 Attorney Docket No.: 283912000140 Apo-E, apolipophorin III, myoglobin, or hemoglobin. In some embodiments, the additional amphipathic helix sequence is synthetic or not naturally occurring. In some embodiments, the additional amphipathic helix sequence comprises at least one (e.g., 1, 2, 3, 4, 5, or more) α- helixes or fragments thereof. In some embodiments, the MSP comprises an additional helix sequence, such as one, two, or three of any of first, second, or third helix sequences. In some embodiments, the additional amphipathic helix sequence is added to the N-terminus of an apolipoprotein (e.g., ApoA-I). In some embodiments, the additional amphipathic helix sequence is added to the C-terminus of an apolipoprotein (e.g., ApoA-I). In some embodiments, the additional amphipathic helix sequence is inserted between the original helixes of an apolipoprotein (e.g., ApoA-I). In some embodiments, the additional amphipathic helix sequences is added after the last Q residue of H3 into the original MSP1 sequence (e.g., SEQ ID NO: 2). In some embodiments, each additional amphipathic helix sequence has about 10 to about 30 (including for example any of about 15 to about 25, about 20, or about 22) amino acids. In some embodiments, the total additional amphipathic helix sequence is about any of 20-25, 40-45, 60- 65 amino acids long. In some embodiments, the MSP does not comprise the GLOB domain of SEQ ID NO: 19. In some embodiments, an amphipathic helix sequence of helix 4 (H4) can be inserted to MSP1D1 to generate MSP1E1D1, as defined by SEQ ID NO: 16. In some embodiments, 2 amphiphilic helices of Helix 4 (H4) and Helix 5 (H5) can be inserted into MSP1D1, generating MSP1E2D1 (SEQ ID NO: 17). In some embodiments, three amphiphilic helices, H4, H5 and H6, can be inserted into MSP1D1, generating MSP1E3D1 defined by SEQ ID NO: 8. In some embodiments, an additional 9 amphipathic helices is added to MSP1D1 (SEQ ID NO: 3) with a GT peptide linker to generate an extended MSP—MSP2N2 (SEQ ID NO: 18). [0104] In some embodiments, the MSP comprises an additional helix sequence, such as one, two, or three of any of first, second, or third helix sequences. In some embodiments, the additional amphipathic helix sequence is added to the N-terminus of an apolipoprotein (e.g., ApoA-I). In some embodiments, the additional amphipathic helix sequence is added to the C- terminus of an apolipoprotein (e.g., ApoA-I). In some embodiments, the additional amphipathic helix sequence is inserted between the helixes of an apolipoprotein (e.g., ApoA-I). In some embodiments, each additional amphipathic helix sequence has about 10 to about 30 (including, for example, any of about 15 to about 25, about 24, about 20, about 22, about 11) amino acids. ny-2692819 Attorney Docket No.: 283912000140 In some embodiments, the total additional amphipathic helix sequence is about any of about 20- 25, about 40-46, about 60-68, or about 100-200 amino acids long. In some embodiments, the MSP is MSP1E1, MSP1E2, or MSP1E3. MSP1E1, MSP1E2, and MSP1E3 are all extended membrane scaffold proteins, and are obtained via insertion of one, two, or three extra 22-mer amphipathic helices after the last Q residue of H3 into the original MSP1 sequence. In some embodiments, MSP1E1 comprises the amino acid sequence of SEQ ID NO: 5. In some embodiments, MSP1E2 comprises the amino acid sequence of SEQ ID NO: 6. In some embodiments, MSP1E3 comprises the amino acid sequence of SEQ ID NO: 7. In some embodiments, the MSP has at least about 70% (e.g., at least about any of 80%, 85%, 90%, 95%, 99%, or 100%) sequence identity to any of SEQ ID NOs: 5-7. [0105] In some embodiments, the MSP comprises both an additional amphipathic helix sequence and a truncation relative to ApoA-I. In some embodiments, the MSP is MSP1E3D1. In some embodiments, MSP1E3D1 comprises the amino acid sequence of SEQ ID NO: 8. In some embodiments, the MSP has at least about 70% (e.g., at least about any of 80%, 85%, 90%, 95%, 99%, or 100%) sequence identity to SEQ ID NO: 8. In some embodiments, the MSP is a variant of MSP1E3D1 that does not comprise the N-terminal His-tag portion (his-tag removed variant) (hereinafter referred to as MSP1E3D1(-)), which comprises the amino acid sequence of SEQ ID NO: 15. [0106] Detailed preparation procedures and descriptions of MSP1D1, MSP1D2, MSP1E1, MSP1E2, MSP1E3, and MSP1E3D1 are disclosed in Denisov et al J Am Chem Soc. 2004 Mar 24. 126(11):3477-87, the content of which is incorporated herein by reference in its entirety. [0107] MSP2N2 is generated by extending the His-tagged MSP1D1, adding a GT spacer and an additional Helix2 through Helix10. Detailed composition and preparation procedures for MSP2N2 are described in Grinkova et al. Protein Engineering Design and Selection 201023, 843-848, the content of which is incorporated herein by reference in its entirety. [0108] In some embodiments, the histidine tag is removed from MSP1D1, MSPE1D1, MSP1E2D1, MSPE3D1 or MSP2N2 to generate MSP1D1(-), MSPE1D1(-), MSP1E2D1(-), MSPE3D1(-) or MSP2N2(-), respectively, by cleaving with TEV protease. It is understood that TEV protease cleavage at the boxed area shown in Table 1 can result in a residual glycine (G) ny-2692819 Attorney Docket No.: 283912000140 amino acid residue on the N-terminal of the resulting MSP protein. These variants are herein referred to as “his-tag removed variants.” [0109] In some embodiments, the MSP is a variant of Apo-C. The Apo-C family comprises three closely related proteins: ApoC-I, ApoC-II, and ApoC-III, that are mostly made by the liver and, to a lesser degree, in the intestine. In some embodiments, ApoC-I, ApoC-II, and ApoC-III comprises the amino acid sequence of SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11, respectively. In some embodiments, the MSP has at least about 70% (e.g., at least about any of 80%, 85%, 90%, 95%, 99%, or 100%) sequence identity to any of SEQ ID NOs: 9-11. [0110] In some embodiments, the MSP is a variant of Apo-E. ApoE is responsible for the uptake and transport of cholesterol in the blood. In some embodiments, Apo-E comprises the amino acid sequence of SEQ ID NO: 12. In some embodiments, the MSP has at least about 70% (e.g., at least about any of 80%, 85%, 90%, 95%, 99%, or 100%) sequence identity to SEQ ID NO: 12. [0111] In some embodiments, the MSP is a variant of Apo-M. Apo-M is found associated with high density lipoproteins and to a lesser extent with low density lipoproteins and triglyceride-rich lipoproteins. In some embodiments, Apo-M comprises the amino acid sequence of SEQ ID NO: 13. In some embodiments, the MSP has at least about 70% (e.g., at least about any of 80%, 85%, 90%, 95%, 99%, or 100%) sequence identity to SEQ ID NO: 13. b. Phospholipid [0112] Phospholipids generally refer to a class of lipid with a hydrophilic head of phosphate moiety and two hydrophobic tails derived from fatty acids, joined by an alcohol or glycerol backbone. [0113] In some embodiments, the phospholipid is a sphingolipid. Sphingolipids typically refers to a class of lipids containing a backbone of sphingoid bases, which are a set of aliphatic amino alcohols that includes sphingosine. In some embodiments, the sphingolipid is sphingosine-1- phosphate. [0114] In some embodiments, the phospholipid comprises one or more acyl chains. In some embodiments, each acyl chain independently has a length of about 5 to about 30 carbon units, such as about 10 to about 22 carbon units or about 10 to about 16 carbon units. In some ny-2692819 Attorney Docket No.: 283912000140 embodiments, the phospholipid has a glycerol backbone. In some embodiments, the phospholipid is phosphatidylcholine (PC). PC generally refers to the class of phospholipids that comprise choline as a head group, glycerophosphoric unit, and a variety of fatty acids. In some embodiments, the phospholipid comprises a PC selected from the group consisting of 1-oleoyl-2- palmitoyl-sn-glycero-3-phosphocholine (OPPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 2-dierucoyl-sn-glycero-3- phosphocholine (DEPC), 1-palmitoyl-2-oleoyl-sn-glycero-3- phosphorylcholine (POPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2- dilauroyl-sn-glycero-3-phosphocholine (DLPC), dimyristoylphosphatidylcholine (DMPC), 1,2- distearoyl-sn-glycero-3-phosphocholine (DSPC), and any combination thereof. In some embodiments, the phospholipid is selected from the group consisting of DMPC, POPC, DPPC, DSPC, and any combination thereof. In some embodiments, the phospholipid is DMPC, DPPC, or POPC. In some embodiments, the phospholipid is a single type of PC. In some embodiments, the phospholipid comprises a mixture of at least two types of PC. In some embodiments, the phospholipid is selected from the group consisting of DMPC, DMPS, POPC, DPPC, DSPC, POPS, PiP2, and any combination thereof. In some embodiments, the phospholipid is DMPC, DPPC, or POPC. In some embodiments, the phospholipid is a single type of PC. In some embodiments, the phospholipid comprises a mixture of at least two types of PC. [0115] In some embodiments, the one or more acyl chains of the phospholipid are fully saturated. In some embodiments, the phospholipid is selected from the group consisting of DMPC, DSPC, DPPC, and DMPS. In some embodiments, the phospholipid is DMPC. DMPC is a synthetic phospholipid that comprises two myristoyl fatty acids attached in ester linkage to the first and second carbon of glycerol, and choline attached through a phosphodiester linkage to the third carbon of the glycerol. In some embodiments, the phospholipid is DPPC. DPPC is a synthetic phospholipid that comprises two palmitoyl fatty acids attached in ester linkage to the first and second carbon of glycerol, and choline attached through a phosphodiester linkage to the third carbon of the glycerol. In some embodiments, the phospholipid is 1,2-dimyristoyl-sn- glycero-3-phospho-L-serine (DMPS), such as a 14:0 DMPS. In some embodiments, the phospholipid is DSPC. DSPC is a synthetic phospholipid that comprises two stearoyl fatty acids attached in ester linkage to the first and second carbon of glycerol, and choline attached through a phosphodiester linkage to the third carbon of the glycerol. ny-2692819 Attorney Docket No.: 283912000140 [0116] In some embodiments, the one or more acyl chains of the phospholipid are unsaturated. In some embodiments, the phospholipid is POPC, phosphatidylinositol 4,5-bisphosphate (PiP2), or palmitoyl-oleoyl phosphatidylserine (POPS). In some embodiments, the phospholipid is POPC. POPC is a synthetic phospholipid that comprises two fatty acids, palmitic acid and oleic acid, attached in ester linkage to the first and second carbon of glycerol, respectively, and choline attached through a phosphodiester linkage to the third carbon of the glycerol. In some embodiments, the phospholipid is PiP2, which could also be referred to as PI(4,5)P2. In some embodiments, the PiP2 is 18:1 PI(4,5)P2 or 18:0-20:4 PI(4,5)P2. In some embodiments, the PiP2 is 18:1 PI(4,5)P2. In some embodiments, the PiP2 is 18:0-20:4 PI(4,5)P2. In some embodiments, the phospholipid is DOPC. DOPC is a synthetic phospholipid that comprises two oleoyl fatty acids attached in ester linkage to the first and second carbon of glycerol, and choline attached through a phosphodiester linkage to the third carbon of the glycerol. In some embodiments, the phospholipid is DEPC. DEPC is a synthetic phospholipid that comprises two erucoyl fatty acids attached in ester linkage to the first and second carbon of glycerol, and choline attached through a phosphodiester linkage to the third carbon of the glycerol. [0117] In some embodiments, the phospholipid is 1,2-dimyristoyl-sn-glycero-3-phospho-L- serine (DMPS), such as a 14:0 DMPS. In some embodiments, the phospholipid is DSPC. DSPC is a synthetic phospholipid that comprises two stearoyl fatty acids attached in ester linkage to the first and second carbon of glycerol, and choline attached through a phosphodiester linkage to the third carbon of the glycerol. [0118] In some embodiments, the phospholipid is PiP2, which could also be referred to as PI(4,5)P2. In some embodiments, the PiP2 is 18:1 PI(4,5)P2 or 18:0-20:4 PI(4,5)P2. In some embodiments, the PiP2 is 18:1 PI(4,5)P2. In some embodiments, the PiP2 is 18:0-20:4 PI(4,5)P2. [0119] In some embodiments, the phospholipid is cationic, anionic, or zwitterionic, or any combination thereof. c. MSP to Phospholipid ratio [0120] The stoichiometry of MPS and phospholipid is crucial for the formulation, functions, shape homogeneity and size distribution of the nanodiscs used herein. ny-2692819 Attorney Docket No.: 283912000140 [0121] In some embodiments, the molar ratio of phospholipid to MSP referred herein is the molar ratio (e.g., average or median molar ratio) of phospholipid to MSP in the pharmaceutical composition. In some embodiments, the molar ratio of phospholipid to MSP referred herein is the molar ratio (e.g., average or median molar ratio) of phospholipid to MSP in the nanodisc. [0122] In some embodiments, without being bound by any scientific theory, the amount of phospholipid needed to saturate a nanodisc (i.e., phospholipid-MSP saturation ratio NS) may depend on the identity of the MSP and the phospholipid. In some embodiments, phospholipids may comprise a phosphatidyl choline (PC) head group and more than 12 hydrocarbon tails. In some embodiments, without being bound by any scientific theory, phospholipids may have the tendency to condense into a bilayer structure. In some embodiments, without being bound by any scientific theory, the MSP may contact with a bilayer formed by the phospholipids, wherein the hydrophobic part of the MSP stays in proximity to the hydrophobic tails of the phospholipids and the hydrophilic part of the MSP stays in proximity to a solvent in the pharmaceutical composition. In some embodiments, without being bound by any scientific theory, the MSP is a variant of ApoA-I, and the amino acids forming the globular domain and those of the first 11 amino acids of Helix 1 (H1, Table 1) are not in contact with the lipids (Denisov et. Al, J Am Chem Soc. 2004 Mar 24. 126(11):3477-87). [0123] Variants of the MSPs without the globular domain, and/or without the H1 domain or portion thereof, and/or without any amino or carboxy terminal tag and protease cleavage sites, such as the MSP1D1(-), MSPE1D1(-), MSPE2D1(-), MSPE3D1(-) and MSP2N2(-) provided herein, may have the same molar ratio of the phospholipid to the MSP in the pharmaceutical composition needed to saturate the nanodisc, compared to their parental protein, e.g., MSP1D1, MSPE1D1, MSPE2D1, MSPE3D1, and MSP2N2, respectively. [0124] In some embodiments, at least about 85% (e.g., at least about any of 90%, 95%, 96%, 97%, 98%, 99%, or 100%) of the plurality of nanodiscs in the pharmaceutical composition are fully-lipidated. “Fully-lipidated”, as used herein, refers to the nanodisc wherein the molar ratio of the phospholipid to the MSP in the pharmaceutical composition is at or slightly above the phospholipid-MSP saturation ratio NS, such as equal to or 0-10% above the phospholipid-MSP saturation ratio NS. In some embodiments, the NS for exemplary lipid and MSP combinations are shown in Table 2. ny-2692819 Attorney Docket No.: 283912000140 [0125] In some embodiments, the nanodisc comprises a phospholipid and a MSP, wherein at least about 85% (e.g., at least about any of 90%, 95%, 96%, 97%, 98%, 99%, or 100%) of the plurality of nanodiscs are under-lipidated. “Under-lipidated”, as used herein, refers to the nanodisc wherein the molar ratio of the phospholipid to the MSP in the pharmaceutical composition is less than the phospholipid-MSP saturation ratio NS, such as less than about 95% (e.g., less than about any of 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, or 40%) of the phospholipid-MSP saturation ratio NS. In some embodiments, the NS for exemplary lipid and MSP combinations are shown in Table 2. In some embodiments, the molar ratio of the phospholipid to the MSP in the pharmaceutical composition is less than a phospholipid-MSP saturation ratio NS. [0126] In some embodiments, without being bound by any scientific theory, the phospholipid- MSP saturation ratio NS may be calculated according to Denisov et al. (J Am Chem Soc.2004 Mar 24. 126(11):3477-87) and Denisov and Sligar (Chem Rev.2017 Mar 22; 117(6): 4669– 4713), the contents of each of which are incorporated herein by reference in their entirety. Denisov et al. discloses that in a helical amino acid MSP comprising an amphipathic helical polypeptide with a length of M amino acid residues that form a lipid binding scaffold, the number of lipids needed to saturate the nanodisc (phospholipid-MSP saturation ratio NS) is a function of the length M as well as the mean area occupied by a single phospholipid type. In some embodiments, NS may be calculated by Equation 1.1: NS = (M*L - 2*Π∗r)2 / 4*Π∗AL (Equation 1.1) wherein M is the number of amino acid residues in the helical belt of the MSP around a bilayer of the phospholipids, L is the helical pitch per MSP residue (e.g., about 0.15 nm), and r is the mean radius of the MSP α-helix (e.g., about 0.55 nm). In some embodiments, AL of Equation 1.1 is a constant depending on the phospholipid, e.g., may be the area occupied by a single phospholipid in a bilayer (in nm2). In some embodiments, without being bound by any scientific theory, the area AL may depend on the identity of the lipid used as well as the temperature of assembly relative to the bulk bilayer phase transition temperature. [0127] In some embodiments, M can be calculated according to Equation 1.2: (Equation 1.2)
