Nanodisc for removing lipid accumulation in eye

Abstract

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

Description

[0001] Cross-reference to related applications

[0002] This application claims priority to U.S. Provisional Patent Application No. 63 / 450,945, filed March 8, 2023, and U.S. Provisional Patent Application No. 63 / 610,922, filed December 15, 2023, the entire contents of each of which are incorporated herein by reference.

[0003] Reference to electronic sequence listing

[0004] The contents of the electronic serial number (283912000140SEQLIST.xml; size: 24,766 bytes; and creation date: March 7, 2024) are incorporated herein by reference in their entirety. Technical Field

[0005] This disclosure generally relates to methods for removing lipids (e.g., cholesterol) from the eyes of an individual (e.g., a human) or preventing lipid accumulation in the eyes of an individual, and methods for preventing or treating eye diseases in an individual characterized by lipid accumulation. This disclosure also provides pharmaceutical compositions comprising a plurality of nanodiscs, and methods for using and manufacturing them. Background Technology

[0006] Nanodisks containing membrane scaffold proteins (MSPs) and phospholipids provide a natural lipid-like environment for the incorporation of membrane proteins, and they have become a valuable platform for membrane biophysics research and therapeutic applications.

[0007] The accumulation of lipids (e.g., cholesterol) has become a major cause of many eye diseases, including neurodegeneration, entropy, macular degeneration, and inflammation in the eye. Among these diseases, age-related macular degeneration (AMD) affects approximately 14% to 24% of people aged 65 to 74 and approximately 35% of those over 75, affecting about 200 million people worldwide, and has become a leading cause of legal blindness in developed countries. Despite progress in mechanistic studies of the role of lipids in these eye diseases, the development of effective methods to remove lipids or prevent their accumulation in the eye remains an unmet need. This application addresses this and other needs. Summary of the Invention

[0008] In one aspect, this article provides a method for removing lipids from or preventing lipid accumulation in the eye of an individual, the method comprising administering to the eye of the individual an effective amount of a pharmaceutical composition comprising a plurality of nanodiscs, wherein the nanodiscs comprise membrane scaffold proteins (MSPs) and phospholipids.

[0009] In one aspect, provided herein is a method of preventing or treating an eye disease characterized by lipid accumulation in an individual, the method comprising administering to the eye of the individual an effective amount of a pharmaceutical composition comprising a plurality of nanodiscs, wherein the nanodiscs comprise a MSP and a phospholipid.

[0010] In some embodiments, the MSP is selected from the group consisting of apolipoprotein A-I (ApoA-I), ApoA-II, ApoC, ApoE, ApoM, and variants 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 a truncation relative to the N-terminus 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 variants thereof that are free of the N-terminal His-tag portion (His-tag free variants). In some embodiments, the MSP is: (i) a His-tag free variant of MSP1D1 comprising the amino acid sequence of SEQ ID NO: 14; or (ii) a His-tag free variant of MSP1E3D1 comprising the amino acid sequence of SEQ ID NO: 15.

[0011] In some embodiments, the phospholipid comprises DMPC, DPPC, DMPS, DSPC, POPC, POP S, PiP2, or a combination thereof. In some embodiments, the phospholipid is phosphatidylcholine (PC). In some embodiments, the phospholipid is selected from the group consisting of DMPC, POPC, DPPC, DSPC, and combinations thereof.

[0012] 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 embodiments, the molar ratio of phospholipid to MSP in the pharmaceutical composition is less than the phospholipid-MSP saturation ratio N S In some embodiments, the molar ratio of phospholipid to MSP in the pharmaceutical composition is less than the phospholipid-MSP saturation ratio N S about 95%.

[0013] In some embodiments, the MSP is MSP1D1, MSP1, or a His-tag removed variant thereof, and the molar ratio of phospholipid to MSP in the pharmaceutical composition is about 10: 1 to about 90: 1. In some embodiments, the MSP is MSP1D1, MSP1, or a His-tag removed variant thereof, wherein the phospholipid is POPC, and the molar ratio of POPC to MSP in the pharmaceutical composition is about 10: 1 to about 65: 1. In some embodiments, the MSP is MSP1D1, or a His-tag removed variant thereof, and the molar ratio of POPC to MSP in the pharmaceutical composition is about 65: 1. In some embodiments, the MSP is MSP1D1, or a His-tag removed variant thereof, the phospholipid is POPC, and the N S In some embodiments, the molar ratio of POPC to MSP in the pharmaceutical composition is less than about 58: 1. In some embodiments, the molar ratio of POPC to MSP in the pharmaceutical composition is about 50: 1 or about 32.5: 1.

[0014] In some embodiments, the MSP is MSP1D1, MSP1, or a His-tag removed variant thereof, wherein the phospholipid is DMPC, and the molar ratio of DMPC to MSP in the pharmaceutical composition is about 10: 1 to about 85: 1. In some embodiments, the MSP is MSP1D1, or a His-tag removed variant thereof, the phospholipid is DMPC, and the molar ratio of DMPC to MSP in the pharmaceutical composition is about 85: 1. In some embodiments, the MSP is MSP1D1, or a His-tag removed variant thereof, the phospholipid is DMPC, and the N S In some embodiments, the molar ratio of DMPC to MSP in the pharmaceutical composition is less than about 78: 1. In some embodiments, the molar ratio of DMPC to MSP in the pharmaceutical composition is about 65: 1 to about 75: 1. In some embodiments, the molar ratio of DMPC to MSP in the pharmaceutical composition is about 40: 1 to about 60: 1. In some embodiments, the molar ratio of DMPC to MSP in the pharmaceutical composition is about 46: 1 to about 55: 1. In some embodiments, the MSP is MSP1, or a His-tag removed variant thereof, and the molar ratio of DMPC to MSP is about 80: 1.

[0015] In some embodiments, the MSP is MSP1D1, MSP1, or a His-label-removed variant thereof, the phospholipid is DPPC, and the molar ratio of DPPC to MSP in the pharmaceutical composition is from about 10:1 to about 90:1. In some embodiments, the MSP is MSP1 or a His-label-removed variant thereof, the phospholipid is DPPC, and the molar ratio of DPPC to MSP in the pharmaceutical composition is from about 90:1. In some embodiments, the MSP is MSP1D1 or a His-label-removed variant thereof, the phospholipid is DPPC, and N... S The molar ratio is approximately 81. In some embodiments, the molar ratio of DPPC to MSP in the pharmaceutical composition is less than approximately 75:1.

[0016] In some embodiments, the MSP is MSP1E3D1, MSP1E3, or a His-label-removed variant thereof, and the molar ratio of phospholipid to MSP in the pharmaceutical composition is from about 50:1 to about 200:1. In some embodiments, the MSP is MSP1E3 or a His-label-removed variant thereof, wherein the phospholipid is POPC, and the molar ratio of POPC to MSP in the pharmaceutical composition is from about 130:1. In some embodiments, the MSP is MSP1E3D1 or a His-label-removed variant thereof, the phospholipid is POPC, and N S The molar ratio is approximately 126. In some embodiments, the molar ratio of POPC to MSP in the pharmaceutical composition is less than approximately 118:1. In some embodiments, the MSP is MSP1E3D1 or a variant thereof with the His tag removed, the phospholipid is DMPC, and N... S The molar ratio of DMPC to MSP in the pharmaceutical composition is approximately 167. In some embodiments, the molar ratio of DMPC to MSP in the pharmaceutical composition is less than approximately 158:1. In some embodiments, the molar ratio of DMPC to MSP in the pharmaceutical composition is approximately 50:1 to approximately 160:1. In some embodiments, the molar ratio of DMPC to MSP in the pharmaceutical composition is approximately 50:1 to approximately 100:1. In some embodiments, the molar ratio of DMPC to MSP in the pharmaceutical composition is approximately 87:1 to approximately 96:1, approximately 58:1 to approximately 68:1, approximately 63:1, or approximately 62:1. In some embodiments, the MSP is MSP1E3 or a His-label-removed variant thereof, the phospholipid is DMPC, and the molar ratio of DMPC to MSP in the pharmaceutical composition is approximately 150:1. In some embodiments, the MSP is MSP1E3 or a His-label-removed variant thereof, the phospholipid is DPPC, and the molar ratio of DPPC to MSP in the pharmaceutical composition is approximately 170:1. In some implementations, the MSP is MSP1E3D1 or a variant thereof with the His tag removed, the phospholipid is DPPC, and N Sabout 167. In some embodiments, the molar ratio of DPPC to MSP in the pharmaceutical composition is less than about 158: 1.

[0017] In some embodiments, the MSP is MSP1E1D1 or a His-tag removed variant thereof, the phospholipid is DMPC, and the N S about 107. In some embodiments, the molar ratio of DMPC to MSP in the pharmaceutical composition is less than about 100: 1. In some embodiments, the molar ratio of DMPC to MSP in the pharmaceutical composition is about 60: 1 to about 65: 1.

[0018] In some embodiments, the diameter of the nanodisc in the pharmaceutical composition is about 2 nm to about 20 nm. In some embodiments, the lipid used to remove or prevent 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.

[0019] In some embodiments, the nanodisc increases the rate of cholesterol efflux by at least about 2-fold compared to the untreated state or the rate of cholesterol efflux achieved 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 accumulation of lipid is selected from the group consisting of age-related macular degeneration (AMD), photoreceptor neurodegeneration, optic nerve atrophy, vision loss, hemianopsia, visual agnosia, strabismus, retinal neurovascular disorders, lipid keratopathy, corneal lipidosis, and combinations thereof. In some embodiments, the eye disease characterized by accumulation of lipid 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 in the eye of the individual by at least about 5% or prevents accumulation of lipid in the eye of the individual. BRIEF DESCRIPTION OF DRAWINGS

[0020] The accompanying drawings illustrate certain embodiments of the features and advantages of the present disclosure. These embodiments are not intended to limit the scope of the appended claims in any way.

[0021] Figure 1 Exemplary chromatograms showing nanodisc preparation are shown.

[0022] Figure 2A Chromatograms showing POPC MSP1D1 nanodisc formation at different POPC to MSP1D1 ratios are shown. The results indicate that a ratio of 65: 1 forms fully lipidated nanodiscs. Figure 2BA chromatogram showing DMPC MSP1D1 nanodisc formation at different ratios of DMPC to MSP1D1 is shown. The results indicate that fully lipidated nanodiscs form at a ratio of 85: 1 DMPC to MSP1D1.

[0023] Figure 3 A percentage of cholesterol efflux from macrophages in vitro from various nanodiscs using different ratios of lipid to MSP is shown. Lipid A refers to DMPC and lipid B refers to POPC.

[0024] Figure 4 It is shown that DMPC MSP1D1 nanodiscs with a ratio of lipid to MSP of 85: 1 have the ability to remove almost twice as much cholesterol from macrophages as naturally occurring ApoA-I after 4 hours of incubation.

[0025] Figures 5A-5C A percentage of cholesterol efflux from human retinal pigment epithelial (RPE) cells in vitro using POPC nanodiscs with a ratio of POPC to MSP1D1 of 65: 1 (MSP1D1 lipid B), DMPC nanodiscs with a ratio of DMPC to MSP1D1 of 85: 1 (MSP1D1 lipid A), and MSP1D1 protein alone after 2 hours (top row), Figure 5A ), 4 hours (middle row), Figure 5B ), and 6 hours (bottom row) Figure 5C of incubation is shown.

[0026] Figure 6A and Figure 6B Fluorescence images are shown after intravitreal delivery of GFP-labeled exemplary nanodiscs in vivo into a mouse eye. From left to right, Figure 6A Fluorescence images are shown at 15 minutes, 30 minutes, 60 minutes, 6 hours, and 24 hours after delivery of GFP-labeled exemplary nanodiscs in vivo into a mouse eye. Figure 6B The first row from left to right in shows fluorescence images at 7 minutes, 7 minutes, 10 minutes, 15 minutes, and 20 minutes after delivery of GFP-labeled exemplary nanodiscs in vivo into a mouse eye. Figure 6B The second row from left to right in shows fluorescence images at 30 minutes, 30 minutes, 33 minutes, and 47 minutes after delivery of GFP-labeled exemplary nanodiscs in vivo into a mouse eye. Figure 6B The third row from left to right in shows fluorescence images at 6 hours, 6 hours, and 24 hours after delivery of GFP-labeled exemplary nanodiscs in vivo into a mouse eye.

[0027] Figure 7A is a schematic of a timeline depicting intravitreal injection and tissue harvesting of fluorescently labeled exemplary nanodiscs. Figure 7BRepresentative images of immunostained retinal sections taken from injected mice are shown, depicting retinal neurons (solid arrows) and fluorescently labeled exemplary nanodisks (open arrows). Figure 7B Fluorescence images at 0 minutes, 15 minutes, 30 minutes, 1 hour, 6 hours, and 24 hours after delivery of fluorescently labeled exemplary nanodisks into the mouse eye are shown from left to right. In Figure 7B In the middle, the image shows fluorescence signal at the retinal pigment epithelium (RPE), outer nuclear layer (ONL), and inner nuclear layer (INL).

[0028] Figure 8A is a schematic depicting the timeline of laser injury-induced choroidal neovascularization (CNV), intravitreal injection of exemplary nanodisks, intravenous injection of FITC dextran, and tissue harvest. Figure 8B Representative confocal images of CNV regions labeled by FITC dextran and outlined in circles after laser injury in mice are shown. Figure 8C Dot plots showing CNV area in mice receiving intravitreal injection of exemplary nanodisks as 0 (“Cntl”), 1 (“Nanodisks”), or 2 (“Nanodisks + d3”) are shown. Data (n = 8 to 10 mice / group) are presented as mean (± SEM).