Figure imgf000051_0001
ny-2692819 Attorney Docket No.: 283912000140 wherein r is the mean radius of the MSP α-helix (e.g., about 0.55 nm or about 5.5 Å), L is the helical pitch per MSP residue (e.g., about 0.15 nm or about 1.5 Å), AL is the mean surface area per phospholipid, N is the number of phospholipid per one bilayer of the phospholipids. In some embodiments, M can be referred to from Table 2. [0128] In some embodiments, the phospholipid-MSP saturation ratio NS may be calculated by Equation 1.1, wherein AL is about 0.52 for DPPC and DMPC, and AL is about 0.69 for POPC. [0129] In some embodiments, the length of the helical belt that contacts the lipids (M) is the same for the MSP variants of with the H(0.5) helical sequence, compared to their parental MSP protein containing H1 (see Table 1). In some embodiments, this helical belt length (M) may also be the same with or without an added histidine or other tag. For example, the helical belt length M of MSP1D1, MSPE1D1, MSPE2D1, MSPE3D1, and MSP2N2 for calculating NS is the same as the helical belt length M of MSP1D1(-), MSPE1D1(-), MSPE2D1(-), MSPE3D1(-) and MSP2N2(-) for calculating NS, respectively, as the tag and protease cleavage site is not involved in stabilizing the resulting nanodisc. In some embodiments, the number of lipids (DMPC, DPPC and POPC) needed to saturate the MSP1D1, MSPE1D1, MSPE2D1, MSPE3D1 nanodiscs may be calculated by Equation 1.1 and the calculation results are summarized in Table 2. Table 2. NS values for exemplary nanodiscs
Figure imgf000052_0001
[0130] In some embodiments, without being bound by any scientific theory, the stoichiometry of MSP and phospholipid is crucial for the functions, shape homogeneity and size distribution of the nanodiscs. In some embodiments, the molar ratio of the phospholipid to MSP in the pharmaceutical composition is less than the phospholipid-MSP saturation ratio NS, such as less than about 95% (e.g., less than about any of 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, or 40%) of the phospholipid-MSP saturation ratio NS. In some embodiments, the NS for exemplary lipid and MSP are shown in Table 2. ny-2692819 Attorney Docket No.: 283912000140 [0131] In some embodiments, the MSP is MSP1D1 or MSP1, and the molar ratio of phospholipid to the MSP (e.g., in the nanodisc, or in the pharmaceutical composition) is from about 5:1 to about 120:1 (including for example any of about 10:1 to about 100:1, about 10:1 to about 90:1, about 20:1 to about 90:1, about 10:1 to about 85:1, about 30:1 to about 85:1, about 30:1 to about 65:1, about 32.5:1 to about 65:1, about 10:1 to about 65:1, about 10:1 to about 50:1, about 20:1 to about 65:1, and about 25:1 to about 50:1). In some embodiments, the molar ratio of phospholipid to the MSP (e.g., MSP1D1 or MSP1) (e.g., in the nanodisc, or in the pharmaceutical composition) is about any of 10:1, 15:1, 20:1, 25:1, 30:1, 32.5:1, 35:1, 40:1, 45:1, 50:1, 55:1, 60:1, 65:1, 70:1, 75:1, 80:1, 85:1, 90:1, and 95:1. In some embodiments, the MSP is MSP1D1 or MSP1, and the molar ratio of phospholipid to MSP (e.g., in the nanodisc, or in the pharmaceutical composition) is from about 10:1 to about 90:1. [0132] In some embodiments, the MSP is MSP1D1 or MSP1, the phospholipid is POPC, and the molar ratio of POPC to the MSP (e.g., in the nanodisc, or in the pharmaceutical composition) is any of about 10:1 to about 90:1, about 32.5:1 to about 85:1, about 50:1 to about 65:1, about 10:1 to about 65:1, about 10:1 to about 85:1, about 32.5:1, about 50:1, or about 65:1. In some embodiments, the MSP is MSP1, the phospholipid is POPC, and the molar ratio of POPC to MSP1D1 (e.g., in the nanodisc, or in the pharmaceutical composition) is about 32.5:1. In some embodiments, the MSP is MSP1D1, the phospholipid is POPC, and the molar ratio of POPC to MSP1 (e.g., in the nanodisc, or in the pharmaceutical composition) is about 50:1. In some embodiments, the MSP is MSP1D1 or MSP1, the phospholipid is POPC, and the molar ratio of POPC to MSP1D1 or MSP1 (e.g., in the nanodisc, or in the pharmaceutical composition) is about 65:1. In some embodiments, the MSP is MSP1D1, MSP1, or His-tag removed variant thereof, the phospholipid is POPC, and the molar ratio of POPC to the MSP (e.g., in the nanodisc, or in the pharmaceutical composition) is from about 10:1 to about 65:1. [0133] In some embodiments, the MSP is MSP1D1 (e.g., SEQ ID NO: 3) or His-tag removed variant thereof (e.g., SEQ ID NO: 14), the phospholipid is POPC, and at least about 85% (e.g., at least about any of 90%, 95%, 96%, 97%, 98%, 99%, or 100%) of the plurality of nanodiscs are fully-lipidated. In some embodiments, the NS is about 62. In some embodiments, the MSP is MSP1D1 or His-tag removed variant thereof, the phospholipid is POPC, wherein the molar ratio of POPC to the MSP (e.g., in the nanodisc, or in the pharmaceutical composition) is about 65:1. ny-2692819 Attorney Docket No.: 283912000140 [0134] In some embodiments, the MSP is MSP1D1 (e.g., SEQ ID NO: 3) or His-tag removed variant thereof (e.g., SEQ ID NO: 14), the phospholipid is POPC, and at least about 85% (e.g., at least about any of 90%, 95%, 96%, 97%, 98%, 99%, or 100%) of the plurality of nanodiscs in the pharmaceutical composition are under-lipidated. In some embodiments, the molar ratio of POPC to MSP1D1 or His-tag removed variant thereof in the pharmaceutical composition is less than a phospholipid-MSP saturation ratio NS of about 62:1, such as less than about 95% (e.g., less than about any of 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, or 40%) of the phospholipid-MSP saturation ratio NS of about 62:1. In some embodiments, the molar ratio of POPC to MSP1D1 or His-tag removed variant thereof in the pharmaceutical composition is less than or equal to 60:1, such as less than or equal to about any of 58:1, 55:1, 50:1, 45:1, 40:1, 35:1, 32.5:1, 30:1, or less. In some embodiments, the molar ratio of POPC to MSP1D1 in the pharmaceutical composition is about 10:1 to about 60:1, such as any of about 20:1 to about 60:1, about 30:1 to about 55:1, about 40:1 to about 55:1, about 58:1, about 55:1, about 50:1, about 45:1, about 40:1, about 35:1, about 32.5:1, or about 30:1. In some embodiments, the MSP is MSP1D1 or His-tag removed variant thereof, the phospholipid is POPC, and the molar ratio of POPC to the MSP in the pharmaceutical composition is less than about 58:1, such as about 50:1 or about 32.5:1. In some embodiments, the MSP is MSP1D1 comprising the amino acid sequence of SEQ ID NO: 3. In some embodiments, the MSP is an MSP1D1 His-tag removed variant comprising the amino acid sequence of SEQ ID NO: 14. [0135] In some embodiments, the MSP is MSP1D1 or MSP1, the phospholipid is DMPC, and the molar ratio of DMPC to MSP (e.g., in the nanodisc, or in the pharmaceutical composition) is any of about 10:1 to about 85:1, about 10:1 to about 120:1, about 50:1 to about 100:1, about 50:1 to about 90:1, about 70:1 to about 90:1, about 80:1, or about 85:1. In some embodiments, the MSP is MSP1, the phospholipid is DMPC, and the molar ratio of DMPC to MSP1 (e.g., in the nanodisc, or in the pharmaceutical composition) is about 80:1. In some embodiments, the MSP is MSP1 or His-tag removed variant thereof, the phospholipid is DMPC, wherein the molar ratio of DMPC to the MSP1 or His-tag removed variant thereof e.g., in the nanodisc, or in the pharmaceutical composition) is about 80:1. In some embodiments, the MSP is MSP1D1, the phospholipid is DMPC, and the molar ratio of DMPC to MSP1D1 (e.g., in the nanodisc, or in the pharmaceutical composition) is about 85:1. In some embodiments, the MSP is MSP1D1, MSP1, ny-2692819 Attorney Docket No.: 283912000140 or His-tag removed variant thereof, the phospholipid is DMPC, wherein the molar ratio of DMPC to the MSP in the pharmaceutical composition is from about 10:1 to about 85:1. [0136] In some embodiments, the MSP is MSP1D1 (e.g., SEQ ID NO: 3) or His-tag removed variant thereof (e.g., SEQ ID NO: 14), the phospholipid is DMPC, and at least about 85% (e.g., at least about any of 90%, 95%, 96%, 97%, 98%, 99%, or 100%) of the plurality of nanodiscs are fully-lipidated. In some embodiments, the NS is about 81. In some embodiments, the MSP is MSP1D1 or His-tag removed variant thereof, the phospholipid is DMPC, wherein the molar ratio of DMPC to the MSP in the pharmaceutical composition is about 85:1. [0137] In some embodiments, the MSP is MSP1D1 (e.g., SEQ ID NO: 3) or His-tag removed variant thereof (e.g., SEQ ID NO: 14), the phospholipid is DMPC, wherein at least about 85% (e.g., at least about any of 90%, 95%, 96%, 97%, 98%, 99%, or 100%) of the plurality of nanodiscs in the pharmaceutical composition are under-lipidated. In some embodiments, the molar ratio of DMPC to MSP1D1 or His-tag removed variant thereof in the pharmaceutical composition is less than a phospholipid-MSP saturation ratio NS of about 81:1, such as less than about 95% (e.g., less than about any of 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, or 40%) of the phospholipid-MSP saturation ratio NS of about 81:1. In some embodiments, the molar ratio of DMPC to MSP1D1 or His-tag removed variant thereof in the pharmaceutical composition is less than or equal to about 78:1, such as less than or equal to about any of 77:1, 76:1, 75:1, 74:1, 72:1, 70:1, 65:1, 60:1, 55:1, 51:1, 50:1, 45:1, 40:1, or less. In some embodiments, the molar ratio of DMPC to MSP1D1 or His-tag removed variant thereof in the pharmaceutical composition is about 10:1 to about 80:1, such as any of about 10:1 to about 78:1, about 20:1 to about 78:1, about 20:1 to about 78:1, about 30:1 to about 78:1, about 70:1 to about 80:1, about 74:1 to about 77:1, about 40:1 to about 60:1, about 65:1 to about 75:1, about 70:1 to about 75:1, about 72:1 to about 78:1, about 46:1 to about 55:1, about 75:1, about 54:1 to about 56:1, about 65:1, about 60:1, about 55:1, about 51:1, about 50:1, about 45:1, about 40:1, about 35:1, or about 30:1. In some embodiments, the molar ratio of DMPC to MSP1D1 or His-tag removed variant thereof in the pharmaceutical composition is about 65:1 to about 75:1. [0138] In some embodiments, the MSP is MSP1D1 (e.g., SEQ ID NO: 3) or His-tag removed variant thereof (e.g., SEQ ID NO: 14), the phospholipid is DMPC, wherein the molar ratio of the ny-2692819 Attorney Docket No.: 283912000140 phospholipid to the MSP in the pharmaceutical composition is about 72:1 to about 78:1, such as about 75:1. [0139] In some embodiments, the MSP is MSP1D1 (e.g., SEQ ID NO: 3) or His-tag removed variant thereof (e.g., SEQ ID NO: 14), the phospholipid is DMPC, wherein the molar ratio of the phospholipid to the MSP in the pharmaceutical composition is from about 40:1 to about 60:1, such as about 46:1 to about 55:1, or about 51:1. [0140] In some embodiments, the MSP is MSP1D1 or MSP1, the phospholipid is DPPC, and the molar ratio of DPPC to the MSP (e.g., in the nanodisc, or in the pharmaceutical composition) is any of about 10:1 to about 120:1, about 10:1 to about 100:1, about 50:1 to about 100:1, about 70:1 to about 100:1, about 80:1 to about 100:1, about 80:1, about 85:1, or about 90:1. In some embodiments, the MSP is MSP1, the phospholipid is DPPC, and the molar ratio of DPPC to MSP1 (e.g., in the nanodisc, or in the pharmaceutical composition) is about 90:1. In some embodiments, the MSP is MSP1D1, MSP1, or His-tag removed variant thereof, the phospholipid is DPPC, wherein the molar ratio of DPPC to the MSP (e.g., in the nanodisc, or in the pharmaceutical composition) is from about 10:1 to about 90:1. [0141] In some embodiments, the MSP is MSP1D1 (e.g., SEQ ID NO: 3) or His-tag removed variant thereof (e.g., SEQ ID NO: 14), the phospholipid is DPPC, wherein at least about 85% (e.g., at least about any of 90%, 95%, 96%, 97%, 98%, 99%, or 100%) of the plurality of nanodiscs in the pharmaceutical composition are fully-lipidated. In some embodiments, the NS is about 81. In some embodiments, the MSP is MSP1 or His-tag removed variant thereof, the phospholipid is DPPC, wherein the molar ratio of DPPC to the MSP in the pharmaceutical composition is about 90:1. [0142] In some embodiments, the MSP is MSP1D1 (e.g., SEQ ID NO: 3) or His-tag removed variant thereof (e.g., SEQ ID NO: 14), the phospholipid is DPPC, wherein at least about 85% (e.g., at least about any of 90%, 95%, 96%, 97%, 98%, 99%, or 100%) of the plurality of nanodiscs in the pharmaceutical composition are under-lipidated. In some embodiments, the molar ratio of DPPC to MSP1D1 or His-tag removed variant thereof in the pharmaceutical composition is less than a phospholipid-MSP saturation ratio NS of about 81:1, such as less than about 95% (e.g., less than about any of 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, or 40%) of the phospholipid-MSP saturation ratio NS of about 81:1. In some embodiments, the ny-2692819 Attorney Docket No.: 283912000140 molar ratio of DPPC to MSP1D1 or His-tag removed variant thereof in the pharmaceutical composition is less than or equal to about 78:1, such as less than or equal to about any of 77:1, 76:1, 75:1, 74:1, 72:1, 70:1, 65:1, 60:1, 55:1, 51:1, 50:1, 45:1, 40:1, or less. In some embodiments, the molar ratio of DPPC to MSP1D1 or His-tag removed variant thereof in the pharmaceutical composition is about 10:1 to about 80:1, such as any of about 10:1 to about 78:1, about 20:1 to about 78:1, about 20:1 to about 78:1, about 30:1 to about 78:1, about 70:1 to about 80:1, about 74:1 to about 77:1, about 40:1 to about 60:1, about 65:1 to about 75:1, about 70:1 to about 75:1, about 72:1 to about 78:1, about 46:1 to about 55:1, about 75:1, about 54:1 to about 56:1, about 65:1, about 60:1, about 55:1, about 51:1, about 50:1, about 45:1, about 40:1, about 35:1, or about 30:1. In some embodiments, the MSP is MSP1D1 or His-tag removed variant thereof, the phospholipid is DPPC, the molar ratio of DPPC to the MSP in the pharmaceutical composition is less than about 75:1. [0143] In some embodiments, the MSP is MSP1E3, and the molar ratio of phospholipid to MSP1E3 (e.g., the molar ratio of phospholipid to MSP1E3 in the nanodisc, or in the pharmaceutical composition) is from about 50:1 to about 250:1 (including for example any of about 50:1 to about 200:1, about 100:1 to about 200:1, about 100:1 to about 180:1, about 100:1 to about 170:1, about 130:1 to about 180:1, or about 130:1 to about 170:1). In some embodiments, the molar ratio of phospholipid to MSP1E3 (e.g., in the nanodisc, or in the pharmaceutical composition) is about any of 50:1, 100:1, 110:1, 120:1, 130:1, 140:1, 150:1, 160:1, 170:1, 180:1, 190:1, 200:1, 210:1, 220:1, 230:1, 240:1, or 250:1. In some embodiments, at least about 85% (e.g., at least about any of 90%, 95%, 96%, 97%, 98%, 99%, or 100%) of the plurality of nanodiscs in the pharmaceutical composition are fully-lipidated. In some embodiments, at least about 85% (e.g., at least about any of 90%, 95%, 96%, 97%, 98%, 99%, or 100%) of the plurality of nanodiscs in the pharmaceutical composition are under-lipidated. [0144] In some embodiments, the MSP is MSP1E3, MSP1E3D1, or His-tag removed variant thereof, and the molar ratio of the phospholipid to the MSP (e.g., in the nanodisc, or in the pharmaceutical composition) is from about 50:1 to about 200:1, such as any of about 50:1 to about 100:1, about 100:1 to about 200:1, about 100:1 to about 180:1, about 100:1 to about 170:1, about 130:1 to about 180:1, or about 130:1 to about 170:1. ny-2692819 Attorney Docket No.: 283912000140 [0145] In some embodiments, the MSP is MSP1E3, the phospholipid is POPC, and the molar ratio of POPC to MSP1E3 (e.g., in the nanodisc, or in the pharmaceutical composition) is about 100:1 to about 250:1 (including for example any of about 100:1 to about 200:1, about 120:1 to about 190:1, about 120:1 to about 180:1, and about 130:1 to about 170:1). In some embodiments, the MSP is MSP1E3, the phospholipid is POPC, and the molar ratio of POPC to MSP1E3 (e.g., in the nanodisc, or in the pharmaceutical composition) is about any of 100:1, 110:1, 120:1, 130:1, 140:1, 150:1, 160:1, and 170:1. In some embodiments, the molar ratio of POPC to MSP1E3 (e.g., in the nanodisc, or in the pharmaceutical composition) is about 130:1. In some embodiments, the MSP is MSP1E3 or His-tag removed variant thereof, the phospholipid is POPC, wherein the molar ratio of POPC to the MSP (e.g., in the nanodisc, or in the pharmaceutical composition) is about 130:1. [0146] In some embodiments, the MSP is MSP1E3, the phospholipid is DMPC, and the molar ratio of DMPC to MSP1E3 (e.g., in the nanodisc, or in the pharmaceutical composition) is about 100:1 to about 200:1 (including for example about any of about 130:1 to about 190:1, or about 120:1 to about 170:1). In some embodiments, the molar ratio of DMPC to MSP1E3 (e.g., in the nanodisc, or in the pharmaceutical composition) is about any of 100:1, 110:1, 120:1, 130:1, 140:1, 150:1, 160:1, and 170:1. In some embodiments, the molar ratio of DMPC to MSP1E3 (e.g., in the nanodisc, or in the pharmaceutical composition) is about 150:1. In some embodiments, the MSP is MSP1E3 or His-tag removed variant thereof, the