[0029] Figure 9A is a schematic depicting the timeline of high-fat diet administration, intravitreal injection, baseline (TO) and follow-up (Tl) imaging, and tissue harvest of Abca1 / g1-rod / -rod mice. Figure 9B and Figure 9C Line plots showing full-field scotopic (A) Figure 9B ) and mild bleaching recovery (B) Figure 9C ) response amplitudes and ratios in vehicle (“Vehicle”) and exemplary nanodisk-treated (“Disk”) mouse groups are shown. Data (Vehicle, n = 3; Disk, n = 4) are presented as mean (± SEM). Figure 9D Representative images of hematoxylin and eosin-stained (H&E) sections of the entire eye (left) and retina (right) are shown. Figure 9E Representative images of immunostained retinal sections showing retinal neurons (solid arrows) and complement-3 (open arrows) expression in the central (left) and intermediate peripheral (right) retina are shown. Figure 9FRepresentative baseline (top) and follow-up (bottom) optical coherence tomography (OCT) images of a live mouse retinal cross-section showing the incidence of an intra- (asterisk and arrow in top image) and extra-foveal abnormality (arrow in lower left image) and absence of an intra-foveal abnormality (arrow in lower right image) and retinal pigment epithelial detachment (arrow in lower right image) from baseline (top image).

[0030] Figure 10A A schematic diagram depicting the schedule of high-fat diet administration, intravitreal injection, baseline (TO) and follow-up (Tl) imaging and blood collection, and tissue harvest of Abca1 / g1-rod / -rod mice is shown. Figure 10B and Figure 10C A line graph showing the full-field scotopic (A) and mild bleaching recovery (B) response amplitudes and ratios in vehicle (“Vehicle”) and exemplary nanodisc-treated (“Disc”) mouse groups is shown. Figure 10B ) and mild bleaching recovery (B) response amplitudes and ratios in vehicle (“Vehicle”) and exemplary nanodisc-treated (“Disc”) mouse groups is shown. Figure 10C The y-axis shows amplitude (pV). Data (Vehicle, n=3; Disc, n=4) is presented as mean (±SEM). Figure 10D A box plot showing the cholesterol ester (CE) proportion of retinal lipids measured in vehicle-treated and nanodisc-treated mice is shown. Data (Vehicle, n=3; Disc, n=4) is presented as median with interquartile range, minimum, and maximum indicated.

[0031] Figure 11A A chromatogram of preparative size exclusion chromatography (SEC) of exemplary DMPC / MSP1D1(-) nanodiscs prepared at a 30: 1 ratio is shown. (-) indicates removal of His tag from MSP1D1. The dark gray shaded region of the peak (upper panel) corresponds to under-lipidated nanodiscs with approximately 51 lipids per MSP1D1(-) (lower panel). Figure 11B A chromatogram of preparative SEC of exemplary DMPC / MSP1E3D1(-) nanodiscs prepared at a 50: 1 ratio is shown. The single highlighted peak (upper panel) was collected, which corresponds to under-lipidated nanodiscs with approximately 62 lipids per MSP1E3D1(-).

[0032] Figure 12 Gas chromatography (GC) results for determining the phospholipid to MSP ratio of exemplary DMPC / MSP1D1(-) nanodiscs prepared at a 30: 1 ratio are shown.

[0033] Figures 13A-13D Nanodiscs for DMPC / MSP1D1(-) are about 81 : 1 (preparation mixture 80: 1; final nanodiscs 81 ± 3: 1) Figure 13Ananodiscs (preparation mix 55:1; final nanodiscs 75 ± 1.5:1) of DMPC / MSP1D1(-) at about 75:1 (preparation mix 55:1; final nanodiscs 75 ± 1.5:1) Figure 13B nanodiscs (preparation mix 40:1; final nanodiscs 51 ± 1:1) of DMPC / MSP1D1(-) at about 51:1 (preparation mix 40:1; final nanodiscs 51 ± 1:1) Figure 13C nanodiscs (preparation mix 30:1; final nanodiscs 51 ± 1:1) of DMPC / MSP1D1(-) at about 51:1 (preparation mix 30:1; final nanodiscs 51 ± 1:1) Figure 13D correspondence between the phospholipid / MSP1D1(-) ratio in the preparation mix and the final nanodiscs.

[0034] Figures 14A-14C nanodiscs (preparation mix 90:1; final nanodiscs 93 ± 3:1) of DMPC / MSP1E3D1(-) at about 93:1 (preparation mix 90:1; final nanodiscs 93 ± 3:1) Figure 14A nanodiscs (preparation mix 60:1; final nanodiscs 63 ± 4:1) of DMPC / MSP1E3D1(-) at about 63:1 (preparation mix 60:1; final nanodiscs 63 ± 4:1) Figure 14B nanodiscs (preparation mix 50:1; final composition 62 ± 0.5:1) of DMPC / MSP1E3D1(-) at about 62:1 (preparation mix 50:1; final composition 62 ± 0.5:1) Figure 14C correspondence between the phospholipid / MSP1E3D1(-) ratio in the preparation mix and the final nanodiscs.

[0035] Figure 15A Cholesterol efflux assay results using various under-lipidated nanodiscs are shown. The phospholipid to MSP ratio was measured from the final nanodiscs (not the preparation mix). 2.5 μg of sample was used. Controls included fully lipidated nanodiscs of DMPC:MSP1D1(-)=81:1, fully lipidated nanodiscs of DMPC:MSP1D1=83:1, lipid-free MSP1D1(-), lipid-free Apo-AI, lipid-free HDL, a positive control (from the assay kit), and PBS buffer blank (not shown). Figure 15 shows the cholesterol efflux dose dependence for 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 removal of the N-terminal His tag.

[0036] Figure 16 SEC chromatogram of exemplary under-lipidated DMPC / MSP1D1(-) nanodiscs prepared at a 30:1 ratio (final composition ratio of about 51:1) after freezing and thawing without cryoprotectant, compared to before freezing.

[0037] Figure 17Cholesterol efflux results are shown for exemplary under-lipidated DMPC:MSP1D1(-) nanodiscs prepared at a 30:1 ratio (final composition ratio of about 51:1) compared to controls including fully lipidated DMPC:MSP1D1 nanodiscs prepared at 85:1 (final composition ratio of 83:1), HDL, and PBS solution, before and after freezing and thawing. DETAILED DESCRIPTION

[0038] The present application provides a method of removing or preventing the accumulation of a lipid (e.g., cholesterol) in an eye of an individual (e.g., a human), and / or preventing or treating an eye disease characterized by accumulation of a lipid in an individual, the method 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 application is based, at least in part, on the surprising finding that the nanodiscs described herein i) are able to reach the posterior segment of the eye with superior penetration; ii) have good PK properties and do not stay in the eye for an extended period of time (e.g., > 24 hours); iii) can effectively remove lipids; iv) do not require the aid of any targeting moieties or cell penetrating peptides (CPPs); and v) are effective and safe in treating eye diseases characterized by accumulation of lipids in vivo (e.g., age-related macular degeneration (AMD). Thus, the nanodiscs and methods described herein are particularly useful for treating a wide range of eye diseases characterized by accumulation of lipids, including macular degeneration and atrophy in the eye.

[0039] I. Definitions

[0040] As used herein, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise.

[0041] The term “about” indicates and covers the recited value and ranges above and below that value. In certain embodiments, the term “about” means ±10%, ±5%, or ±1% of the indicated value. In certain embodiments, the term “about” means ±one standard deviation of the indicated value.