phospholipid is DMPC, wherein the molar ratio of DMPC to the MSP (e.g., in the nanodisc, or in the pharmaceutical composition) is about 150:1. [0147] In some embodiments, the MSP is MSP1E3, the phospholipid is DPPC, and the molar ratio of DPPC to MSP1E3 (e.g., in the nanodisc, or in the pharmaceutical composition) is about 120:1 to about 220:1 (including for example about any of about 140:1 to about 200:1, or about 150:1 to about 190:1). In some embodiments, the molar ratio of DPPC to MSP1E3 (e.g., in the nanodisc, or in the pharmaceutical composition) is about any of 130:1, 140:1, 150:1, 160:1, 170:1, 180:1, 190:1, 200:1, 210:1, 220:1, and 230:1. In some embodiments, the molar ratio of DPPC to MSP1E3 (e.g., in the nanodisc, or in the pharmaceutical composition) is about 170:1. In some embodiments, the MSP is MSP1E3 or His-tag removed variant thereof, the phospholipid is DPPC, wherein the molar ratio of DPPC to the MSP (e.g., in the nanodisc, or in the pharmaceutical composition) is about 170:1. ny-2692819 Attorney Docket No.: 283912000140 [0148] In some embodiments, the MSP is MSP1E3D1 (e.g., SEQ ID NO: 8) or His-tag removed variant thereof (e.g., SEQ ID NO: 15), the phospholipid is DMPC, wherein at least about 85% (e.g., at least about any of 90%, 95%, 96%, 97%, 98%, 99%, or 100%) of the plurality of nanodiscs in the pharmaceutical composition are fully-lipidated. In some embodiments, the NS is about 167. [0149] In some embodiments, the MSP is MSP1E3D1 (e.g., SEQ ID NO: 8) or His-tag removed variant thereof (e.g., SEQ ID NO: 15), the phospholipid is DMPC, wherein at least about 85% (e.g., at least about any of 90%, 95%, 96%, 97%, 98%, 99%, or 100%) of the plurality of nanodiscs in the pharmaceutical composition are under-lipidated. In some embodiments, the MSP is MSP1E3D1 or His-tag removed variant thereof, the phospholipid is DMPC, wherein the molar ratio of the phospholipid to the MSP in the pharmaceutical composition is less than a phospholipid-MSP saturation ratio NS of about 167:1, such as less than about 95% (e.g., less than about any of 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, or 40%) of the phospholipid-MSP saturation ratio NS of about 167:1. In some embodiments, the molar ratio of DMPC to MSP1E3D1 or His-tag removed variant thereof in the pharmaceutical composition is less than or equal to about 160:1, such as less than or equal to about any of 158:1, 155:1, 150:1, 145:1, 140:1, 135:1, 130:1, 125:1, 120:1, 115:1, 110:1, 105:1, 100:1, 95:1, 90:1, 85:1, 80:1, 75:1, or less. In some embodiments, the molar ratio of DMPC to MSP1E3D1 or His-tag removed variant thereof in the pharmaceutical composition is about 50:1 to about 160:1, such as any of about 50:1 to about 150:1, about 60:1 to about 140:1, about 60:1 to about 130:1, about 60:1 to about 120:1, about 60:1 to about 100:1, about 60:1 to about 70:1, about 90:1 to about 100:1, about 50:1 to about 100:1, about 87:1 to about 96:1, about 58:1 to about 68:1, about 60:1, about 62:1, about 63:1, about 65:1, about 70:1, about 80:1, about 90:1, about 93:1, about 100:1, or about 110:1. In some embodiments, the MSP is MSP1E3 or His-tag removed variant thereof, the phospholipid is DMPC, and the molar ratio of DMPC to the MSP in the pharmaceutical composition is about 150:1. [0150] In some embodiments, the MSP is MSP1E3D1 (e.g., SEQ ID NO: 8) or His-tag removed variant thereof (e.g., SEQ ID NO: 15), the phospholipid is DMPC, wherein the molar ratio of DMPC to the MSP in the pharmaceutical composition is about 87:1 to about 96:1, such as about 93:1, or about 58:1 to about 68:1, such as about 63:1, or about 62:1. ny-2692819 Attorney Docket No.: 283912000140 [0151] In some embodiments, the MSP is MSP1E3D1, the phospholipid is DMPC, and the molar ratio of the phospholipid to MSP in the pharmaceutical composition is about 58:1 to about 68:1, such as about 62:1. In some embodiments, the MSP is MSP1E3D1. In some embodiments, the MSP is an MSP1E3D1 His-tag removed variant comprising the amino acid sequence of SEQ ID NO: 15. [0152] In some embodiments, the MSP is MSP1E3D1 (e.g., SEQ ID NO: 8) or His-tag removed variant thereof (e.g., SEQ ID NO: 15), the phospholipid is DPPC, wherein at least about 85% (e.g., at least about any of 90%, 95%, 96%, 97%, 98%, 99%, or 100%) of the plurality of nanodiscs in the pharmaceutical composition are fully-lipidated. In some embodiments, the NS is about 167. In some embodiments, the MSP is MSP1E3 or His-tag removed variant thereof, the phospholipid is DPPC, wherein the molar ratio of DPPC to the MSP in the pharmaceutical composition is about 170:1. [0153] In some embodiments, the MSP is MSP1E3D1 (e.g., SEQ ID NO: 8) or His-tag removed variant thereof (e.g., SEQ ID NO: 15), the phospholipid is DPPC, wherein at least about 85% (e.g., at least about any of 90%, 95%, 96%, 97%, 98%, 99%, or 100%) of the plurality of nanodiscs in the pharmaceutical composition are under-lipidated. In some embodiments, the MSP is MSP1E3D1 or His-tag removed variant thereof , the phospholipid is DPPC, wherein the molar ratio of DPPC to the MSP in the pharmaceutical composition is less than a phospholipid- MSP saturation ratio NS of about 167:1, such as less than about 95% (e.g., less than about any of 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, or 40%) of the phospholipid-MSP saturation ratio NS of about167:1. In some embodiments, the molar ratio of DPPC to MSP1E3D1 or His-tag removed variant thereof in the pharmaceutical composition is less than or equal to about 160:1, such as less than or equal to about any of 158:1, 155:1, 150:1, 145:1, 140:1, 135:1, 130:1, 125:1, 120:1, 115:1, 110:1, 105:1, 100:1, 95:1, 90:1, 85:1, 80:1, 75:1, or less. In some embodiments, the molar ratio of DPPC to MSP1E3D1 or His-tag removed variant thereof in the pharmaceutical composition is about 70:1 to about 160:1, such as any of about 80:1 to about 150:1, about 70:1 to about 140:1, about 70:1 to about 130:1, about 80:1 to about 120:1, about 80:1 to about 100:1, about 80:1, about 90:1, about 100:1 about 87:1 to about 96:1, about 58:1 to about 68:1, about 60:1, about 62:1, about 63:1, about 65:1, about 70:1, about 80:1, about 90:1, about 93:1, about 100:1, or about 110:1. In some embodiments, the MSP is MSP1E3 or His-tag removed variant thereof, the phospholipid is DPPC, wherein the molar ratio of DPPC to ny-2692819 Attorney Docket No.: 283912000140 the MSP in the pharmaceutical composition is less than about 158:1. In some embodiments, the MSP is MSP1E3D1. In some embodiments, the MSP is an MSP1E3D1 His-tag removed variant comprising the amino acid sequence of SEQ ID NO: 15. [0154] In some embodiments, the MSP is MSP1E3D1 (e.g., SEQ ID NO: 8) or His-tag removed variant thereof (e.g., SEQ ID NO: 15), the phospholipid is POPC, wherein at least about 85% (e.g., at least about any of 90%, 95%, 96%, 97%, 98%, 99%, or 100%) of the plurality of nanodiscs are fully-lipidated. In some embodiments, the NS is about 126. [0155] In some embodiments, the MSP is MSP1E3D1 (e.g., SEQ ID NO: 8) or His-tag removed variant thereof (e.g., SEQ ID NO: 15), the phospholipid is POPC, wherein at least about 85% (e.g., at least about any of 90%, 95%, 96%, 97%, 98%, 99%, or 100%) of the plurality of nanodiscs are under-lipidated. In some embodiments, the MSP is MSP1E3D1 or His-tag removed variant thereof, the phospholipid is POPC, wherein the molar ratio of POPC to the MSP in the pharmaceutical composition is less than a phospholipid-MSP saturation ratio NS of about 126: 1, such as less than about 95% (e.g., less than about any of 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, or 40%) of the phospholipid-MSP saturation ratio NS of about 126:1. In some embodiments, the molar ratio of POPC to MSP1E3D1 or His-tag removed variant thereof in the pharmaceutical composition is less than or equal to about 120:1, such as less than or equal to about any of 118:1, 115:1, 110:1, 105:1, 100:1, 95:1, 90:1, 85:1, 80:1, 75:1, 70:1, 65:1, or less. In some embodiments, the molar ratio of POPC to MSP1E3D1 or His-tag removed variant thereof in the pharmaceutical composition is about 60:1 to about 120:1, such as any of about 70:1 to about 120:1, about 70:1 to about 110:1, about 70:1 to about 100:1, about 80:1 to about 110:1, about 80:1 to about 100:1, about 80:1, about 90:1, about 100:1, or about 110:1. In some embodiments, the MSP is MSP1E3D1 or His-tag removed variant thereof, the phospholipid is POPC, wherein the molar ratio of POPC to the MSP in the pharmaceutical composition is less than about 118:1. In some embodiments, the MSP is MSP1E3D1. In some embodiments, the MSP is an MSP1E3D1 His-tag removed variant comprising the amino acid sequence of SEQ ID NO: 15. [0156] In some embodiments, the MSP is MSP1E1D1 (e.g., SEQ ID NO: 16) or His-tag removed variant thereof, the phospholipid is DMPC, wherein at least about 85% (e.g., at least ny-2692819 Attorney Docket No.: 283912000140 about any of 90%, 95%, 96%, 97%, 98%, 99%, or 100%) of the plurality of nanodiscs in the pharmaceutical composition are fully-lipidated. In some embodiments, the NS is about 107. [0157] In some embodiments, the MSP is MSP1E1D1 (e.g., SEQ ID NO: 16) or His-tag removed variant thereof, the phospholipid is DMPC, wherein at least about 85% (e.g., at least about any of 90%, 95%, 96%, 97%, 98%, 99%, or 100%) of the plurality of nanodiscs in the pharmaceutical composition are under-lipidated. In some embodiments, the MSP is MSP1E1D1 or His-tag removed variant thereof, the phospholipid is DMPC, wherein the molar ratio of the phospholipid to MSP in the pharmaceutical composition is less than a phospholipid-MSP saturation ratio NS of about 107:1, such as less than about 95% (e.g., less than about any of 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, or 40%) of the phospholipid-MSP saturation ratio NS of about 107:1. In some embodiments, the molar ratio of DMPC to MSP1D1E1 or His- tag removed variant thereof in the pharmaceutical composition is less than or equal to about 105:1, such as less than or equal to about any of 100:1, 95:1, 90:1, 85:1, 80:1, 75:1, 70:1, 65:1, 60:1, 55:1, 50:1, 45:1, 40:1, or less. In some embodiments, the molar ratio of DMPC to MSP1E1D1 or His-tag removed variant thereof in the pharmaceutical composition is about 30:1 to about 100:1, such as any of about 40:1 to about 90:1, about 50:1 to about 80:1, about 50:1 to about 70:1, about 60:1 to about 70:1, about 60:1 to about 65:1, about 70:1, about 65:1, about 60:1, or about 55:1. In some embodiments, the molar ratio of DMPC to MSP1E1D1 or His-tag removed variant thereof in the pharmaceutical composition is about 60:1, about 62:1, about 64:1 to about 66:1, or about 68:1. In some embodiments, the MSP is MSP1E1D1. In some embodiments, the MSP is a His-tag removed variant of MSP1E1D1. [0158] In some embodiments, the MSP is MSP1E1D1 (e.g., SEQ ID NO: 16) or His-tag removed variant thereof, the phospholipid is DPPC, wherein at least about 85% (e.g., at least about any of 90%, 95%, 96%, 97%, 98%, 99%, or 100%) of the plurality of nanodiscs in the pharmaceutical composition are fully-lipidated. In some embodiments, the NS is about 107. [0159] In some embodiments, the MSP is MSP1E1D1 (e.g., SEQ ID NO: 16) or His-tag removed variant thereof, the phospholipid is DPPC, wherein at least about 85% (e.g., at least about any of 90%, 95%, 96%, 97%, 98%, 99%, or 100%) of the plurality of nanodiscs in the pharmaceutical composition are under-lipidated. In some embodiments, the MSP is MSP1E1D1 or His-tag removed variant thereof, the phospholipid is DPPC, wherein the molar ratio of the ny-2692819 Attorney Docket No.: 283912000140 phospholipid to MSP in the pharmaceutical composition is less than a phospholipid-MSP saturation ratio NS of about 107:1, such as less than about 95% (e.g., less than about any of 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, or 40%) of the phospholipid-MSP saturation ratio NS of about 107:1. In some embodiments, the molar ratio of DPPC to MSP1D1E1 or His- tag removed variant thereof in the pharmaceutical composition is less than or equal to about 105:1, such as less than or equal to about any of 100:1, 95:1, 90:1, 85:1, 80:1, 75:1, 70:1, 65:1, 60:1, 55:1, 50:1, 45:1, 40:1, or less. In some embodiments, the molar ratio of DPPC to MSP1E1D1 or His-tag removed variant thereof is about 30:1 to about 100:1, such as any of about 40:1 to about 90:1, about 50:1 to about 80:1, about 50:1 to about 70:1, about 60:1 to about 70:1, about 60:1 to about 65:1, about 70:1, about 65:1, about 60:1, or about 55:1. In some embodiments, the molar ratio of DPPC to MSP1E1D1 or His-tag removed variant thereof in the pharmaceutical composition is about 60:1, 62:1, about 64:1 to about 66:1, or about 68:1. In some embodiments, the MSP is MSP1E1D1. In some embodiments, the MSP is a His-tag removed variant of MSP1E1D1. [0160] In some embodiments, the MSP is MSP1E1D1 (e.g., SEQ ID NO: 16) or His-tag removed variant thereof, the phospholipid is POPC, wherein at least about 85% (e.g., at least about any of 90%, 95%, 96%, 97%, 98%, 99%, or 100%) of the plurality of nanodiscs in the pharmaceutical composition are fully-lipidated. In some embodiments, the NS is about 80. [0161] In some embodiments, the MSP is MSP1E1D1 (e.g., SEQ ID NO: 16) or His-tag removed variant thereof, the phospholipid is POPC, and at least about 85% (e.g., at least about any of 90%, 95%, 96%, 97%, 98%, 99%, or 100%) of the plurality of nanodiscs in the pharmaceutical composition are under-lipidated. In some embodiments, the MSP is MSP1E1D1 (or His-tag removed variant thereof, the phospholipid is POPC, wherein the molar ratio of the phospholipid to the MSP in the pharmaceutical composition is less than a phospholipid-MSP saturation ratio NS of about 80:1, such as less than about 95% (e.g., less than about any of 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, or 40%) of the phospholipid-MSP saturation ratio NS of about 80:1. In some embodiments, the molar ratio of POPC to MSP1E1D1 or His-tag removed variant thereof in the pharmaceutical composition is less than or equal to about 76:1, such as less than or equal to about any of 76:1, 70:1, 65:1, 60:1, 55:1, 50:1, 45:1, 40:1, or less. In some embodiments, the molar ratio of POPC to MSP1D1E1 or His-tag removed variant thereof in the pharmaceutical composition is about 10:1 to about 75:1, such as any of about 20:1 ny-2692819 Attorney Docket No.: 283912000140 to about 75:1, about 20:1 to about 60:1, about 30:1 to about 60:1, about 40:1 to about 60:1, about 65:1, about 60:1, about 55:1, about 50:1, about 45:1, about 40:1, about 35:1, or about 30:1. In some embodiments, the molar ratio of POPC to MSP1E1D1 or His-tag removed variant thereof in the pharmaceutical composition is about 50:1, about 54:1 to about 56:1, or about 55:1. In some embodiments, the MSP is MSP1E1D1. In some embodiments, the MSP is a His-tag removed variant of MSP1E1D1. [0162] In some embodiments, the MSP is MSP1E2D1 (e.g., SEQ ID NO: 17) or His-tag removed variant thereof, the phospholipid is DMPC, wherein at least about 85% (e.g., at least about any of 90%, 95%, 96%, 97%, 98%, 99%, or 100%) of the plurality of nanodiscs in the pharmaceutical composition are fully-lipidated. In some embodiments, the NS is about 135. [0163] In some embodiments, the MSP is MSP1E2D1 (e.g., SEQ ID NO: 17) or His-tag removed variant thereof, the phospholipid is DMPC, wherein at least about 85% (e.g., at least about any of 90%, 95%, 96%, 97%, 98%, 99%, or 100%) of the plurality of nanodiscs in the pharmaceutical composition are under-lipidated. In some embodiments, the MSP is MSP1E2D1 or His-tag removed variant thereof, the phospholipid is DMPC, wherein the molar ratio of the phospholipid to the MSP in the pharmaceutical composition is less than a phospholipid-MSP saturation ratio NS of about 135:1, such as less than about 95% (e.g., less than about any of 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, or 40%) of the phospholipid-MSP saturation ratio NS of about 135:1. In some embodiments, the molar ratio of DMPC to MSP1E2D1 or His- tag removed variant thereof in the pharmaceutical composition is less than or equal to about 128:1, such as less than or equal to about any of 125:1, 120:1, 115:1, 110:1, 105:1, 100:1, 95:1, 90:1, 85:1, 80:1, 75:1, 70:1, 65:1, 60:1, 55:1, or less. In some embodiments, the molar ratio of DMPC to MSP1E2D1 or His-tag removed variant thereof in the pharmaceutical composition is about 50:1 to about 130:1, such as any of about 50:1 to about 120:1, about 60:1 to about 120:1, about 70:1 to about 110:1, about 70:1 to about 100:1, about 70:1, about 75:1, about 80:1, or about 85:1. In some embodiments, the MSP is MSP1E2D1. In some embodiments, the MSP is a His-tag removed variant of MSP1E2D1. [0164] In some embodiments, the MSP is MSP1E2D1 (e.g., SEQ ID NO: 17) or His-tag removed variant thereof, the phospholipid is DPPC, wherein at least about 85% (e.g., at least ny-2692819 Attorney Docket No.: 283912000140 about any of 90%, 95%, 96%, 97%, 98%, 99%, or 100%) of the plurality of nanodiscs in the pharmaceutical composition are fully-lipidated. In some embodiments, the NS is about 135. [0165] In some embodiments, the MSP is MSP1E2D1 (e.g., SEQ ID NO: 17) or His-tag removed variant thereof, the phospholipid is DPPC, wherein at least about 85% (e.g., at least about any of 90%, 95%, 96%, 97%, 98%, 