[0042] The terms “effective amount” and “pharmaceutically effective amount” refer to an amount of an agent that is sufficient to provide a desired biological result. The result can be a reduction (e.g., a reduction of any of at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or 100%) and / or alleviation of a sign, symptom, or cause of a disease or disorder, or any other desired alteration in a biological system. An appropriate effective amount in any individual case can be determined by one of ordinary skill in the art using routine experimentation.

[0043] 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 or predisposed to treatment for a disorder or its sequelae. Individuals can 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.

[0044] The term "treatment" generally refers to clinical intervention designed to alter the natural course of the individual or cell being treated, and includes both therapeutic treatment and prophylactic or preventative measures. Desirable effects of treatment include decreasing the rate of disease progression, ameliorating or palliating a disease state, and remission or improved prognosis. An individual is successfully "treated" if one or more symptoms associated with a disease or condition are reduced or eliminated, including, but not limited to, reducing the frequency and / or severity of signs and / or symptoms caused by the disease, increasing the quality of life of individuals suffering from the disease, decreasing the dosage of other medications required to treat the disease, and / or prolonging survival of individuals. Treatment can be prophylactic (to prevent or delay onset of a disease, or to prevent the appearance of clinical or subclinical symptoms of the disease) or therapeutic (to alleviate or reduce symptoms appearing after the disease has manifested).

[0045] As used herein, "percent (%) amino acid sequence identity" and "homology" with respect to a peptide, polypeptide, or antibody sequence is 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 necessary, to achieve the maximum percent sequence identity, but 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 ™ (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring, including any algorithms required for performing alignments over the full length of the sequences being compared.

[0046] As used herein, "amphipathic" molecules refer to molecules that have both hydrophilic and hydrophobic properties. Examples include phospholipids, cholesterol, glycolipids, and fatty acids.

[0047] It is understood that embodiments of the application described herein include "consisting" and / or "consisting essentially of embodiments.

[0048] II. Methods of removing or preventing accumulation of lipids, and methods of treating eye diseases

[0049] In one aspect, provided herein is a method of removing or preventing accumulation of a lipid in an eye of an individual, the method comprising administering to the eye of the individual an effective amount of a pharmaceutical composition comprising a nanodisc, wherein the nanodisc comprises a MSP and a phospholipid. In one aspect, provided herein is a method of removing or preventing accumulation of a lipid in an eye of an individual, the method 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-l. In some embodiments, the MSP is selected from the group consisting of MSP1E1, MSP1E2, MSP1E3, MSP1, MSP1D1, MSP1E1D1, MSP1E2D1, MSP1E3D1, MSP2N2, and variants thereof that are free of the N-terminal His-tag portion (His-tag free variants). 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) a His-tag free variant of MSP1D1 comprising the amino acid sequence of SEQ ID NO: 14; or (ii) a His-tag free variant of MSP1E3D1 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 PC, such as selected from the group consisting of DMPC, POPC, DPPC, DSPC, and combinations thereof.

[0050] In some embodiments, provided herein is a method of removing or preventing accumulation of a lipid in an eye of an individual, the method 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 or preventing accumulation of a lipid in an eye of an individual, the method 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 phospholipid to MSP in the pharmaceutical composition is less than the phospholipid-MSP saturation ratio N S In some embodiments, the molar ratio of phospholipid to MSP in the pharmaceutical composition is less than the phospholipid-MSP saturation ratio N S of about 95%.

[0051] In some embodiments, the MSP is MSP1D1, MSP1, or a His tag-removed variant thereof, and the molar ratio of phospholipid to MSP in the pharmaceutical composition is 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 about 10: 1 to about 90: 1. In some embodiments, the MSP is MSP1D1, MSP1, or a His tag-removed variant thereof, the phospholipid is POPC, and the molar ratio of POPC to MSP in the pharmaceutical composition is 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 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 a His tag-removed variant thereof, the phospholipid is POPC, and the molar ratio of POPC to MSP in the pharmaceutical composition is about 65: 1. In some embodiments, the MSP is MSP1D1 or a His tag-removed variant thereof, the phospholipid is POPC, and the molar ratio of POPC to MSP1D1 or a His tag-removed variant thereof in the pharmaceutical composition is about 50: 1 or about 32.5: 1. S In some embodiments, the MSP is MSP1D1 or a His tag-removed variant thereof, the phospholipid is POPC, and the molar ratio of POPC to MSP1D1 or a His tag-removed variant thereof in the pharmaceutical composition is less than about 58: 1. In some embodiments, the MSP is MSP1D1 or a His tag-removed variant thereof, the phospholipid is POPC, and the molar ratio of POPC to MSP1D1 or a His tag-removed variant thereof in the pharmaceutical composition is about 50: 1 or about 32.5: 1.

[0052] In some embodiments, the MSP is MSP1D1, MSP1, or a His-label-removed variant thereof, and the phospholipid is DMPC, wherein the molar ratio of DMPC to 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 a His-label-removed variant thereof, the phospholipid is DMPC, and the molar ratio of DMPC to MSP in the pharmaceutical composition is from about 85:1. In some embodiments, the MSP is MSP1D1, the phospholipid is DMPC, and the molar ratio of DMPC to MSP1D1 is from about 85:1. In some embodiments, the molar ratio of DMPC to MSP1D1 in the pharmaceutical composition is from about 30:1 to about 80:1, such as from about 40:1 to about 60:1, from about 46:1 to about 55:1, from about 51:1, from about 55:1, or from about 50:1. In some embodiments, the MSP is MSP1D1 or a variant thereof with its His tag removed, the phospholipid is DMPC, and the N... S The molar ratio is approximately 81. In some embodiments, the MSP is MSP1, the phospholipid is DMPC, and the molar ratio of DMPC to MSP1 is approximately 80:1. In some embodiments, the MSP is MSP1D1 or a variant thereof with the His tag removed, the phospholipid is DMPC, and the molar ratio of DMPC to MSP in the pharmaceutical composition is approximately 40:1 to approximately 60:1, such as approximately 46:1 to approximately 55:1, approximately 51:1, approximately 55:1, or approximately 50:1.

[0053] In some embodiments, MSP is MSP1D1, MSP1, or a His-label-removed variant thereof, wherein the phospholipid is DPPC, and the molar ratio of DPPC in the pharmaceutical composition to MSP1D1, MSP1, or a His-label-removed variant thereof is about 10:1 to about 90:1. In some embodiments, MSP is MSP1 or a His-label-removed variant thereof, the phospholipid is DPPC, and the molar ratio of DPPC in the pharmaceutical composition to MSP1D1, MSP1, or a His-label-removed variant thereof is about 90:1. In some embodiments, MSP is MSP1D1 or a His-label-removed variant thereof, the phospholipid is DPPC, and N... S The molar ratio is approximately 81. In some embodiments, the molar ratio of DPPC to MSP1D1 or a His-labeled variant of the pharmaceutical composition is less than approximately 75:1. In some embodiments, the MSP is MSP1, the phospholipid is DPPC, and the molar ratio of DPPC to MSP1 is approximately 90:1.

[0054] In some embodiments, the MSP is MSP1E3 and the molar ratio of phospholipid to MSP1E3 (e.g., in a nanodisc, or in a pharmaceutical composition) is 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 a nanodisc, or in a 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 a nanodisc, or in a pharmaceutical composition) is about 170:1.

[0055] In some embodiments, the MSP is MSP1E3D1 or a His-tag removed variant thereof, the phospholipid is DMPC, and the molar ratio of DMPC to 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.

[0056] In some embodiments, the nanodisc comprises DMPC and MSP1D1, such as a molar ratio of about 85:1. In some embodiments, the nanodisc comprises DMPC and MSP1D1, optionally wherein the molar ratio of DMPC to MSP1D1 in the pharmaceutical composition is about 46:1 to about 55:1, about 51:1, about 55:1, or about 50:1. Accordingly, in some embodiments, there is provided a method of removing or preventing accumulation of a lipid (e.g., cholesterol) in an eye of an individual (e.g., a human), the method comprising administering to the eye of the individual an effective amount of a pharmaceutical composition comprising a plurality of nanodiscs, wherein the nanodiscs comprise DMPC and MSP1D1, and the molar ratio of DMPC to MSP1D1 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, the nanodisc comprises POPC and MSP1D1, such as a molar ratio of about 65:1, about 50:1, or about 32.5:1. Accordingly, in some embodiments, there is provided a method of removing or preventing accumulation of a lipid (e.g., cholesterol) in an eye of an individual (e.g., a human), the method 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 in a molar ratio of about 85:1, about 51:1, about 55:1, or about 50:1.

[0058] In some embodiments, the nanodiscs comprise 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. Accordingly, in some embodiments, a method of removing or preventing accumulation of a lipid (e.g., cholesterol) in an eye of an individual (e.g., a human) is provided, the method comprising administering to the eye of the individual an effective amount of a pharmaceutical composition comprising a plurality of nanodiscs, wherein the nanodiscs comprise 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.

[0059] In some embodiments, a method of removing or preventing accumulation of a lipid (e.g., cholesterol) in an eye of an individual (e.g., a human) is provided, the method comprising administering to the eye of the individual an effective amount of a pharmaceutical composition comprising a plurality of nanodiscs, wherein the nanodiscs comprise 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.

[0060] In some embodiments, a method of removing or preventing accumulation of a lipid (e.g., cholesterol) in an eye of an individual (e.g., a human) is provided, the method comprising administering to the eye of the individual an effective amount of a pharmaceutical composition comprising a plurality of nanodiscs, wherein the nanodiscs comprise DMPC and MSP1D1 (e.g., SEQ ID NO: 3) or a 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 a 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.

[0061] In some embodiments, a method of removing or preventing accumulation of a lipid (e.g., cholesterol) in an eye of an individual (e.g., a human) is provided, the method comprising administering to the eye of the individual an effective amount of a pharmaceutical composition comprising a plurality of nanodiscs, wherein the nanodiscs comprise DMPC and MSP1E3D1 (e.g., SEQ ID NO: 8) or a 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 a 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.

[0062] In some embodiments, the lipid for removal or prevention of accumulation in the eye is cholesterol. In some embodiments, the nanodiscs are administered intravitreally. In some embodiments, the nanodiscs have a diameter of about 2 nm to about 20 nm, such as about 5 nm to about 20 nm or about 10 nm to about 20 nm. In some embodiments, the nanodiscs do not comprise a CPP. In some embodiments, the nanodiscs do not comprise a target protein. In some embodiments, the nanodiscs do not comprise a prophylactic or therapeutic agent. In some embodiments, the nanodiscs increase the rate of cholesterol efflux at least about 2-fold (e.g., at least any of about 2.5, 3, 4, 5, 10, 15-fold or more) compared to the untreated state or the rate of cholesterol efflux achieved by ApoA-I or MSP. In some embodiments, the nanodiscs decrease the lipid in the eye of the individual by at least about 5% (e.g., at least any of about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%) or prevent accumulation of lipid in the eye of the individual.

[0063] 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., a human), comprising administering to the eye of the individual an effective amount of a pharmaceutical composition comprising nanodiscs, wherein the nanodiscs comprise 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., a human), comprising administering to the eye of the individual an effective amount of a pharmaceutical composition comprising a plurality of nanodiscs, wherein the nanodiscs comprise an MSP and a phospholipid. In some embodiments, the MSP is a variant of ApoA-l. In some embodiments, the MSP is selected from the group consisting of MSP1E1, MSP1E2, MSP1E3, MSP1, MSP1D1, MSP1E1D1, MSP1E2D1, MSP1E3D1, MSP2N2, and variants thereof that are free of the N-terminal His-tag portion (His-tag free variants). 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) a His-tag free variant of MSP1D1 comprising the amino acid sequence of SEQ ID NO: 14; or (ii) a His-tag free variant of MSP1E3D1 comprising the amino acid sequence of SEQ ID NO: 15. In some embodiments, the phospholipid comprises DMPC, DPPC, DMPS, DSPC, POPC, POP S, PiP2, or a combination thereof. In some embodiments, the phospholipid is PC, such as selected from the group consisting of DMPC, POPC, DPPC, DSPC, and combinations thereof.

[0064] 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., a human), comprising administering to the eye of the individual an effective amount of a pharmaceutical composition comprising 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 preventing or treating an eye disease characterized by lipid (e.g., cholesterol) accumulation in an individual (e.g., a human), comprising administering to the eye of the individual an effective amount of a pharmaceutical composition comprising 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 phospholipid to MSP in the pharmaceutical composition is less than the phospholipid-MSP saturation ratio N S In some embodiments, the molar ratio of phospholipid to MSP in the pharmaceutical composition is less than the phospholipid-MSP saturation ratio N S of about 95%.

[0065] In some embodiments, the MSP is MSP1D1, MSP1, or a His tag-removed variant thereof, and the molar ratio of phospholipid to MSP in the pharmaceutical composition is 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 about 10: 1 to about 90: 1. In some embodiments, the MSP is MSP1D1, MSP1, or a His tag-removed variant thereof, the phospholipid is POPC, and the molar ratio of POPC to MSP in the pharmaceutical composition is about 10: 1 to about 65: 1. In some embodiments, the phospholipid is POPC, and the molar ratio of POPC to MSP is 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 a His tag-removed variant thereof, the phospholipid is POPC, and the molar ratio of POPC to MSP in the pharmaceutical composition is about 65: 1. In some embodiments, the MSP is MSP1D1 or a His tag-removed variant thereof, the phospholipid is POPC, and the N S about 62. In some embodiments, the molar ratio of POPC to MSP1D1 or a His tag-removed variant thereof in the pharmaceutical composition is less than about 58: 1. In some embodiments, the molar ratio of POPC to MSP1D1 or a His tag-removed variant thereof in the pharmaceutical composition is about 50: 1 or about 32.5: 1.