99%, or 100%) of the plurality of nanodiscs in the pharmaceutical composition are under-lipidated. In some embodiments, the MSP is MSP1E2D1 or His-tag removed variant thereof, the phospholipid is DPPC, wherein the molar ratio of the phospholipid to the MSP in the pharmaceutical composition is less than a phospholipid-MSP saturation ratio NS of about 135:1, such as less than about 95% (e.g., less than about any of 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, or 40%) of the phospholipid-MSP saturation ratio NS of about 135:1. In some embodiments, the molar ratio of DPPC to MSP1E2D1 or His- tag removed variant thereof in the pharmaceutical composition is less than or equal to about 128:1, such as less than or equal to about any of 125:1, 120:1, 115:1, 110:1, 105:1, 100:1, 95:1, 90:1, 85:1, 80:1, 75:1, 70:1, 65:1, 60:1, 55:1, or less. In some embodiments, the molar ratio of DPPC to MSP1E2D1 or His-tag removed variant thereof in the pharmaceutical composition is about 50:1 to about 130:1, such as any of about 50:1 to about 120:1, about 60:1 to about 120:1, about 70:1 to about 110:1, about 70:1 to about 100:1, about 70:1, about 75:1, about 80:1, or about 85:1. In some embodiments, the MSP is MSP1E2D1. In some embodiments, the MSP is a His-tag removed variant of MSP1E2D1. [0166] In some embodiments, the MSP is MSP1E2D1 (e.g., SEQ ID NO: 17) or His-tag removed variant thereof, the phospholipid is POPC, wherein at least about 85% (e.g., at least about any of 90%, 95%, 96%, 97%, 98%, 99%, or 100%) of the plurality of nanodiscs in the pharmaceutical composition are fully-lipidated. In some embodiments, the NS is about 102. [0167] In some embodiments, the MSP is MSP1E2D1 (e.g., SEQ ID NO: 17) or His-tag removed variant thereof, the phospholipid is POPC, and at least about 85% (e.g., at least about any of 90%, 95%, 96%, 97%, 98%, 99%, or 100%) of the plurality of nanodiscs in the pharmaceutical composition are under-lipidated. In some embodiments, the MSP is MSP1E2D1 or His-tag removed variant thereof, the phospholipid is POPC, wherein the molar ratio of the phospholipid to the MSP in the pharmaceutical composition is less than a phospholipid-MSP saturation ratio NS of about 102:1, such as less than about 95% (e.g., less than about any of 90%, ny-2692819 Attorney Docket No.: 283912000140 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, or 40%) of the phospholipid-MSP saturation ratio NS of about 102:1. In some embodiments, the molar ratio of POPC to MSP1E2D1 or His- tag removed variant thereof in the pharmaceutical composition is less than about 96:1, such as less than about any of 95:1, 90:1, 85:1, 80:1, 75:1, 70:1, 65:1, 60:1, 55:1, 50:1, or less. In some embodiments, the molar ratio of POPC to MSP1E2D1 or His-tag removed variant thereof in the pharmaceutical composition is about 40:1 to about 95:1, such as any of about 50:1 to about 90:1, about 60:1 to about 90:1, about 70:1 to about 90:1, about 70:1 to about 80:1, about 70:1, about 75:1, about 80:1, or about 85:1. In some embodiments, the MSP is MSP1E2D1. In some embodiments, the MSP is a His-tag removed variant of MSP1E2D1. [0168] In some embodiments, the MSP is MSP2N2 (e.g., SEQ ID NO: 18) or His-tag removed variant thereof, the phospholipid is DMPC, wherein at least about 85% (e.g., at least about any of 90%, 95%, 96%, 97%, 98%, 99%, or 100%) of the plurality of nanodiscs in the pharmaceutical composition are fully-lipidated. In some embodiments, the NS is about 387. [0169] In some embodiments, the MSP is MSP2N2 (e.g., SEQ ID NO: 18) or His-tag removed variant thereof, the phospholipid is DMPC, and at least about 85% (e.g., at least about any of 90%, 95%, 96%, 97%, 98%, 99%, or 100%) of the plurality of nanodiscs in the pharmaceutical composition are under-lipidated.In some embodiments, the MSP is MSP2N2 or His-tag removed variant thereof, the phospholipid is DMPC, wherein the molar ratio of the phospholipid to the MSP in the pharmaceutical composition is less than a phospholipid-MSP saturation ratio NS of about 387:1, such as less than about 95% (e.g., less than about any of 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, or 40%) of the phospholipid-MSP saturation ratio NS of about 387:1. In some embodiments, the molar ratio of DMPC to MSP2N2 or His-tag removed variant thereof in the pharmaceutical composition is less than or equal to about 370:1, such as less than or equal to about any of 367:1, 360:1, 350:1, 340:1, 330:1, 320:1, 310:1, 300:1, 290:1, 280:1, 270:1, 260:1, 250:1, 240:1, 230:1, 220:1, 210:1, 200:1, 190:1, 180:1, 170:1, 160:1, 150:1, 140:1, 130:1, 120:1, 110:1, 100:1, or less. In some embodiments, the molar ratio of DMPC to MSP2N2 or His-tag removed variant thereof in the pharmaceutical composition is about 150:1 to about 350:1, such as any of about 200:1 to about 350:1, about 250:1 to about 350:1, about 250:1 to about 330:1, about 280:1 to about 320:1, about 280:1 to about 300:1, about 280:1, about 290:1, about 300:1, or about 310:1. In some embodiments, the MSP is MSP2N2. In some embodiments, the MSP is a His-tag removed variant of MSP2N2. ny-2692819 Attorney Docket No.: 283912000140 [0170] In some embodiments, the MSP is MSP2N2 (e.g., SEQ ID NO: 18) or His-tag removed variant thereof, the phospholipid is DPPC, and at least about 85% (e.g., at least about any of 90%, 95%, 96%, 97%, 98%, 99%, or 100%) of the plurality of nanodiscs in the pharmaceutical composition are fully-lipidated. In some embodiments, the NS is about 387. [0171] In some embodiments, the MSP is MSP2N2 (e.g., SEQ ID NO: 18) or His-tag removed variant thereof, the phospholipid is DPPC, wherein at least about 85% (e.g., at least about any of 90%, 95%, 96%, 97%, 98%, 99%, or 100%) of the plurality of nanodiscs in the pharmaceutical composition are under-lipidated. In some embodiments, the MSP is MSP2N2 or His-tag removed variant thereof, the phospholipid is DPPC, wherein the molar ratio of the phospholipid to the MSP in the pharmaceutical composition is less than a phospholipid-MSP saturation ratio NS of about 387: 1, such as less than about 95% (e.g., less than about any of 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, or 40%) of the phospholipid-MSP saturation ratio NS of about 387:1. In some embodiments, the molar ratio of DPPC to MSP2N2 or His-tag removed variant thereof in the pharmaceutical composition is less than or equal to about 370:1, such as less than or equal to about any of 367:1, 360:1, 350:1, 340:1, 330:1, 320:1, 310:1, 300:1, 290:1, 280:1, 270:1, 260:1, 250:1, 240:1, 230:1, 220:1, 210:1, 200:1, 190:1, 180:1, 170:1, 160:1, 150:1, 140:1, 130:1, 120:1, 110:1, 100:1, or less. In some embodiments, the molar ratio of DPPC to MSP2N2 or His-tag removed variant thereof in the pharmaceutical composition is about 150:1 to about 350:1, such as any of about 200:1 to about 350:1, about 250:1 to about 350:1, about 250:1 to about 330:1, about 280:1 to about 320:1, about 280:1 to about 300:1, about 280:1, about 290:1, about 300:1, or about 310:1. In some embodiments, the MSP is MSP2N2. In some embodiments, the MSP is a His-tag removed variant of MSP2N2. [0172] In some embodiments, the MSP is MSP2N2 (e.g., SEQ ID NO: 18) or His-tag removed variant thereof, the phospholipid is POPC, and at least about 85% (e.g., at least about any of 90%, 95%, 96%, 97%, 98%, 99%, or 100%) of the plurality of nanodiscs in the pharmaceutical composition are fully-lipidated. In some embodiments, the NS is about 291. [0173] In some embodiments, the MSP is MSP2N2 (e.g., SEQ ID NO: 18) or His-tag removed variant thereof, the phospholipid is POPC, wherein at least about 85% (e.g., at least about any of 90%, 95%, 96%, 97%, 98%, 99%, or 100%) of the plurality of nanodiscs in the pharmaceutical composition are under-lipidated. In some embodiments, the MSP is MSP2N2 or His-tag removed ny-2692819 Attorney Docket No.: 283912000140 variant thereof, the phospholipid is POPC, wherein the molar ratio of the phospholipid to the MSP in the pharmaceutical composition is less than a phospholipid-MSP saturation ratio NS of about 291:1, such as less than about 95% (e.g., less than about any of 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, or 40%) of the phospholipid-MSP saturation ratio NS, which is 291:1. In some embodiments, the molar ratio of POPC to MSP2N2 or His-tag removed variant thereof in pharmaceutical the composition is less than or equal to about 280:1, such as less than or equal to about any of 276:1, 270:1, 260:1, 250:1, 240:1, 230:1, 220:1, 210:1, 200:1, 190:1, 180:1, 170:1, 160:1, 150:1, 140:1, 130:1, 120:1, 110:1, 100:1, or less. In some embodiments, the molar ratio of POPC to MSP2N2 or His-tag removed variant thereof in the pharmaceutical composition is about 100:1 to about 280:1, such as any of about 100:1 to about 260:1, about 120:1 to about 260:1, about 140:1 to about 260:1, about 160:1 to about 240:1, about 180:1 to about 220:1, about 180:1, about 190:1, about 200:1, or about 210:1. In some embodiments, the MSP is MSP2N2. In some embodiments, the MSP is a His-tag removed variant of MSP2N2. d. Characteristics [0174] In some embodiments, the pharmaceutical composition comprises nanodiscs purified by chromatography, such as size exclusion chromatography (SEC). In some embodiments, at least about 85% of the plurality of nanodiscs in the pharmaceutical composition are under- lipidated. In some embodiments, at least about 85% of the plurality of nanodiscs in the pharmaceutical composition are fully-lipidated. [0175] In some embodiments, the nanodisc increases the cholesterol efflux rate by at least about 2 folds (e.g., at least about any of 2.5, 3, 4, 5, 10, 15-fold, or more) compared to an untreated state (e.g., before administering the nanodisc) or the cholesterol efflux rate by ApoA-I or the MSP (e.g., MSP1D1). In some embodiments, the nanodisc increases (e.g., increasing at least about any of 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 10-fold, or more) the cholesterol efflux rate in retinal pigment epithelium cells, or macrophages. In some embodiments, the nanodisc reduces lipid or prevents lipid accumulation in the eye of the individual by at least about 5% (e.g., at least about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%). [0176] In some embodiments, the nanodisc pharmaceutical compositions described herein increase the cholesterol efflux rate by at least about 20% (e.g., at least about any of 30%, 40%, ny-2692819 Attorney Docket No.: 283912000140 50%, 60%, 70%, 80%, 90%, 100%, 2-fold, 2.5-fold, 3-fold, 4-fold, 5-fold, 10-fold, 15-fold, or more) compared to an untreated state (e.g., before administering the nanodiscs) or the cholesterol efflux rate by ApoA-I or a high density lipoprotein (HDL) alone. In some embodiments, the under-lipidated nanodisc pharmaceutical composition described herein increases the cholesterol efflux rate by at least about 20% (e.g., at least about any of 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 2-fold, 2.5-fold, 3-fold, 4-fold, 5-fold, 10-fold, 15-fold, or more) compared to a fully-lipidated nanodisc pharmaceutical composition containing the same MSP and phospholipid (the molar ratio of the phospholipid to the MSP in the pharmaceutical composition is about the phospholipid-MSP saturation ratio NS). [0177] In some embodiments, the nanodisc does not comprise a cell-penetrating peptide (CPP). In some embodiments, the CPP is selected from the group consisting of penetratin, polyarginine (R8), LL-37, transportan, Pep-1, and membrane translocating sequence (MTS). [0178] In some embodiments, the nanodisc does not comprise a target protein. In some embodiments, the nanodisc does not comprise a prophylactic or therapeutic agent. [0179] In some embodiments, the diameter of the nanodisc is any of about 2 nm to about 100 nm, about 5 nm to about 100 nm, about 5 nm to about 75 nm, about 5 nm to about 50 nm, about 5 nm to about 40 nm, about 5 nm to about 30 nm, about 5 nm to about 20 nm, or about 10 nm to about 20 nm. In some embodiments, the diameter of the nanodisc is about 10 nm to about 20 nm. In some embodiments, the average size of the nanodiscs can be tuned by the length of the MSP. In some embodiments, the diameter of the nanodisc allows for the penetration of the nanodisc into or through ocular muscles. In some embodiments, the diameter of the nanodisc allows for the reachability of the nanodisc to the posterior of the eye without CPP. In some embodiments, the diameter of the nanodisc can be tuned by the length of the MSP belt. [0180] In some embodiments, the average diameter of the nanodiscs is any of about 2 nm to about 100 nm, about 5 nm to about 75 nm, about 5 nm to about 50 nm, about 5 nm to about 40 nm, about 5 nm to about 30 nm, about 5 nm to about 20 nm, or about 10 nm to about 20 nm. In some embodiments, the average diameter of the nanodiscs is about 2 nm to about 20 nm, such as about 10 nm to about 20 nm. In some embodiments, the average diameter of the nanodiscs can be tuned by the length of the MSP. ny-2692819 Attorney Docket No.: 283912000140 [0181] In some embodiments, the thickness of the nanodisc is any of about 2 nm to about 20 nm, about 3 nm to about 15 nm, about 3 nm to about 10 nm, about 4 nm to about 7 nm, about 4 nm, about 5 nm, or about 6 nm. In some embodiments, the thickness of the nanodisc can be tuned by the type of phospholipid. [0182] In some embodiments, each nanodisc comprises any of about 50 to about 300, about 100 to about 300, about 100 to about 250, or about 100 to about 200 phospholipid molecules. In some embodiments, each nanodisc comprises any of about 2, 3, 4, or more MSPs. [0183] In some embodiments, the molecular weight of the nanodisc is any of about 100,000 g/mol to about 400,000 g/mol, about 120,000 g/mol to about 400,000 g/mol, about 150,000 g/mol to about 350,000 g/mol, about 150,000 g/mol to about 300,000 g/mol, about 150,000 g/mol to about 250,000 g/mol, about 150,000 g/mol, about 200,000 g/mol, about 250,000 g/mol, about 300,000 g/mol, or about 320,000 g/mol. In some embodiments, the nanodisc comprises MSP in a weight percentage of any of about 15% to about 50%, about 20% to about 40%, about 22% to about 38%, about 24%, about 25%, about 27%, about 29%, about 30%, about 33%, or about 35%. In some embodiments, the nanodisc comprises a phospholipid in a weight percentage of any of about 50% to about 90%, about 60% to about 80%, about 63% to about 77%, about 64%, about 65%, about 66%, about 67%, about 70%, about 73%, or about 75%. In some embodiments, the nanodisc comprises MSP and phospholipid in a weight ratio of about any of 1.5:8.5 to 1:1, 2:8 to 1:1, 2.5:7.5:1.1, 3:7 to 1:1, 1.5:7.5, 2:8, 3:7, 3.5:6.5, 4:6, or 4.5:5.5. In some embodiment, about any of 80% (w/w), 85% (w/w), 90% (w/w), 92% (w/w), 94% (w/w), 96% (w/w), 99% (w/w), or 99.5% (w/w) of the total amount of protein in the naodisc is MSP. [0184] In some embodiments, the polydispersity index (a measure of the heterogeneity) of the size of the nanodiscs in the pharmaceutical composition is less than about 0.5, such as less than about any of 0.4, 0.3, 0.2, 0.1, 0.09, 0.08, 0.07, 0.06, 0.05, 0.04, 0.03, 0.02, or 0.01. In some embodiments, the size and size distribution of the nanodiscs in the pharmaceutical composition may be determined by combines multi-angle light scattering with size-exclusion chromatography (SEC-MALS). In some embodiments, at least about 60%, such as at least about any of 70%, 75%, 80%, 85%, 90%, 95%, or 99% of the nanodiscs in the pharmaceutical composition have a size of about 2 nm to about 20 nm. In some embodiments, at least about 60%, such as at least about any of 70%, 75%, 80%, 85%, 90%, 95%, or 99% of the nanodiscs remain to have sizes of ny-2692819 Attorney Docket No.: 283912000140 about 2 nm to about 20 nm after being frozen and thawed for one or more times, such as being frozen at -80°C and thawed with or without a cryoprotectant. In some embodiments, at least about 60%, such as at least about any of 70%, 75%, 80%, 85%, 90%, 95%, or 99% of the nanodiscs in the pharmaceutical composition have sizes substantially unchanged (e.g., a size change of the nanodiscs in the pharmaceutical composition of less than about any of 20%, 15%, 10%, 5%, 2%, or 1%) after being frozen and thawed for one or more times, such as being frozen at -80 °C and thawed with or without a cryoprotectant. [0185] In some embodiments, the polydispersity index (a measure of the heterogeneity) of the lipid content of the nanodiscs in the pharmaceutical composition is less than about 0.5, such as less than about any of 0.4, 0.3, 0.2, 0.1, 0.09, 0.08, 0.07, 0.06, 0.05, 0.04, 0.03, 0.02, or 0.01. In some embodiments, at least about 60%, such as at least about any of 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% of the nanodiscs have molar ratio between phospholipid and MSP of less than about 95% (e.g., less than about any of 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, or 40%) of the phospholipid- MSP saturation ratio NS. In some embodiments, at least about 60%, such as at least about any of 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% of the nanodiscs have molar ratio between phospholipid and MSP of less than about 95% (e.g., less than about any of 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, or 40%) of the phospholipid- MSP saturation ratio NS after being frozen and thawed for one or more times, such as being frozen at -80°C and thawed, with or without a cryoprotectant. In some embodiments, at least about 60%, such as at least about any of 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% of the nanodiscs have molar ratio between phospholipid and MSP remains substantially unchanged (e.g., a size change of the nanodiscs in the pharmaceutical composition of less than about any of 20%, 15%, 10%, 5%, 2%, or 1%) after being frozen and thawed for one or more times, such as being frozen at -80°C and thawed with or without a cryoprotectant. [0186] In some embodiments, the molar ratio between phospholipid and MSP in the pharmaceutical composition remains substantially unchanged (e.g., a size change of the nanodiscs in the pharmaceutical composition of less than about any of 20%, 15%, 10%, 5%, 2%, or 1%) after being storied under room temperature for at least about 1 hour, such as at least about any of 2, 5, 10, 12, 15, 20, or 24 hours, or 2, 3, 4, 5, 6, 7, 8, 15, 30, 40, 50, 100 days. In some embodiments, the molar ratio between phospholipid and MSP remains substantially unchanged (e.g., a size change of the nanodiscs in the pharmaceutical composition of less than about any of ny-2692819 Attorney Docket No.: 283912000140 20%, 15%, 10%, 5%, 2%, or 1%) after being storied under -80°C for at least about 1 month, such as at least about any of 2, 3, 4, 5, 6, 7, 8, 15, 30, 40, 50, 100 months. e. Method of preparation [0187] Nanodiscs can be formed by the interaction between the MSP and the phospholipid. Method of preparing nanodiscs is known in the art, and description of the exemplary methods can be found in Timothy H. Bayburt, Yelena V. Grinkova, and Stephen G. Sligar. Self-Assembly of Discoidal Phospholipid Bilayer Nanoparticles with Membrane Scaffold Proteins, Nano Letters 20022 (8), 853-856. DOI: 10.1021/nl025623k, the content of which is incorporated herein by reference in its entirety. Also see Examples 1 and 8-10 for exemplary methods. [0188] In some embodiments, there is provided a method of preparing a pharmaceutical composition comprising a plurality of nanodiscs (e.g., any of the nanodisc pharmaceutical compositions described herein, e.g., fully-lipidated or under-lipidated), the method comprising: (1) incubating the MSP and the phospholipid to obtain a preparation mixture, wherein the phospholipid is solubilized by a detergent or organic compound prior to the incubation; and (2) removing the detergent or organic compound from the preparation mixture, thereby obtaining the nanoparticles. In some embodiments, the molar ratio of the phospholipid to the MSP in the preparation mixture is less than (e.g., less than about any of 95%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, or lower) or equal to the molar ratio of the phospholipid to the MSP in the pharmaceutical composition. In some embodiments, the method further comprises purifying the obtained nanodiscs, e.g., by SEC (one or more times). In some embodiments, the method further comprises verifying the obtained nanodisc pharmaceutical composition, e.g., after the final purification step. In some embodiments, the MSP is any of the MSPs described herein. In some embodiments, the phospholipid is any of the phospholipids described herein. [0189] In some embodiments, the nanodiscs can be synthesized by incubating solubilized phospholipid with MSP. In some embodiments, the phospholipid is solubilized by a detergent or an organic compound. In some embodiments, the solubilized phospholipid is prepared by adding solution of detergent or an organic compound to a thin film of phospholipid. Exemplary detergents or organic compounds include, but are not limited to, alkyglucosides such as n- dodecyl-β-D-maltoside (DDM), octyl-β-glucoside (OG), Triton X-100, 3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate (CHAPS), 2,2-didecylpropane-1,3-bis-β-D- ny-2692819 Attorney Docket No.: 283912000140 maltopyranoside (LMNG), cholate, and any combination thereof. In some embodiments, the phospholipid is solubilized by a cholate salt, such as sodium cholate. In some embodiments, the MSP is provided in a solution comprising a buffer. In some embodiments, the buffer has a pH of about 5 to about 9, such as about 6 to about 8, about 7, or about 7.5. In some embodiments, the MSP in the solution is at least about 0.1 mg/mL, such as at least about any of 0.2 mg/mL, 0.3 mg/mL, 0.4 mg/mL, 0.5 mg/mL, 1 mg/mL, 1.5 mg/mL, 2 mg/mL, or 2.5 mg/mL. In some embodiments, the MSP in the solution is about 0.5 mg/mL to about 10 mg/mL, such as any of about 1 mg/mL to about 8 mg/mL, about 1 mg/mL to about 5 mg/mL, about 1.5 mg/mL to about 6 mg/mL, about 2 mg/mL to about 5 mg/mL, or about 2.5 mg/ml to about 4 mg/mL. [0190] In some embodiments, the method comprises removing the detergent or organic compound from the preparation mixture, thereby obtaining the nanodiscs. In some embodiments, after forming the nanodiscs, the detergent or organic compound (e.g., cholate) is removed, such as by incubation with absorbent polymer beads. In some embodiments, the absorbent polymer beads comprise styrene-divinylbenzene (macroreticular) beads (e.g., Amberlite® XAD-2® Beads). In some embodiments, the formation and size of nanodiscs can be determined by tools known in the art, such as size-exclusion chromatography (SEC) and electron microscopy. [0191] In some embodiments, the method further comprises isolating the nanodiscs. In some embodiments, the nanodiscs are isolated by chromatography. In some embodiments, the method further comprises subjecting the nanodiscs to one or more freeze thaw cycles with or without cryoprotectant. [0192] In some embodiments, the method further comprises purifying the prepared nanodiscs, such as separating nanodiscs with different phospholipid to MSP ratios. In some embodiments, the purification comprises the use of SEC. In some embodiments, the method comprises more than one purification cycles until a desired distribution of phospholipid to MSP ratio (e.g., fully- saturated, or less than phospholipid-polymer saturation ratio NS) is reached. [0193] In some embodiments, the method further comprises measuring the actual phospholipid to MSP ratio in the nanodisc pharmaceutical compositions. In some embodiments, the actual phospholipid to MSP ratio in the nanodisc pharmaceutical compositions can be determined by measuring the fatty acid content using gas chromatography (GC) analysis. ny-2692819 Attorney Docket No.: 283912000140 [0194] In some embodiments, the molar ratio of phospholipid to the MSP in the preparation mixture (e.g., mixture of method of preparation step (1) described above) is the same as that in the obtained nanodisc pharmaceutical composition. In some embodiments, the molar ratio of phospholipid to the MSP in the preparation mixture (e.g., mixture of method of preparation step (1) described above) is less than (e.g., less than about any of 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, or 40% of) that in the obtained nanodisc pharmaceutical composition. In some embodiments, the molar ratio of the phospholipid to the MSP in the preparation mixture is determined such that at least about 60%, such as at least about any of 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100%, of the as-prepared nanodics are under- lipidated, such as having a molar ratio of the phospholipid to the MSP less than about 95% (e.g., less than about any of 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, or 40%) of the phospholipid-MSP saturation ratio NS. In some embodiments, the molar ratio of the phospholipid to the MSP in the preparation mixture is determined such that at least about 60%, such as at least about any of 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100%, of the as-prepared nanodics are fully-lipidated. [0195] In some embodiments, provided herein are pharmaceutical compositions comprising nanodiscs prepared according to any of the methods of preparation detailed herein. [0196] In some embodiments, the pharmaceutical composition further comprises a prophylactic or therapeutic agent. In some embodiments, the pharmaceutical composition does not comprise a prophylactic or therapeutic agent. Routes of administration and Dose [0197] Nanodics or pharmaceutical compositions thereof described herein can be administered by any suitable administration routes. In some embodiments, the nanodics or pharmaceutical compositions thereof are administered via local ocular administration. [0198] In some embodiments, the suitable administration routes include, but are not limited to, intravitreal, topical, periocular injections, intra- or periocular implants, intravitreal implants, and suprachoroidal implants or particles or polymeric composition, or any releasing systems such as emulsions, solid non-biodegradable or degradable implants or tablets, or mini pumps. In some embodiments, the nanodiscs or pharmaceutical compositions thereof are directly administered to ny-2692819 Attorney Docket No.: 283912000140 the eye by ocular tissue injection such as periocular, conjunctival, subtenon, intracameral, intravitreal, intraocular, subretinal, subconjunctival, retrobulbar, suprachoroidal or intracanalicular injections. In some embodiments, the nanodiscs or pharmaceutical compositions thereof are administered by direct application to the eye using a catheter or other placement device such as a retinal pellet, intraocular insert, suppository or an implant comprising a porous, non-porous, or gelatinous material. In some embodiments, the nanodiscs or pharmaceutical compositions thereof are administered by topical ocular drops or ointments. In some embodiments, the nanodiscs or pharmaceutical compositions thereof are administered by a slow release device in the cul-de-sac or implanted adjacent to the sclera (transscleral) or in the sclera (intrascleral) or suprachoroidal or within the eye. Intracameral injection may be through the cornea into the anterior chamber to allow the agent to reach the trabecular meshwork. Intracanalicular injection may be into the venous collector channels draining Schlemm's canal or into Schlemm's canal. In some embodiments, the nanodiscs or pharmaceutical compositions thereof are administered intravitreally. [0199] In some embodiments, the pharmaceutical composition comprising a nanodisc (e.g., a plurality of nanodiscs) may be in the form of isotonic, sterile, saline solutions (monosodium or disodium phosphate, sodium, potassium, calcium or magnesium chloride and the like or mixtures of such salts), sterile aqueous dispersions (e.g., water-oil emulsion), or dry, especially freeze- dried compositions which upon addition, depending on the case, of sterilized water or physiological saline, permit the constitution of injectable solutions. [0200] In some embodiments, the pharmaceutical composition comprising a nanodisc (e.g., a plurality of nanodiscs) and a pharmaceutically acceptable excipient is administered as an ophthalmic eye drop. Suitable pharmaceutically acceptable excipient include, but are not limited to, ophthalmologically acceptable preservatives, solvents, surfactants, viscosity enhancers, penetration enhancers, buffers, isotonic agents, stabilizer, pH regulators, or water to form an aqueous, sterile ophthalmic suspension or solution. In some embodiments, the retention of the pharmaceutical composition can be further improved by viscosity building agents, such as hydroxymethyl cellulose, hydroxyethyl cellulose, hydroxypropyl methylcellulose, methylcellulose, carboxymethylcellulose, polyvinylpyrrolidone, hyaluronic acid, or the like. ny-2692819 Attorney Docket No.: 283912000140 [0201] In some embodiments, the pharmaceutical composition comprising a nanodisc (e.g., a plurality of nanodiscs) and a pharmaceutically acceptable excipient is administered as an ophthalmic ointment composition. In some embodiments, the pharmaceutically acceptable excipient comprises a preservative in an appropriate vehicle, such as mineral oil, liquid lanolin, or white petrolatum. [0202] In some embodiments, the pharmaceutical composition is an anhydrous dosage form comprising a nanodisc. Such an anhydrous dosage form can be prepared using anhydrous or low moisture containing ingredients and low moisture or low humidity conditions. [0203] In some embodiments, the pharmaceutical composition further comprises a buffer. In some embodiments, the buffer is an isotonic buffer. In some embodiments, the isotonic buffer is phosphate-buffer saline (PBS). In some embodiments, the isotonic buffer does not comprise tromethamine (tris). [0204] In some embodiments, the pharmaceutical composition further comprises a preservative. Suitable preservatives include, but are not limited to, sodium bisulfite, sodium bisulfate, sodium thiosulfate, benzalkonium chloride, chlorobutanol, thimerosal, phenylmercuric acetate, phenylmercuric nitrate, methylparaben, polyvinyl alcohol and phenylethyl alcohol. [0205] In some embodiments, the excipient can be selected by one of ordinary skill in the art. Exemplary excipients include, for example, those described in the Handbook of Pharmaceutical Excipients, Rowe et al. (Eds.) 6th Ed. (2009), the content of which is incorporated by reference herein in its entirety. [0206] In some embodiments, a doctor can determine the dosage which they consider most appropriate according to a preventive or curative treatment and according to the age, weight, condition, and other factors specific to the individual to be treated. [0207] In some embodiments, the frequency and dosage may also vary according to factors specific for each individual depending on the specific therapy (e.g., therapeutic or prophylactic), the route of administration, as well as age, body, weight, response, and the past medical history of the individual. In some embodiments, effective doses may be extrapolated from dose- response curves derived from in vitro or animal model test systems. ny-2692819 Attorney Docket No.: 283912000140 [0208] In some embodiments, dose of the nanodiscs or pharmaceutical compositions thereof, when administered to remove lipid or prevent lipid (e.g., cholesterol) accumulation in the eye, or to prevent or treat an eye disease characterized by lipid (e.g., cholesterol) accumulation in an individual, is about 50 μg/kg to about 50 mg/kg (including for example any of about 50 μg/kg to about 30 mg/kg, about 70 μg/kg to about 20 mg/kg, about 100 μg/kg to about 10 mg/kg, about 100 μg/kg to about 500 μg /kg, or about 200 μg/kg to about 500 μg /kg) of the individual weight. In some embodiments, dose of the nanodiscs or pharmaceutical compositions thereof, when administered to remove lipid or prevent lipid (e.g., cholesterol) accumulation in the eye, or to prevent or treat an eye disease characterized by lipid (e.g., cholesterol) accumulation in an individual, is about 0.1 μg/kg to about 20 µg/kg (including for example any of about 0.5 μg/kg to about 18 mg/kg, about 1 μg/kg to about 15 mg/kg, about 1 μg/kg to about 10 mg/kg, about 1.5 μg/kg to about 9 μg /kg, or about 2 μg/kg to about 9 μg /kg) of the individual weight. In some embodiments, dose of the nanodiscs or pharmaceutical compositions thereof for methods described herein is about 1 µg to about 50 mg (including for example any of about 1 µg to about 30 mg, about 1 µg to about 20 mg, about 1 µg to about 10 mg, about 10 µg to about 20 mg, about 10µg, or about 15 mg). In some embodiments, dose of the nanodiscs or pharmaceutical compositions thereof for methods described herein is about 1 µg to about 1000 µg (including for example any of about 5 µg to about 800 mg, about 5 µg to about 600 mg, about 8 µg to about 600 mg, or about 10 µg to about 600 mg). [0209] In some embodiments, the dose of the nanodiscs or pharmaceutical compositions thereof can be administered according to a suitable schedule, for example, any of about every day, about every two days, about every three days, about every four days, about every five days, about every six days, about once a week, about once every two weeks, about once every three weeks, and about once a month. In some embodiments, the administration of the nanodiscs or pharmaceutical compositions thereof can be repeated about every three days. In some embodiments, the administration of the nanodiscs or pharmaceutical compositions thereof can be repeated about every 2 to 4 weeks. In some embodiments, the administration of the nanodiscs or pharmaceutical compositions thereof can be repeated about every 3 weeks. In some embodiments, administration of the nanodiscs or pharmaceutical compositions thereof may be repeated, such as separated by at least 1 day, 2 days, 3 days, 5 days, 10 days, 15 days, 30 days, 45 days, 2 months, 75 days, 3 months, or 6 months. ny-2692819 Attorney Docket No.: 283912000140 [0210] It may be necessary to use dosages of the nanodiscs or pharmaceutical compositions thereof outside the ranges disclosed herein in some cases, as will be apparent to those of ordinary skill in the art. Furthermore, it is noted that the clinician or treating physician will know how and when to interrupt, adjust, or terminate therapy in conjunction with the individual’s response. ENUMERATED EMBODIMENTS Embodiment 1. A method of removing lipid or preventing lipid accumulation in the eye of an individual, comprising administering to the eye of the individual an effective amount of a pharmaceutical composition