[0066] In some embodiments, the MSP is MSP1D1, MSP1, or a His-tag removed variant thereof, the phospholipid is DMPC, and the molar ratio of DMPC to MSP in the pharmaceutical composition is about 10:1 to about 85:1. In some embodiments, the MSP is MSP1D1, MSP1, or a His-tag removed variant thereof, the phospholipid is DMPC, and the molar ratio of DMPC to MSP in the pharmaceutical composition is about 10:1 to about 85:1. In some embodiments, the MSP is MSP1D1, MSP1, or a His-tag removed variant thereof, the phospholipid is DMPC, and the molar ratio of DMPC to 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 a His-tag removed variant thereof, the phospholipid is DMPC, and the molar ratio of DMPC to MSP1D1 in the pharmaceutical composition 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 a His-tag removed variant thereof, the phospholipid is DMPC, and the molar ratio of DMPC to MSP in the pharmaceutical composition is less than about 78:1. In some embodiments, the molar ratio of DMPC to MSP in the pharmaceutical composition is about 65:1 to about 75:1. In some embodiments, the molar ratio of DMPC to MSP1D1, MSP1, or a His-tag removed variant thereof in the pharmaceutical composition is about 40:1 to about 60:1. In some embodiments, the molar ratio of DMPC to MSP in the pharmaceutical composition is about 46:1 to about 55:1, about 51:1, about 55:1, or about 50:1. S In some embodiments, the MSP is MSP1D1, MSP1, or a His-tag removed variant thereof, the phospholipid is DMPC, and the molar ratio of DMPC to MSP in the pharmaceutical composition is about 10:1 to about 85:1. In some embodiments, the MSP is MSP1D1, MSP1, or a His-tag removed variant thereof, the phospholipid is DMPC, and the molar ratio of DMPC to MSP in the pharmaceutical composition is about 10:1 to about 85:1. In some embodiments, the MSP is MSP1D1, MSP1, or a His-tag removed variant thereof, the phospholipid is DMPC, and the molar ratio of DMPC to 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 a His-tag removed variant thereof, the phospholipid is DMPC, and the molar ratio of DMPC to MSP1D1 in the pharmaceutical composition 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 a His-tag removed variant thereof, the phospholipid is DMPC, and the molar ratio of DMPC to MSP in the pharmaceutical composition is less than about 78:1. In some embodiments, the molar ratio of DMPC to MSP in the pharmaceutical composition is about 65:1 to about 75:1. In some embodiments, the molar ratio of DMPC to MSP1D1, MSP1, or a His-tag removed variant thereof in the pharmaceutical composition is about 40:1 to about 60:1. In some embodiments, the molar ratio of DMPC to MSP 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 MSP is MSP1D1, MSP1, or a His-tag removed variant thereof, the phospholipid is DPPC, and the molar ratio of DPPC to MSP in the pharmaceutical composition is about 10: 1 to about 90: 1. In some embodiments, the MSP is MSP1, or a His-tag removed variant thereof, the phospholipid is DPPC, and the molar ratio of DPPC to MSP in the pharmaceutical composition is about 90: 1. In some embodiments, the MSP is MSP1D1, or a His-tag removed variant thereof, the phospholipid is DPPC, and the molar ratio of DPPC to MSP in the pharmaceutical composition is about 81. In some embodiments, the molar ratio of DPPC to 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. S In some embodiments, the molar ratio of DPPC to 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.

[0068] In some embodiments, the MSP is MSP1E3D1, MSP1E3, or a His-tag removed variant thereof, and the molar ratio of phospholipid to MSP in the pharmaceutical composition is about 50: 1 to about 200: 1. In some embodiments, the MSP is MSP1E3, and the molar ratio of phospholipid to MSP1E3 (e.g., in a nanodisc, or in a pharmaceutical composition) is 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 a nanodisc, or in a pharmaceutical composition) is about 130: 1. In some embodiments, the MSP is MSP1E3, or a His-tag removed variant thereof, the phospholipid is POPC, and the molar ratio of POPC to MSP1E3D1, or a His-tag removed variant thereof, in the pharmaceutical composition is about 130: 1. In some embodiments, the MSP is MSP1E3D1, or a His-tag removed variant thereof, the phospholipid is POPC, and the molar ratio of POPC to MSP1E3D1, or a His-tag removed variant thereof, in the pharmaceutical composition is about 126. S In some embodiments, the molar ratio of POPC to MSP1E3D1, or a 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 a His-tag removed variant thereof, and the molar ratio of DPPC to MSP1E3 is about 170: 1.

[0069] In some embodiments, the MSP is MSP1E3D1, or a His-tag removed variant thereof, the phospholipid is DMPC, and the molar ratio of DMPC to 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.

[0070] In some embodiments, the nanodiscs comprise DMPC and MSP1D1, such as at a molar ratio of about 85: 1. In some embodiments, the nanodiscs comprise DMPC and MSP1D1 (e.g., SEQ ID NO: 3) or a 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 a 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. Accordingly, in some embodiments, there is provided a method of preventing or treating an eye disease characterized by lipid (e.g., cholesterol) accumulation in an eye of an individual (e.g., a human), the method comprising administering to the eye of the individual an effective amount of a pharmaceutical composition comprising a plurality of nanodiscs, wherein the nanodiscs comprise DMPC and MSP1D1 (e.g., SEQ ID NO: 3) or a His tag-removed variant thereof (e.g., SEQ ID NO: 14), and the molar ratio of DMPC to MSP1D1 (e.g., SEQ ID NO: 3) or a 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.

[0071] In some embodiments, the nanodiscs comprise POPC and MSP1D1 (e.g., SEQ ID NO: 3), such as at a molar ratio of about 65: 1, about 50: 1, or about 32.5: 1. Accordingly, in some embodiments, there is provided a method of preventing or treating an eye disease characterized by lipid (e.g., cholesterol) accumulation in an eye of an individual (e.g., a human), the method comprising administering to the eye of the individual an effective amount of a pharmaceutical composition comprising nanodiscs, wherein the nanodiscs comprise 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.

[0072] In some embodiments, the nanodisc comprises DMPC and MSP1E3D1 (e.g., SEQ ID NO: 8) or a 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 a 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. Accordingly, in some embodiments, there is provided a method of preventing or treating an eye disease characterized by lipid (e.g., cholesterol) accumulation in an eye of an individual (e.g., a human), the method comprising administering to the eye of the individual an effective amount of a pharmaceutical composition comprising a plurality of nanodiscs, wherein the nanodiscs comprise DMPC and MSP1E3D1 (e.g., SEQ ID NO: 8) or a 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 a 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.

[0073] In some embodiments, there is provided a method of preventing or treating an eye disease characterized by lipid (e.g., cholesterol) accumulation in an eye of an individual (e.g., a human), the method comprising administering to the eye of the individual an effective amount of a pharmaceutical composition comprising nanodiscs, wherein the nanodiscs comprise 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.

[0074] In some embodiments, there is provided a method of preventing or treating an eye disease characterized by lipid (e.g., cholesterol) accumulation in an eye of an individual (e.g., a human), the method comprising administering to the eye of the individual an effective amount of a pharmaceutical composition comprising a plurality of nanodiscs, wherein the nanodiscs comprise DMPC and MSP1D1 (e.g., SEQ ID NO: 3) or a 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 a 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.