comprising a nanodisc, wherein the nanodisc comprises a membrane scaffold protein (MSP) and a phospholipid. Embodiment 2. A method of preventing or treating an eye disease characterized by lipid accumulation in an individual, comprising administering to the eye of the individual an effective amount of a pharmaceutical composition comprising a nanodisc, wherein the nanodisc comprises an MSP and a phospholipid. Embodiment 3. The method of embodiment 1 or 2, wherein the MSP comprises a variant of an apolipoprotein selected from the group consisting of apolipoprotein A-I (ApoA-I), ApoA-II, ApoC, ApoE, and ApoM. Embodiment 4. The method of any one of embodiments 1-3, wherein the MSP is a variant of ApoA-I. Embodiment 5. The method of embodiment 4, wherein the MSP comprises one or more amphipathic helix sequences relative to ApoA-I. Embodiment 6. The method of embodiment 5, wherein the one or more amphipathic helix sequences are derived from ApoA-I. Embodiment 7. The method of embodiment 5 or 6, wherein the MSP is MSP1E1, MSP1E2, or MSP1E3. Embodiment 8. The method of any one of embodiments 4-7, wherein the MSP comprises an N- terminus truncation relative to ApoA-I. ny-2692819 Attorney Docket No.: 283912000140 Embodiment 9. The method of embodiment 8, wherein the MSP is MSP1, MSP1D1, MSP1D2, or MSPE3D1. Embodiment 10. The method of any one of embodiments 1-9, wherein the phospholipid is a phosphatidylcholine (PC). Embodiment 11. The method of embodiment 10, wherein the phospholipid is selected from the group consisting of dimyristoylphosphatidylcholine (DMPC), palmitoyloleoylphosphatidylcholine (POPC), dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylcholine (DSPC), and a combination thereof. Embodiment 12. The method of any one of embodiment 10 or 11, wherein the phospholipid is DMPC, DPPC, or POPC. Embodiment 13. The method of any one of embodiments 1-12, wherein the MSP is MSP1D1 or MSP1, and wherein the molar ratio of phospholipid to MSP is from about 10:1 to about 90:1. Embodiment 14. The method of embodiment 13, wherein the phospholipid is POPC, and wherein the molar ratio of POPC to MSP is from about 10:1 to about 65:1. Embodiment 15. The method of embodiment 14, wherein the MSP is MSP1D1, and wherein the molar ratio of POPC to MSP1D1 is about 50:1 or about 32.5:1. Embodiment 16. The method of embodiment 14, wherein the MSP is MSP1D1 or MSP1, and wherein the molar ratio of POPC to MSP1D1 or MSP1 is about 65:1. Embodiment 17. The method of embodiment 13, wherein the phospholipid is DMPC, and wherein the molar ratio of DMPC to MSP is from about 10:1 to about 85:1. Embodiment 18. The method of embodiment 17, wherein the MSP is MSP1D1, and wherein the molar ratio of DMPC to MSP1D1 is about 85:1. Embodiment 19. The method of embodiment 17, wherein the MSP is MSP1, and wherein the molar ratio of DMPC to MSP1 is about 80:1. Embodiment 20. The method of embodiment 13, wherein the MSP is MSP1, the phospholipid is DPPC, and wherein the molar ratio of DPPC to MSP1 is about 90:1. Embodiment 21. The method of any one of embodiments 1-12, wherein the MSP is MSP1E3, and wherein the molar ratio of phospholipid to MSP1E3 is from about 100:1 to about 200:1. ny-2692819 Attorney Docket No.: 283912000140 Embodiment 22. The method of embodiment 21, wherein the phospholipid is POPC, and wherein the molar ratio of POPC to MSP1E3 is about 130:1. Embodiment 23. The method of embodiment 21, wherein the phospholipid is DMPC, and wherein the molar ratio of DMPC to MSP1E3 is about 150:1. Embodiment 24. The method of embodiment 21, wherein the phospholipid is DPPC, and wherein the molar ratio of DPPC to MSP1E3 is about 170:1. Embodiment 25. The method of any one of embodiments 1-24, wherein the nanodisc is about 5 nm to about 20 nm in diameter. Embodiment 26. The method of any one of embodiments 1-25, wherein the lipid for removal or prevention from accumulation in the eye is cholesterol. Embodiment 27. The method of any one of embodiments 1-26, wherein the nanodisc does not comprise a cell-penetrating peptide (CPP). Embodiment 28. The method of embodiment 27, wherein the CPP is selected from the group consisting of penetratin, polyarginine (R8), LL-37, transportan, Pep-1, and membrane translocating sequence (MTS). Embodiment 29. The method of any one of embodiments 1-28, wherein the nanodisc does not comprise a target protein. Embodiment 30. The method of any one of embodiments 1-29, wherein the nanodisc does not comprise a prophylactic or therapeutic agent. Embodiment 31. The method of any one of embodiments 26-30, wherein the nanodisc increases cholesterol efflux rate by at least about 2-fold compared to an untreated state, or the cholesterol efflux rate by ApoA-I or the MSP. Embodiment 32. The method of any one of embodiments 1-31, wherein the pharmaceutical composition is administered via intravitreal injection. Embodiment 33. The method of any one of embodiments 2-32, wherein the eye disease characterized by lipid accumulation is selected from the group consisting of age-related macular degeneration (AMD), photoreceptor neurodegeneration, optic nerve atrophy, loss of acuity, ny-2692819 Attorney Docket No.: 283912000140 hemianopia, visual agnosia, strabismus, retinal neurovascular disorder, lipid keratopathy, corneal lipidosis, and a combination thereof. Embodiment 34. The method of embodiment 33, wherein the eye disease characterized by lipid accumulation is AMD. Embodiment 35. The method of embodiment 34, wherein the AMD is dry AMD. Embodiment 36. The method of embodiment 35, wherein the dry AMD is geographic atrophy (GA). Embodiment 37. The method of any one of embodiments 1-36, wherein the nanodisc reduces lipid or prevents lipid accumulation in the eye of the individual by at least about 5%. EXAMPLES Example 1: Preparation of Nanodisc [0211] Nanodiscs were assembled from a phospholipid, a MSP, and a detergent. See Sligar Lab, Nanodisc Technology: Protocols for Preparation of Nanodiscs (2008) University of Illinois at Urbana Champaign as an exemplary method, the content of which is incorporated herein by reference in its entirety [0212] Briefly, a frozen solution containing MSP in 20 mM tromethamine (Tris) (pH 7.4), 0.1 M NaCl, 0.5 mM EDTA, 0.01% NaN3 was thawed and filtered through a 0.22 micron filter. The concentration of MSP was determined spectrophotometrically using the molar extinction coefficient ε280=21,000 M-1cm-1. [0213] Phospholipid stocks were prepared in chloroform at 50-100 mM for long-term storage at –20ºC in 4 ml glass vials with Teflon-lined screw caps. Concentration of the stock solution was determined by phosphate analysis, the desired amount of chloroform lipid stock was dispensed into a disposable glass culture tube, and the solvent was dried up using a gentle stream of nitrogen gas in a fume hood. The tube was rotated at an angle to obtain a thin film on the lower walls. Residual solvent was removed by placing the tube in a vacuum desiccator under high vacuum for at least 4 hours. ny-2692819 Attorney Docket No.: 283912000140 [0214] Buffer comprising a detergent and sodium cholate was added to the tube so that the ratio of cholate to lipid in the mixture was 2:1. The tube was vortexed, heated under hot tap water, and sonicated in an ultrasonic bath until the solution was clear and no lipid remained on the walls of the tube. MSP was added to the solution and incubated for at least 15 minutes. [0215] To remove the cholate and initiate self-assembly of the nanodisc, 0.5-0.8 g of Amberlite XAD 2 beads (Sigma) were added per every ml of the reconstitution mixture. The suspension was placed on the orbital shaker and incubated overnight. The optimal lipid to MSP ratios and incubation temperature for various lipids, and the optimized incubation temperatures and minimum incubation times are shown in Table B-1. Table B-1
Figure imgf000082_0001
[0216] Samples were removed from Amberlite XAD 2 beads, filtered through a 0.22 micron filter, and fractionated on a Superdex 200 increase 10/300 GL column (GE Healthcare) in MSP Standard Buffer with a flow rate of 0.75 ml/min. Samples were filtered prior to injection and fractions were collected every minute. FIG. 1 displays an example chromatogram (MSP1D1+DMPC) of the reaction. Example 2A: Nanodisc preparation at different lipid to MSP ratios [0217] Nanodiscs were constructed using the same methods as Example 1. However, MSP1D1, was added to the sodium cholate lipid mixture at a lipid to MSP ratio of 10:1, 32.5:1, 50:1, and 65:1 for POPC lipids, and 20:1, 30:1, 40:1, 50:1, 60:1, and 85:1 for DMPC lipids. No lipid addition served as control (lipid free). [0218] As shown in FIG.2A, a POPC to MSP1D1 ratio of 65:1 resulted in a fully lipidated (10 nm diameter) nanodisc. As shown in FIG. 2B, at a molar ratio of 85:1 DMPC to MSP1D1, a fully lipidated nanodisc was formed. ny-2692819 Attorney Docket No.: 283912000140 Example 3: Exemplary nanodiscs alter ability to efflux cholesterol from macrophages in vitro [0219] Nanodiscs containing DMPC (Lipid A) or POPC (Lipid B) at different lipid concentrations were formed with MSP1D1 using the methods of Examples 1 and 2. J774 cells derived from a murine macrophage cell line were plated and radiolabeled with 74 kBq of 3H- cholesterol per millimeter. ABCA1 was up-regulated by means of a 6-hour incubation with 0.3 mM 8-(4-chlorophenylthio)-cAMP. Subsequently, the nanodiscs and efflux medium were added and incubated for 4 hours. [0220] All steps were performed in the presence of the acyl-coenzyme A cholesterol acyltransferase inhibitor CP113,818 (2 μg/mL). Liquid scintillation counting was used to quantify the efflux of radioactive cholesterol from the cells. The quantity of radioactive cholesterol incorporated into cellular lipids was calculated by means of isopropanol extraction of control wells not exposed to nanodiscs/media. The percentage of cholesterol efflux was calculated by the following formula: ^^^^^^^^^^ ^^ 3 ^ ^ℎ^^^^^^^^^ ! "^# ^" − ^^^^^^^^^^ ^^ 3 ^ ^ℎ^^^^^^^^^ ! ^^^^" ^^^^ "^# ^" %^^^ = 3 × 100 ^^^^^^^^ ^^ ^ ^ℎ^^^^^^^^^ ! ^^^^^ ^%^^&^^^# ^^^^^^ ^ℎ^ ^^^^^% ^^^' [0221] All experiments were performed in duplicate, and performed according to published literature (see Mehta, Nehal N et al. “Abnormal lipoprotein particles and cholesterol efflux capacity in patients with psoriasis.” Atherosclerosis vol.224,1 (2012): 218-21. doi:10.1016/j.atherosclerosis.2012.06.068, the content of which is incorporated herein by reference in its entirety). [0222] As shown in FIG.3, the POPC (Lipid B) containing nanodiscs with lipid to MSP1D1 ratios of 50:1 and 32.5:1 had higher cholesterol efflux percentage than fully lipidated POPC (Lipid B) nanodiscs with a lipid to MSP1D1 ratio of 65:1. Nanodiscs containing POPC at lipid to MSP1D1 ratios from 32.5:1 to 65:1 all demonstrated better cholesterol efflux properties compared to nanodiscs containing DMPC at lipid to MSP1D1 ratio of 85:1. Further, all tested nanodiscs demonstrated at least about 2-fold cholesterol efflux of that achieved by MSP1D1 protein per se. ny-2692819 Attorney Docket No.: 283912000140 Example 4: Exemplary nanodisc removes cholesterol from J774 murine macrophages in vitro [0223] A nanodisc with a DMPC to MSP1D1 ratio of 85:1 was formed using the methods of Examples 1 and 2. The methods of Example 3 were repeated to measure cholesterol efflux by such exemplary nanodisc in comparison to naturally occurring ApoA-I. As shown in FIG.4, the percent of cholesterol efflux with the exemplary nanodisc was more than two times that of naturally occurring ApoA-I. Example 5: Exemplary nanodisc removes cholesterol from human retinal pigment epithelial cells in vitro [0224] Nanodiscs with a POPC to MSP1D1 ratio of 65:1 (MSP1D1 Lipid B) and nanodiscs with a DMPC to MSP1D1 ratio of 85:1 (MSP1D1 Lipid A) were formed using the methods of Examples 1 and 2. The methods of measuring cholesterol efflux in Example 3 were conducted on human retinal pigment epithelial (RPE) cells (Neuromics) to measure cholesterol efflux at 2, 4 and 6 hours of incubation. Passive efflux (no protein or nanodisc added) and MSP1D1 protein alone were used for comparison. [0225] FIGs.5A-5C show percent cholesterol efflux from RPE cells after incubation for 2 hours (FIG.5A), 4 hours (FIG.5B), and 6 hours (FIG.5C). These results collectively demonstrate that nanodiscs with POPC to MSP1D1 ratio of 65:1 and nanodiscs with DMPC to MSP1D1 ratio of 85:1 are both capable of continuously promoting cholesterol efflux from RPE cells for at least 6 hours, which were significantly more than that achieved by MSP1D1 protein alone. Further, nanodiscs with DMPC to MSP1D1 ratio of 85:1 seemed to efflux cholesterol faster than nanodiscs with POPC to MSP1D1 ratio of 65:1 during 2-4 hour incubation window, but nanodiscs with POPC to MSP1D1 ratio of 65:1 caught up speed by 6 hour. Example 6: In vivo delivery of exemplary nanodisc in murine eyes [0226] To determine the penetrability and persistence of the exemplary nanodiscs in the eye in vivo, nanodiscs comprising MSP1D1 and DMPC in a molar ratio of 1:85 was prepared, with the MSP1D1 linked to a green fluorescent label via a cysteine residue engineered into the MSP1D1 protein. ny-2692819 Attorney Docket No.: 283912000140 [0227] The nanodiscs were prepared the day before the procedure and stored at 4°C. [0228] 6-week old C57Bl/6J mice were anesthetized with 87.6mg/kg Ketamine plus 10mg/kg Xylazine cocktail via IP injection and administered 0.5% proparacaine hydrochloride ophthalmic drops for pain management. The prepared nanodiscs were administered at a dose of 7 µg/eye in a volume of 1.14 µl via intravitreal injection. A subset of mice had the left eye dilated with 1% Tropicamide ophthalmic solution, followed by application of 2.5% Gonak hypromellose demulcent solution and noninvasive fundus imaging using Phoenix Micron III imaging system at various time points. At times indicated in FIGs. 6A-6B, mice were sacrificed and eyes were enucleated. Eyes were fixed in 10% Neutral buffered formalin for 2 hrs at room temperature (RT) before being transferred to graded sucrose (10% followed by 30%) overnight. The fixed, cryopreserved eyes were mounted in optimal cutting temperature (O.C.T.) and slides were prepared by cutting sections (20 μM) on a Lieca 1850 cryostat. Coverslips were applied using Vectasheild antifade mounting medium with Dapi to counterstain nuclei and to avoid additional processing steps associated with other counterstaining methods. The prepared slides were imaged on an Olympus FV1000 confocal microscope using system-optimized z step distances for each respective objective. This allowed for direct visualization of fluorescently labeled nanodiscs following the successful removal of cholesterol from the retina. [0229] As indicated by FIG. 6A and FIG. 6B, the nanodisc was successfully delivered to the murine eye via intravitreal injection, and the nanodisc was able to enter from the vitreous, got to the retinal space, into the RPE, and into the ocular muscle by 6 hours. By 24 hours, the disc appeared to have left the eye. These results indicate that penetrability of exemplary nanodiscs are excellent in vivo, and they do not dwell in the eye for an extended period of time (> 24 hours). Example 7: In vivo safety and efficacy of exemplary nanodisc in murine eyes [0230] To determine in vivo safety and efficacy of the nanodiscs in the eye, an exemplary nanodisc comprising MSP1D1 and DMPC at a molar ratio of 1:85 was prepared, with the MSP1D1 linked to a green fluorescent label via a cysteine residue engineered into the MSP1D1 protein. [0231] The fluorescence-labelled exemplary nanodisc was intravitreally delivered into the eyes of anesthetized mice, and its path and rate of diffusion within the eye was visualized by imaging ny-2692819 Attorney Docket No.: 283912000140 post-mortem retinal tissue along a 24-hour post-injection time course (FIG. 7A). As shown in FIG.7B, the fluorescence signal was detectable within 15 minutes of injection and diffused rapidly across the retina in a vitreous-to-RPE (retinal pigment epithelium) gradient, indicating that the nanodisc can be delivered into the eye and traffic through the retina effectively to target tissues. These results collectively demonstrated that the exemplary nanodisc can be distributed to RPE. [0232] Next, a model of ‘wet’ AMD mice was used, which had a laser-induced injury of the Bruch’s membrane to cause choroidal neovascularization (CNV). The exemplary nanodisc was intravitreally injected in tandem with, or 3 days after (‘+d3’), laser injury to examine the resulting CNV area size (FIG.8A). Importantly, the mean CNV area was similar across control and exemplary nanodisc -treated mice, suggesting that the exemplary nanodisc does not exacerbate prominent features of AMD in the mouse (FIG. 8B and FIG. 8C). These studies were key in showing that the exemplary nanodisc can be delivered efficiently to the retina and does not exacerbate features of neovascular AMD in the mouse, unlike the currently approved complement inhibitors for the treatment of geographic atrophy (GA). [0233] To determine whether the exemplary nanodisc could reduce lipid burden in the eye, a transgenic mouse species was implemented, which allows for specific deletion of Abca1 and Abcg1 cholesterol efflux transporters in rod photoreceptors (Abca1/g1-rod/-rod), resulting in lipid accumulation in