[0075] In some embodiments, a method of preventing or treating an eye disease characterized by accumulation of a lipid (e.g., cholesterol) in an eye of an individual (e.g., a human) is provided, the method comprising administering to the eye of the individual an effective amount of a pharmaceutical composition comprising a plurality of nanodiscs, wherein the nanodiscs comprise DMPC and MSP1E3D1 (e.g., SEQ ID NO: 8) or a 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 a 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.

[0076] In some embodiments, the lipid accumulated in the eye is cholesterol. In some embodiments, the eye disease characterized by accumulation of a lipid is photoreceptor neurodegeneration, a retinal neurovascular disorder, age-related macular degeneration (AMD), or atrophy. In some embodiments, the eye disease characterized by accumulation of a lipid is AMD, such as dry AMD. In some embodiments, the dry AMD is geographic atrophy (GA). In some embodiments, the eye disease characterized by accumulation of a lipid is wet AMD. In some embodiments, the eye disease is choroidal neovascularization (CNV). In some embodiments, the nanodiscs are administered intravitreally. In some embodiments, the nanodiscs have a diameter of 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 some embodiments, the nanodiscs do not comprise a CPP. In some embodiments, the nanodiscs do not comprise a target protein. In some embodiments, the nanodiscs do not comprise a prophylactic or therapeutic agent. In some embodiments, the nanodiscs increase the rate of cholesterol efflux at least about 2-fold (e.g., at least any of about 2.5, 3, 4, 5, 10, 15, or more) compared to an untreated state or the rate of cholesterol efflux achieved by ApoA-I or MSP. In some embodiments, the nanodiscs reduce the lipid in the eye of the individual by at least about 5% (e.g., at least any of about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%) or prevent accumulation of the lipid in the eye of the individual.

[0077] In some embodiments, a method of preventing or treating AMD in an eye of an individual (e.g., a human) is provided, the method comprising administering to the eye of the individual an effective amount of a pharmaceutical composition comprising nanodiscs, wherein the nanodiscs comprise MSP and a phospholipid. In some embodiments, a method of preventing or treating AMD in an eye of an individual (e.g., a human) is provided, the method comprising administering to the eye of the individual an effective amount of a pharmaceutical composition comprising nanodiscs, wherein the nanodiscs comprise DMPC and MSP1D1 (e.g., SEQ ID NO: 3) at a molar ratio of about 85: 1. In some embodiments, a method of preventing or treating AMD in an eye of an individual is provided, the method comprising administering to the eye of the individual an effective amount of a pharmaceutical composition comprising a plurality of nanodiscs, wherein the nanodiscs comprise DMPC and MSP1D1 (e.g., SEQ ID NO: 3) or a 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 a 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, a method of preventing or treating AMD in an eye of an individual is provided, the method comprising administering to the eye of the individual an effective amount of a pharmaceutical composition comprising a plurality of nanodiscs, wherein the nanodiscs comprise DMPC and MSP1E3D1 (e.g., SEQ ID NO: 8) or a 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 a 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, a method of preventing or treating AMD in an eye of an individual (e.g., a human) is provided, the method comprising administering to the eye of the individual an effective amount of a pharmaceutical composition comprising nanodiscs, wherein the nanodiscs comprise 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 or prevents accumulation of a lipid (e.g., cholesterol) in the eye of the individual. In some embodiments, the nanodiscs are administered intravitreally.In some embodiments, the nanodisc has a diameter of 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 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 the rate of cholesterol efflux at least about 2-fold (e.g., any of at least about 2.5, 3, 4, 5, 10, 15, or more) compared to an untreated state or the rate of cholesterol efflux achieved by ApoA-l or MSP. In some embodiments, the nanodisc reduces lipid in the eye of the individual by at least about 5% (e.g., any of at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%) or prevents accumulation of lipid in the eye of the individual. In some embodiments, the AMD is GA, and the nanodisc reduces the total area of GA lesions by at least about 5% (e.g., any of at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%). In some embodiments, the nanodisc slows progression of the total area of GA lesions in the eye of the individual by at least about 5% (e.g., any of at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%). In some embodiments, the total area of GA lesions is determined by autofluorescence imaging.

[0078] In some embodiments, the lipid for removal or prevention of accumulation in the eye is selected from the group consisting of a fat, a sterol, a phospholipid, and any combination thereof. In some embodiments, the lipid comprises a cholesteryl ester (ChE), a wax ester (WE), a diester, a triacylglycerol (TG), free cholesterol, free fatty acid (FA), (O-acyl)-omega-hydroxy fatty acid (OAHFA), or any combination thereof. In some embodiments, the lipid is a fatty acid or derivative thereof, including triglycerides, diglycerides, monoglycerides, and phospholipids. In some embodiments, the lipid for removal or prevention of accumulation in the eye comprises two or more of the lipids described herein. In some embodiments, the lipid for removal or prevention of accumulation in the eye is cholesterol.

[0079] In some embodiments, the eye disease characterized by lipid accumulation is selected from the group consisting of AMD, photoreceptor neurodegeneration, optic nerve atrophy, vision loss, hemianopsia, visual agnosia, strabismus, retinal neurovascular disorder, lipid corneal disorder, 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 branched RVO), retinopathy (e.g., diabetic retinopathy (DR), including in patients with DME, proliferative vitreoretinopathy (PVR), Purtscher retinopathy, and radiation retinopathy), retinal artery occlusion (RAO, e.g., central and branched RAO), retinal vein occlusion (RVO, e.g., central RVO, including central RVO with cystoid macular edema (CME), and branched RVO, including branched RVO with CME), retinitis (e.g., Coats’ disease (exudative retinitis) and retinitis pigmentosa (RP)), chorioretinitis, choroiditis (e.g., progressive chorioiditis), uveitis (including anterior uveitis, intermediate uveitis, posterior uveitis with or without CME, panuveitis, and non-infectious uveitis), retinal detachment (e.g., in von Hippel-Lindau disease), retinal pigment epithelial (RPE) detachment, rod or / and cone cell dystrophy, and any combination thereof.

[0080] In some embodiments, the eye disease characterized by lipid accumulation is photoreceptor neurodegeneration or retinal neurovascular disorder.

[0081] In some embodiments, the eye disease characterized by lipid accumulation is AMD. In some embodiments, the AMD is dry AMD. Dry AMD is typically characterized by the accumulation of yellowish deposits called drusen under the retina, and typically affects vision in both eyes, although vision loss usually occurs in one eye before the other. A later stage of dry AMD is known as geographic atrophy (GA), in which the macular area wastes away (atrophies), leading to severe vision loss.

[0082] Dry AMD can progress to wet AMD. Wet AMD is typically characterized by the growth of leaky blood vessels under the macula. This growth of leaky blood vessels is known as choroidal neovascularization. The blood 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, which 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, wet AMD includes CNV and / or RAP.