the RPE and Bruch’s membrane and retinal neurodegeneration similar to patients with early to intermediate dry AMD. To accelerate lipid accumulation, the mice was provided with high-fat diet food for the duration of the study. As shown in FIG.9A, for the first cohort of mice (Study 1), baseline (T0) optical coherence tomography (OCT) fundus images of the mouse retina were collected prior to high-fat diet induction and intravitreal delivery of the exemplary nanodisc (‘Disc’ in FIG.9B and FIG.9C) or a blank injection (‘Vehicle’ in FIG.9B and FIG. 9C). A second intravitreal injection was administered 3 weeks later. After 6 weeks of high-fat diet exposure, follow-up OCT fundus imaging (T1) and blood sample collection were performed followed by electroretinography (ERG) visual function testing and tissue harvest. First, full-field scotopic and light bleach recovery ERG diagnostics that measure photoreceptor and RPE function, respectively, revealed no differences between vehicle and exemplary nanodisc-injected mice, demonstrating safety of the exemplary nanodisc on retinal electrical signaling (FIG. 9B and FIG.9C). Further confirmation of safety was demonstrated by ny-2692819 Attorney Docket No.: 283912000140 examination of retinal histology where retinal inflammation or differences in retinal integrity among mouse groups was the same (FIG.9D). Additionally, retina section immunostaining revealed complement 3 (C3) staining was detectable in the mouse retina but similarly expressed across vehicle and exemplary nanodisc-treated mice, indicating that the exemplary nanodisc did not exacerbate expected complement signaling in this dry AMD model (FIG.9E). RPE disruption and hypertransmission, ellipsoid zone abnormalities, inner retinal subsidence, and hyperreflective foci are primary anatomical changes linked to AMD progression are identified with noninvasive imaging. Thus, follow-up (T1) optical coherence tomography (OCT) images were examined for potential structural changes resulting from the genetic mutation and high-fat diet. As expected, numerous incidences of inner segment/outer segment (IS/OS) junction abnormalities and RPE disruptions including pigment epithelial detachments (PED) were found in images of vehicle-injected mice following 6 weeks of high-fat diet absent in baseline T0 images (FIG.9F). However, these abnormalities were rarely detected or absent in exemplary nanodisc-treated mouse eyes, indicating ameliorating features of AMD on OCT imaging. [0234] To replicate this study, and to further accentuate the high-fat diet-induced phenotype, a second cohort of mice (Study 2) on a high-fat diet for 6 weeks was tested but did not perform injections of the exemplary nanodisc or vehicle until 2- and 4-weeks after diet induction (FIG. 10A). At 6 weeks, visual function and again was evaluated, and no differences was found in full- field scotopic and light-bleach recovery responses between vehicle and exemplary nanodisc- treated mouse groups, confirming the safety of the nanodiscs (FIG. 10B and FIG.10C). In analyzing follow-up OCT imaging, several more lesions in the IS/OS junction and RPE were observed in vehicle-injected mice, but far fewer lesions were observed in mice injected with the exemplary nanodisc. Furthermore, greater numbers of lesions in both groups were observed compared to the first study, suggesting that exposing Abca1/g1-rod/-rod mice to a high-fat diet for 2 weeks prior to initial intravitreal injection worsened the disease (Table B-2 below, which summarizes cumulative incidence of retinal lesions in vehicle and exemplary nanodisc-treated mice via OCT imaging examination across two studies). Regardless, treatment of mice with the exemplary nanodisc resulted in reduced retinal lesions in both studies by nearly four times compared to vehicle. Table B-2 Cumulative incidence of retinal lesions ny-2692819 Attorney Docket No.: 283912000140
Figure imgf000088_0001
[0235] Lastly, lipidomics analyses were performed on retinal samples to examine potential changes in individual lipid species in the mouse eye following treatment with the exemplary nanodisc. Cholesteryl ester (CE) composition, a major component of drusen-causing AMD, was found to decrease in the retinas of nanodisc-treated mice, suggesting that the treatment reduces cholesterol load in diseased mice with features of AMD. Additionally, taken together with the improvement in OCT lesions, this reduction in cholesteryl ester, the main lipid species found in drusen, provides strong support of both the mechanism of action as well as scientific promise of the nanodisc provided herein. Example 8: Preparation of Exemplary Under-lipidated Nanodiscs [0236] Under-lipidated nanodiscs were prepared from a phospholipid, an MSP, and an organic compound following the protocol provided below. Materials [0237] MSP1D1(-) refers to MSP1D1 with the N-terminal Histidine affinity tag removed, which was used for the preparation herein. MSP1D1(-) comprises the sequence of SEQ ID NO: 14. MSP1D1 refers to the N-terminal Histidine affinity tag not-removed format, comprising SEQ ID NO: 3. Concentration was measured by UV-Vis Spectroscopy, for which Extinction Coefficient at 280 nm = 18450 M-1 (0.84/mg/ml). [0238] Phospholipid (e.g., DMPC, DPPC) was dissolved in chloroform and the concentration was measured by determination of total phosphorous (see, e.g., Chen, Toribara, and Warner (1956) Anal. Chem.28:1756-1758, the content of which is incorporated herein by reference in its entirety). [0239] Other reagents used for the preparation included Phosphate Buffered Saline (PBS), 200 mM Sodium Cholate detergent, Superdex S-200 Size Exclusion Column, methanol, ethyl acetate, ethanol, hexane, concentrated hydrochloric acid, and water. Preparation of DMPC/MSP1D1(-) Nanodisc ny-2692819 Attorney Docket No.: 283912000140 [0240] All manipulations were performed at room temperature (20 – 22°C). [0241] DMPC/chloroform stock was added to a glass tube so that the final ratio of DMPC to MSP1D1(-) in the preparation mixture was 30:1. DMPC was dried under a stream of nitrogen while rotating the glass tube, forming a thin film on the sides of the tube. The tube containing DMPC was then placed in a desiccator and vacuum was applied for a minimum of 4 hours, for instance, overnight. [0242] DMPC was then hydrated in water. To determine the amount of water needed, the desired final sodium cholate concentration was first determined. The final cholate to lipid ratio was determined to be greater than 2. One exemplary concentration was 17 mM cholate. The amount of water and 200 mM sodium cholate were determined such that the final concentration of Sodium Cholate was 17 mM Sodium Cholate. The amount of protein was then determined such that the final protein concentration was about 3.5 mg/ml. Only water was added to the dried DMPC at this time. [0243] The water/DMPC mixture was vortexed until the solution was milky white.200 mM sodium cholate was then added to the hydrated DMPC to give a final cholate concentration of 17 mM in the assembly mixture. The sodium cholate/DMPC mixture was fully solubilized by placing the tube in a sonicating water bath for 5 min, followed by gentle heating at ~ 40°C, repeated for 2 times. MSP1D1(-) was then added to the sodium cholate/DMPC mixture and mixed well, arriving at a preparation mixture with a protein concentration of about 3.5 mg/ml. The preparation mixture was incubated for 10 – 20 minutes at room temperature. [0244] The assembly of the nanodisc was initiated by addition of Amberlite XAD-2 resin, where Amberlite absorbed the cholate detergent. The amount of packed Amberlite XAD-2 resin gave at least 50% of the volume of the assembly mixture, for example, for a 5 ml preparation mixture, Amberlite resin was added to the preparation mixture until the volume was 7.5 ml. The assembly mixture containing Amberlite resin was gently agitated by placing on a platform shaker or a tube rotator for at least four hours at room temperature. The supernatant containing assembled nanodiscs was recovered from the Amberlite XAD resin using a pipette and placed in a new tube. The Amberlite XAD resin was washed 2 times with PBS, and the washing solution containing residual assembled nanodiscs was added to a sample tube. Large nanodiscs were removed from the sample by passing through a 0.2 micron filter. Depending on the nanodisc ny-2692819 Attorney Docket No.: 283912000140 sample volume, this could also be done by centrifugal filters (Amicon Ultra Free), or a syringe equipped with a 0.2 micron filter. [0245] Another under-lipidated nanodisc comprising DMPC and MSP1D1(-) was similarly made with molar ratio of DMPC to MSP1D1(-) in the preparation mixture of about 55:1. A third under-lipidated nanodisc comprising DMPC and MSP1D1(-) was similarly made with molar ratio of DMPC to MSP1D1(-) in the preparation mixture of about 40:1. [0246] Under-lipidated nanodisc comprising DMPC and MSP1E3D1(-) was similarly prepared with a different lipid to MSP molar ratio in the preparation mixture. The lipid to MSP molar ratios used for various DMPC/MSP1E3D1(-) compositions were also detailed in Example 12. MSP1E3D1(-) refers to MSP1E3D1 with the N-terminal Histidine affinity tag removed, which was used for the preparation herein. MSP1E3D1(-) comprises the sequence of SEQ ID NO: 15. MSP1E3D1 refers to the N-terminal Histidine affinity tag not-removed format, comprising SEQ ID NO: 8. To make under-lipidated nanodisc, the final ratio of DMPC to MSP1E3D1(-) in the preparation mixture was about 50:1. [0247] Another under-lipidated nanodisc comprising DMPC and MSP1E3D1(-) was similarly made with molar ratio of DMPC to MSP1E3D1(-) in the preparation mixture of about 90:1. [0248] Nanodiscs comprising other MSPs (or other phospholipid/MSP ratios were prepared following similar protocols detailed herein. Example 9: Purification of Exemplary Under-lipidated Nanodiscs [0249] The filtered samples containing DMPC/MSP1D1(-) nanodiscs or DMPC/MSP1E3D1(-) nanodiscs from Example 8 were further purified by size exclusion chromatography (SEC). Control nanodiscs were similarly purified. [0250] For the column: Superdex S-200 increase 10/300 (~25 ml bed volume) was used. Buffer was PBS. Flow rate was 0.75 ml/min. Injection Volume was 0.5 mL. [0251] The chromatogram of the preparative SEC of DMPC/MSP1D1(-) nanodisc (preparation mixture ratio of about 30:1) are shown in FIG.11A. As shown in FIG.11A, two peaks were isolated. The area on the right (dark grey) corresponding to a more under-lipidated (less lipid to protein ratio) nanodisc containing approximately 51 lipids per MSP1D1(-). The peak positioned at 20 minutes (see upper panel of FIG. 11A) is the residual lipid-free protein. ny-2692819 Attorney Docket No.: 283912000140 [0252] The chromatograms of the preparative SEC of DMPC/MSP1E3D1(-) nanodisc (preparation mixture ratio of about 50:1) are shown in FIG. 11B. As shown in FIG. 11B, a single highlighted peak was collected, which comprises an under-lipidated nanodisc with DMPC to MSP1E3D1(-) molar ratio of about 62:1. Example 10: Measurement of the Phospholipid to MSP Ratio [0253] This assay measured the phospholipid to MSP ratio by first extracting the phospholipid, followed by saponification and methylation of the fatty acids for rapid gas chromatography (GC) analysis. The measured quantity of fatty acids/ MSP was twice of that of the starting phospholipid. Since phospholipid DPPC was used as standard, 2 fatty acids per phospholipid was already taken into account. [0254] The phospholipid to MSP ratio in the nanodisc was determined following the procedures detailed below. [0255] The MSP concentration of the nanodisc described herein was measured by UV-Vis spectroscopy. Nanodisc sample was added to a 2 ml GC vial so that there was about 1 µg phospholipid. Each nanodisc sample was run in triplicate. The nanodisc sample was diluted to 100 µL with PBS. 10 µL of standard (DPPC) was added to each nanodisc sample at a known concentration. The total phospholipid was extracted following the modified Bligh and Dyer technique described in conditions E and E’ in the phase diagrams of FIG.7 in Int. J. Mol. Sci.2017, 18(4), 708. [0256] The extracted phospholipids were dried under a stream of nitrogen. The fatty acids were saponified and methylated simultaneously by dissolving the nanodisc sample in 100 µL of toluene.0.75 ml of methanol and 0.15 ml of 8% Hydrochloric Acid/Methanol solution were added to the mixture. (See J Lipid Res.2010; 51(3):635-40)). The mixture was incubated at 100°C for 1 hour. In other batches, the mixture was incubated at 45°C for overnight. [0257] 0.5 ml water and 0.5 hexane were then added to the mixture. The mixture was vortexed, top hexane layer was recovered for GC analysis. A standard curve of known concentrations of DMPC in chloroform was prepared by serial dilution, 10 µL of DPPC standard was added to each dilution, and saponification was performed as described in Section 176. J Lipid Res.2010; 51(3):635-40. ny-2692819 Attorney Docket No.: 283912000140 [0258] The DMPC content was determined from the standard curve by measuring the area ratio of the C14 Fatty Acid Methyl Ester (DMPC) to that of C16 Fatty Acid Methyl Ester (DPPC), and compared to the standard curve. [0259] From the UV-Vis spectroscopy-measured protein concentration and the DMPC concentration, a lipid to MSP ratio of the nanodisc was determined. [0260] An exemplary result generated by the GC method was shown in FIG. 12 using the DMPC/MSP1D1(-) nanodisc (preparation mixture ratio of about 30:1) from Example 8. In FIG. 12, DMPCfame is methyl myristate, and DPPCfame is methly palmitate, i.e., the methylated fatty acids derived from DMPC and DPPC, respectively. [0261] Based on the measurements, as detailed in FIG.11A, starting from a preparation mixture with DMPC to MSP1D1(-) molar ratio of about 30:1, an under-lipidated nanodisc with DMPC to MSP1D1(-) molar ratio of about 72:1 (hereinafter referred to as “DMPC:MSP1D1(-)=72:1 nanodisc”) and an under-lipidated nanodisc with DMPC to MSP1D1(-) molar ratio of about 51:1 (hereinafter referred to as “DMPC:MSP1D1(-)=51:1 nanodisc”) were obtained, wherein the yield of the DMPC:MSP1D1(-)=51:1 nanodisc was optimized. Therefore, this condition was further used to prepare the DMPC:MSP1D1(-)=51:1 nanodisc. [0262] Starting from a preparation mixture with DMPC to MSP1D1(-) molar ratio of about 55:1, an under-lipidated nanodisc with DMPC to MSP1D1(-) molar ratio of about 75:1 was obtained, hereinafter referred to as “DMPC:MSP1D1(-)=75:1 nanodisc.” This condition was further used to prepare the DMPC:MSP1D1(-)=75:1 nanodisc. [0263] Starting from a preparation mixture with DMPC to MSP1D1(-) molar ratio of about 40:1, an under-lipidated nanodisc with DMPC to MSP1D1(-) molar ratio of about 51:1 was obtained, hereinafter referred to as “DMPC:MSP1D1(-)=51:1 nanodisc.” This condition was further used to prepare the DMPC:MSP1D1(-)=51:1 nanodisc. [0264] Starting from a preparation mixture with DMPC to MSP1E3D1(-) molar ratio of about 50:1, an under-lipidated nanodisc with DMPC to MSP1E3D1(-) molar ratio of about 62:1 was obtained, hereinafter referred to as “DMPC:MSP1E3D1(-)=62:1 nanodisc.” ny-2692819 Attorney Docket No.: 283912000140 [0265] Starting from a preparation mixture with DMPC to MSP1E3D1(-) molar ratio of about 60:1, an under-lipidated nanodisc with DMPC to MSP1E3D1(-) molar ratio of about 63:1 was obtained, hereinafter referred to as “DMPC:MSP1E3D1(-)=63:1 nanodisc.” [0266] Starting from a preparation mixture with DMPC to MSP1E3D1(-) molar ratio of about 90:1, an under-lipidated nanodisc with DMPC to MSP1E3D1(-) molar ratio of about 93:1 was obtained, hereinafter referred to as “DMPC:MSP1E3D1(-)=93:1 nanodisc.” [0267] For one control, starting from a preparation mixture with DMPC to MSP1D1 molar ratio of about 85:1, a nanodisc (fully-lipidated) with DMPC to MSP1D1 molar ratio of about 83:1 was obtained, hereinafter referred to as “DMPC:MSP1D1=83:1 nanodisc.” [0268] For another control, starting from a preparation mixture with DMPC to MSP1D1(-) molar ratio of about 80:1, a nanodisc (fully-lipidated) with DMPC to MSP1D1(-) molar ratio of about 81:1 was obtained, hereinafter referred to as “DMPC:MSP1D1(-)=81:1 nanodisc.” Example 11: Correspondence between phospholipid/MSP1D1(-) ratio in the preparation mixture and the final nanodisc [0269] The correspondence between the phospholipid/MSP1D1(-) ratio in the preparation mixture and the final nanodisc was examined. Exemplified compositions comprising a plurality of DMPC/MSP1D1(-) nanodiscs were synthesized and purified following the procedures described in Examples 8 and 9, and measured for ratio as in Example 10. For the nanodisc started with DMPC/MSP1D1(-) preparation mixture ratio of 80:1, 55:1, and 40:1, the collected fraction from the first SEC was repurified with a second SEC (repurification). For the nanodisc started with DMPC/MSP1D1(-) preparation mixture ratio of 40:1, a third SEC purification (repurification 2) was further conducted. For the nanodisc started with DMPC/MSP1D1(-) preparation mixture ratio of 30:1, only one step of SEC was conducted. The lipid to protein ratio was measured in the final nanodisc after the final purification step. The results are shown in FIGs.13A-13D. As shown in FIG.13A, a starting DMPC/MSP1D1(-) ratio of 80:1 in the preparation mixture yielded nanodisc wherein the DMPC/MSP1D1(-) ratio was about 81:1. As shown in FIG. 13B, a starting DMPC/MSP1D1(-) ratio of 55:1 in the preparation mixture yielded nanodisc wherein the DMPC/MSP1D1(-) ratio was about 75:1. As shown in FIG.13C, a starting DMPC/MSP1D1(-) ratio of 40:1 in the preparation mixture yielded nanodisc wherein the ny-2692819 Attorney Docket No.: 283912000140 DMPC/MSP1D1(-) ratio was about 51:1. As shown in FIG.13D, a starting DMPC/MSP1D1(-) ratio of 30:1 in the preparation mixture yielded nanodisc wherein the DMPC/MSP1D1(-) ratio was about 51:1. In addition, the 30:1 (prep. ratio) DMPC to MSP1D1(-) was adequately isolated in a single purification step. These results collectively demonstrated that a low phospholipid/MSP ratio (e.g., a starting DMPC/MSP1D1(-) ratio of less than 81:1 (phospholipid- MSP saturation ratio NS of DMPC and MSP1D1) in the preparation mixture) could yield surprisingly stable under-lipidated compositions. Example 12: Correspondence between phospholipid/MSP1E3D1(-) ratio in the preparation mixture and the final nanodisc composition [0270] The correspondence between the phospholipid/MSP1E3D1(-) ratio in the preparation mixture and the final nanodisc composition was examined. Exemplified nanodiscs comprising a plurality of DMPC/MSP1E3D1(-) nanodiscs were synthesized and purified following the procedures described in Examples 8 and 9, and measured for ratio as in Example 10. Only one step of SEC was conducted. The lipid to protein ratio was measured in the final nanodisc after the final purification step. The results are shown in FIGs. 14A-14C. As shown in FIG.14A, a starting DMPC/MSP1E3D1(-) ratio of 90:1 in the preparation mixture yielded nanodisc wherein the DMPC/MSP1D1(-) ratio was about 93, such as 93±3. As shown in FIG.14B, a starting DMPC/MSP1E3D1(-) ratio of 60:1 in the preparation mixture yielded nanodisc wherein the DMPC/MSP1D1(-) ratio was about 63:1, such as 63±4 :1. As shown in FIG.14C, a starting DMPC/MSP1D1(-) ratio of 50:1 in the preparation mixture yielded nanodisc wherein the DMPC/MSP1D1(-) ratio was about 62:1, such as 62±0.5 :1. These results collectively demonstrated that a low phospholipid/MSP ratio (e.g., a starting DMPC/MSP1E3D1(-) ratio of less than 167:1 (phospholipid-MSP saturation ratio NS of DMPC and MSP1E3D1) in the preparation mixture) could yield surprisingly stable under-lipidated nanodiscs. Example 13: Cholesterol Efflux Assay [0271] The protocol for this assay was performed as described in the product information of Cholesterol Efflux Assay Kit (Cell-based) (ab196985). [0272] Briefly, J774 mouse macrophage cells were cultured and placed in a 96 well tissue culture plate. Cells were labeled with fluorescent cholesterol, rinsed, and incubated overnight. ny-2692819 Attorney Docket No.: 283912000140 The cells were then treated with media containing test nanodiscs generated as described in Examples 1-3 for 4 hours. The media containing test nanodiscs was recovered, and the cholesterol fluorescence was measured in the media (Fmedia). The cells were lysed and the fluorescence remaining in the cells (Fcells) was also measured. % Cholesterol Efflux was defined by: % Efflux = Fmedia/(Fmedia + Fcells) [0273] FIG. 15A and FIG. 15B show efflux assays performed on purified exemplary nanodiscs as described in Examples 8-10. The phospholipid to MSP ratios were ratios measured from the final nanodiscs. Besides fully-lipidated nanodisc controls, MSP1D1(-) (protein only, lipid free), Apo-AI (protein only, lipid free), and high-density lipoprotein (HDL) also served as controls. A positive control from the Cholesterol Efflux Assay Kit (ab196985) was included. PBS served as negative control (not shown). [0274] As shown in FIG.15A, when 2.5 μg of samples were used to incubate with cells labeled with fluorescent cholesterol, the under-lipidated DMPC:MSP1D1(-)=51:1 nanodiscs (e.g., nanodiscs shown in FIG.13D) showed much greater cholesterol efflux rate compared to nanodisc compositions with higher lipid content, e.g., under-lipidated DMPC:MSP1D1(-)=75:1 nanodiscs (e.g., nanodiscs shown in FIG.13B), and fully-lipidated DMPC:MSP1D1(-)=81:1 nanodiscs (e.g., nanodiscs shown in FIG.13A). FIG.15A further shows that removing N- terminal his-tag portion of the MSP protein did not affect cholesterol efflux (compare fully- lipidated DMPC:MSP1D1(-)=81:1 nanodiscs and fully-lipidated DMPC:MSP1D1=83:1 nanodiscs). The phospholipid-MSP saturation ratio NS of a nanodisc comprising DMPC and MSP1E3D1 (or MSP1E3D1(-)) is 167 (see Table 2). Hence the DMPC:MSP1E3D1(-)=62:1 nanodiscs and DMPC:MSP1E3D1(-)=94:1 nanodiscs are very under-lipidated. As shown in FIG. 15A, DMPC:MSP1E3D1(-)=62:1 nanodiscs also showed slightly better cholesterol efflux rate compared to DMPC:MSP1E3D1(-)=94:1 nanodiscs (with higher lipid content). The under- lipidated DMPC:MSP1D1(-)=51:1 nanodiscs, DMPC:MSP1E3D1(-)=62:1 nanodiscs, and DMPC:MSP1E3D1(-)=94:1 nanodiscs all showed much greater cholesterol efflux rate compared to lipid-free MSP1D1(-) protein control, Apo-AI control, and HDL control. FIG. 15B shows a significant increase in cholesterol efflux capacity with increasing dose of nanodiscs (10 µg vs. 2.5 μg). Surprisingly, 2.5 μg under-lipidated DMPC:MSP1D1(-)=51:1 nanodiscs even showed ny-2692819 Attorney Docket No.: 283912000140 stronger cholesterol efflux capacity compared to 10 μg fully-lipidated DMPC:MSP1D1(-)=81:1 nanodiscs, further demonstrating the technical superiority (e.g., cholesterol efflux capacity) of the nanodiscs provided herein, particularly the under-lipidated nanodiscs. Example 14: Stability of Under-Lipidated Nanodisc Compositions to Freeze thaw Cycles [0275] The stability of the prepared nanodiscs from Examples 8-10 was tested. One exemplary DMPC/MSP1D1(-) nanodisc composition tested was the right area portion of the chromatogram peak in FIG. 11A, corresponding to an under-lipidated DMPC:MSP1D1(-)=51:1 nanodiscs. The sample was frozen at -80°C with or without 10% glycerol as a cryoprotectant. Then the frozen samples were removed from -80°C storage and thawed rapidly. Samples were subjected to SEC and compared to the chromatograms taken prior to freezing. The results are shown in FIG.16, indicating that the chromatograms of samples being freeze-and-thawed (without glycerol) and pre-freezing are nearly identical. Chromatograms were also nearly identical after a freeze-thaw cycle between samples frozen with or without cryoprotectant (data not shown). These results indicate that the prepared under-lapidated nanodiscs had surprisingly high stability when subject to freeze-and-thaw cycles. [0276] A freeze-thaw cycle on the under-lipidated nanodiscs had no effect on cholesterol efflux efficiency either. The comparison of cholesterol efflux by the under-lipidated DMPC:MSP1D1(- )=51:1 nanodiscs before and after freeze-and-thaw is shown in FIG.17. As shown in FIG. 17, the cholesterol efflux efficiency of the under-lipidated nanodiscs remained high after a freeze- and-thaw cycle (second column), and remained superior compared to fully-lipidated nanodiscs control with higher DMPC content (DMPC:MSP1D1=83:1 nanodiscs), as well as higher than lipid-free HDL and blank control PBS solution. ny-2692819

Claims

Attorney Docket No.: 283912000140 CLAIMS 1. A method of removing lipid or preventing lipid accumulation in the eye of an individual, comprising administering to the eye of the individual an effective amount of a pharmaceutical composition comprising a plurality of nanodiscs, wherein the nanodisc comprises a membrane scaffold protein (MSP) and a phospholipid. 2. A method of preventing or treating an eye disease characterized by lipid accumulation in an individual, comprising administering to the eye of the individual an effective amount of a pharmaceutical composition comprising a plurality of nanodiscs, wherein the nanodisc comprises an MSP and a phospholipid. 3. The method of claim 1 or 2, wherein the MSP is selected from the group consisting of apolipoprotein A-I (ApoA-I), ApoA-II, ApoC, ApoE, ApoM, and a variant thereof. 4. The method of any one of claims 1-3, wherein the MSP is a variant of ApoA-I. 5. The method of claim 4, wherein the MSP comprises one or more amphipathic helix sequences derived from ApoA-I. 6. The method of claim 4 or 5, wherein the MSP comprises an N-terminus truncation relative to ApoA-I. 7. The method of claim 5 or 6, wherein the MSP is selected from the group consisting of MSP1E1, MSP1E2, MSP1E3, MSP1, MSP1D1, MSP1E1D1, MSP1E2D1, MSP1E3D1, MSP2N2, and N-terminal His-tag portion removed variant thereof (His-tag removed variant). 8. The method of claim 7, wherein the MSP is: (i) an MSP1D1 His-tag removed variant comprising the amino acid sequence of SEQ ID NO: 14; or (ii) an MSP1E3D1 His-tag removed variant comprising the amino acid sequence of SEQ ID NO: 15. 9. The method of any one of claims 1-8, wherein the phospholipid comprises DMPC, DPPC, DMPS, DSPC, POPC, POPS, PiP2, or a combination thereof. ny-2692819 Attorney Docket No.: 283912000140 10. The method of any one of claims 1-9, wherein the phospholipid is a phosphatidylcholine (PC). 11. The method of claim 10, wherein the phospholipid is selected from the group consisting of DMPC, POPC, DPPC, DSPC, and a combination thereof. 12. The method of any one of claims 1-11, wherein at least about 85% of the plurality of nanodiscs in the pharmaceutical composition are fully-lipidated. 13. The method of any one of claims 1-11, wherein at least about 85% of the plurality of nanodiscs in the pharmaceutical composition are under-lipidated. 14. The method of claim 13, wherein the molar ratio of the phospholipid to the MSP in the pharmaceutical composition is less than a phospholipid-MSP saturation ratio NS. 15. The method of claim 14, wherein the molar ratio of the phospholipid to the MSP in the pharmaceutical composition is less than about 95% of the phospholipid-MSP saturation ratio NS. 16. The method of any one of claims 1-15, wherein the MSP is MSP1D1, MSP1, or His-tag removed variant thereof, and wherein the molar ratio of the phospholipid to the MSP in the pharmaceutical composition is from about 10:1 to about 90:1. 17. The method of claim 16, wherein the MSP is MSP1D1, MSP1, or His-tag removed variant thereof, wherein the phospholipid is POPC, and wherein the molar ratio of POPC to the MSP in the pharmaceutical composition is from about 10:1 to about 65:1. 18. The method of claim 17, wherein the MSP is MSP1D1 or His-tag removed variant thereof, and wherein the molar ratio of POPC to the MSP in the pharmaceutical composition is about 65:1. 19. The method of claim 17, wherein the MSP is MSP1D1 or His-tag removed variant thereof, the phospholipid is POPC, and the NS is about 62. 20. The method of claim 19, wherein the molar ratio of POPC to the MSP in the pharmaceutical composition is less than about 58:1. 21. The method of claim 20, wherein the molar ratio of POPC to the MSP in the pharmaceutical composition is about 50:1 or about 32.5:1. ny-2692819 Attorney Docket No.: 283912000140 22. The method of claim 16, wherein the MSP is MSP1D1, MSP1, or His-tag removed variant thereof, wherein the phospholipid is DMPC, and wherein the molar ratio of DMPC to the MSP in the pharmaceutical composition is from about 10:1 to about 85:1. 23. The method of claim 22, wherein the MSP is MSP1D1 or His-tag removed variant thereof, and wherein the molar ratio of DMPC to the MSP in the pharmaceutical composition is about 85:1. 24. The method of claim 22, wherein the MSP is MSP1D1 or His-tag removed variant thereof, the phospholipid is DMPC, and the NS is about 81. 25. The method of claim 24, wherein the molar ratio of DMPC to the MSP in the pharmaceutical composition is less than about 78:1. 26. The method of claim 25, wherein the molar ratio of DMPC to the MSP in the pharmaceutical composition is about 65:1 to about 75:1. 27. The method of claim 25, wherein the molar ratio of DMPC to the MSP in the pharmaceutical composition is from about 40:1 to about 60:1. 28. The method of claim 25 or 27, wherein the molar ratio of DMPC to the MSP in the pharmaceutical composition is about 46:1 to about 55:1. 29. The method of claim 22, wherein the MSP is MSP1 or His-tag removed variant thereof, and wherein the molar ratio of DMPC to the MSP in the pharmaceutical composition is about 80:1. 30. The method of claim 16, wherein the MSP is MSP1D1, MSP1, or His-tag removed variant thereof, wherein the phospholipid is DPPC, and wherein the molar ratio of DPPC to the MSP in the pharmaceutical composition is from about 10:1 to about 90:1. 31. The method of claim 30, wherein the MSP is MSP1 or His-tag removed variant thereof, the phospholipid is DPPC, and wherein the molar ratio of DPPC to the MSP in the pharmaceutical composition is about 90:1. 32. The method of claim 30, wherein the MSP is MSP1D1 or His-tag removed variant thereof, the phospholipid is DPPC, and the NS is about 81. ny-2692819 Attorney Docket No.: 283912000140 33. The method of claim 32, wherein the molar ratio of DPPC to the MSP in the pharmaceutical composition is less than about 75:1. 34. The method of any one of claims 1-15, wherein the MSP is MSP1E3D1, MSP1E3, or His-tag removed variant thereof, and wherein the molar ratio of the phospholipid to the MSP in the pharmaceutical composition is from about 50:1 to about 200:1. 35. The method of claim 34, wherein the MSP is MSP1E3 or His-tag removed variant thereof, wherein the phospholipid is POPC, and wherein the molar ratio of POPC to the MSP in the pharmaceutical composition is about 130:1. 36. The method of claim 34, wherein the MSP is MSP1E3D1 or His-tag removed variant thereof, the phospholipid is POPC, and the NS is about 126. 37. The method of claim 36, wherein the molar ratio of POPC to the MSP in the pharmaceutical composition is less than about 118:1. 38. The method of claim 34, wherein the MSP is MSP1E3D1 or His-tag removed variant thereof, the phospholipid is DMPC, and the NS is about 167. 39. The method of claim 38, wherein the molar ratio of DMPC to the MSP in the pharmaceutical composition is less than about 158:1. 40. The method of claim 39, wherein the molar ratio of DMPC to the MSP in the pharmaceutical composition is from about 50:1 to about 160:1. 41. The method of claim 40, wherein the molar ratio of DMPC to the MSP in the pharmaceutical composition is from about 50:1 to about 100:1. 42. The method of claim 41, wherein the molar ratio of DMPC to the MSP in the pharmaceutical composition is about 87:1 to about 96:1, about 58:1 to about 68:1, about 63:1, or about 62:1. 43. The method of claim 34, wherein the MSP is MSP1E3 or His-tag removed variant thereof, the phospholipid is DMPC, and wherein the molar ratio of DMPC to the MSP in the pharmaceutical composition is about 150:1. ny-2692819 Attorney Docket No.: 283912000140 44. The method of claim 34, wherein the MSP is MSP1E3 or His-tag removed variant thereof, the phospholipid is DPPC, and wherein the molar ratio of DPPC to the MSP in the pharmaceutical composition is about 170:1. 45. The method of claim 34, wherein the MSP is MSP1E3D1 or His-tag removed variant thereof, the phospholipid is DPPC, and the NS is about 167. 46. The method of claim 45, wherein the molar ratio of DPPC to the MSP in the pharmaceutical composition is less than about 158:1. 47. The method of any one of claims 1-15, wherein the MSP is MSP1E1D1 or His-tag removed variant thereof, the phospholipid is DMPC, and the NS is about 107. 48. The method of claim 47, wherein the molar ratio of DMPC to the MSP in the pharmaceutical composition is less than about 100:1. 49. The method of claim 48, wherein the molar ratio of DMPC to the MSP in the pharmaceutical composition is from about 60:1 to about 65:1. 50. The method of any one of claims 1-49, wherein the nanodisc in the pharmaceutical composition is about 2 nm to about 20 nm in diameter. 51. The method of any one of claims 1-50, wherein the lipid for removal or prevention from accumulation in the eye is cholesterol. 52. The method of any one of claims 1-51, wherein the nanodisc does not comprise (i) a cell-penetrating peptide (CPP); (ii) a target protein; and/or (iii) a prophylactic or therapeutic agent. 53. The method of 51 or 52, wherein the nanodisc increases cholesterol efflux rate by at least about 2-fold compared to an untreated state, or the cholesterol efflux rate by ApoA-I or the MSP. 54. The method of any one of claims 1-53, wherein the pharmaceutical composition is administered via intravitreal injection. 55. The method of any one of claims 2-54, wherein the eye disease characterized by lipid accumulation is selected from the group consisting of age-related macular degeneration ny-2692819 Attorney Docket No.: 283912000140 (AMD), photoreceptor neurodegeneration, optic nerve atrophy, loss of acuity, hemianopia, visual agnosia, strabismus, retinal neurovascular disorder, lipid keratopathy, corneal lipidosis, and a combination thereof. 56. The method of claim 55, wherein the eye disease characterized by lipid accumulation is AMD. 57. The method of claim 56, wherein the AMD is dry AMD. 58. The method of claim 57, wherein the dry AMD is geographic atrophy (GA). 59. The method of any one of claims 1-58, wherein the nanodisc reduces lipid or prevents lipid accumulation in the eye of the individual by at least about 5%. ny-2692819
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