[0083] In some embodiments, the methods described herein for removing or preventing accumulation of lipids or for treating an eye disease characterized by accumulation of lipids can achieve one or more of the following effects: i) removing lipids (e.g., cholesterol) in the eye (e.g., by at least any of about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100%); ii) increasing the efflux rate of lipids (e.g., cholesterol) in the eye (e.g., by at least any of about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 1-fold, 2-fold, 2.5-fold, 5-fold, 10-fold, or more); iii) reducing the likelihood of developing an eye disease characterized by accumulation of lipids and / or accumulation of lipids in the eye (e.g., by at least any of about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100%); iv) delaying the onset of an eye disease characterized by accumulation of lipids and / or accumulation of lipids in the eye (e.g., by at least any of about 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 18 months, 24 months, 36 months, or more); v) slowing the progression of an eye disease characterized by accumulation of lipids and / or accumulation of lipids in the eye; and vi) increasing the efflux rate of cholesterol by at least about 2-fold (e.g., by at least any of about 2.5-fold, 3-fold, 4-fold, 5-fold, 10-fold, 15-fold, or more) compared to the untreated state or the efflux rate of cholesterol achieved by ApoA-I or MSP (e.g., MSP1D1) alone.

[0084] In some embodiments, the eye disease characterized by accumulation of lipids is GA, and the methods of preventing or treating GA provided herein achieve one or more of the following effects: i) reducing lipids (e.g., cholesterol) in the eye of the individual by at least about 5% (e.g., by at least any of about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100%) or preventing accumulation of lipids in the eye of the individual; ii) reducing the total area of GA lesions in the eye of the individual by at least about 5% (such as by at least any of about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100%); and iii) slowing the progression of the total area of GA lesions in the eye of the individual by at least about 5% (such as by at least any of about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100%). The total area of GA lesions can be determined by any method known in the art, such as autofluorescence imaging. In some embodiments, the autofluorescence is fundus autofluorescence (FAF).

[0085] Thus in some embodiments, there is provided a method of reducing the total area of GA lesions in an eye of an individual (e.g., a human) and / or slowing the progression of the total area of GA lesions in an eye of an individual (e.g., a human), comprising administering to the eye of the individual an effective amount of a pharmaceutical composition comprising nanodiscs, wherein the nanodiscs comprise MSP and a phospholipid. In some embodiments, there is provided a method of reducing the total area of GA lesions in an eye of an individual (e.g., a human) and / or slowing the progression of the total area of GA lesions in an eye of an individual (e.g., a human), comprising administering to the eye of the individual an effective amount of a pharmaceutical composition comprising nanodiscs, wherein the nanodiscs comprise 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 the total area of GA lesions in an eye of an individual (e.g., a human) and / or slowing the progression of the total area of GA lesions in an eye of an individual (e.g., a human), comprising administering to the eye of the individual an effective amount of a pharmaceutical composition comprising a plurality of nanodiscs, wherein the nanodiscs comprise DMPC and MSP1D1 (e.g., SEQ ID NO: 3) or a 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 a 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 the total area of GA lesions in an eye of an individual (e.g., a human) and / or slowing the progression of the total area of GA lesions in an eye of an individual (e.g., a human), comprising administering to the eye of the individual an effective amount of a pharmaceutical composition comprising a plurality of nanodiscs, wherein the nanodiscs comprise DMPC and MSP1E3D1 (e.g., SEQ ID NO: 8) or a 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 a 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 the total area of GA lesions in an eye of an individual (e.g., a human) and / or slowing the progression of GA lesions in an eye of an individual (e.g., a human), comprising administering to the eye of the individual an effective amount of a pharmaceutical composition comprising nanodiscs, wherein the nanodiscs comprise 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 or prevents accumulation of a lipid (e.g., cholesterol) in the eye of the individual. In some embodiments, the nanodisc is administered intravitreally. In some embodiments, the nanodisc has a diameter of 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 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 the rate of cholesterol efflux at least about 2-fold (e.g., any of at least about 2.5, 3, 4, 5, 10, 15-fold or more) compared to the untreated state or the rate of cholesterol efflux achieved by ApoA-I or MSP. In some embodiments, the nanodisc reduces or prevents accumulation of a lipid in the eye of the individual by at least about 5% (e.g., any of at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%). In some embodiments, the nanodisc reduces the total area of GA lesions by at least about 5% (e.g., any of at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%). In some embodiments, the nanodisc slows progression of the total area of GA lesions in the eye of the individual by at least about 5% (e.g., any of at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%). In some embodiments, the total area of GA lesions is determined by autofluorescence imaging.

[0086] In some embodiments, the methods described herein inhibit neovascularization in the eye of an individual having AMD (e.g., inhibits any of at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100%). In some embodiments, the methods delay the development of AMD or CNV (e.g., delay any of at least about 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 18 months, 24 months, 36 months, or more). In some embodiments, the methods treat and / or reduce the risk of developing retinal neovascularization (RNV) (e.g., reduce any of at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100%). In some embodiments, the methods treat and / or reduce the risk of developing retinal angiomatous proliferation (RAP) (e.g., reduce any of at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100%). Thus, in some embodiments, a method is provided that: i) inhibits neovascularization; ii) delays the development of AMD or CNV; iii) treats and / or reduces the risk of developing RNV and / or RAP in the eye of an individual (e.g., a human); and / or iv) reduces lipid accumulation in the retinal pigment epithelium (RPE) and / or Bruch’s membrane (e.g., reduces any of at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100%); and / or v) reduces retinal neurodegeneration (e.g., reduces any of at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100%); and / or vi) improves inner / outer segment (IS / OS) junction abnormalities; and / or vii) reduces RPE disruption, including pigment epithelium detachment (PED) (e.g., reduces any of at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100%); and / or viii) reduces the cumulative incidence of retinopathy (e.g., reduces any of at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100%); and / or (ix) reduces cholesteryl esters (e.g., reduces any of at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100%), including administering to the eye of an individual an effective amount of a pharmaceutical composition comprising a nanodisc, wherein the nanodisc comprises an MSP and a phospholipid.In some embodiments, a method is provided that: i) inhibits neovascularization; ii) delays the development of AMD or CNV; iii) treats RNV and / or RAP in an eye of an individual (e.g., a human) and / or reduces the risk of developing RNV and / or RAP in an eye of an individual (e.g., a human); and / or iv) reduces lipid accumulation (e.g., reduces by any of at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100%) in the retinal pigment epithelium (RPE) and / or Bruch’s membrane; and / or v) reduces retinal neurodegeneration (e.g., reduces by any of at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100%); and / or vi) improves inner / outer segment (IS / OS) junction abnormalities; and / or vii) reduces RPE disruption, including pigment epithelium detachment (PED) (e.g., reduces by any of at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100%); and / or viii) reduces cumulative incidence of retinopathy (e.g., reduces by any of at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100%); and / or (ix) reduces cholesteryl ester (e.g., reduces by any of at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100%) including administering to the eye of the individual an effective amount of a pharmaceutical composition comprising nanodisks, wherein the nanodisks comprise DMPC and MSP1D1 (e.g., SEQ ID NO: 3) in a molar ratio of about 85: 1.In some embodiments, a method is provided that i) inhibits neovascularization; ii) delays the development of AMD or CNV; iii) treats RNV and / or RAP in the eye of an individual (e.g., a human) and / or reduces the risk of developing RNV and / or RAP in the eye of an individual (e.g., a human); and / or iv) reduces lipid accumulation (e.g., reduces by any of at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100%) in the retinal pigment epithelium (RPE) and / or Bruch’s membrane; and / or v) reduces retinal neurodegeneration (e.g., reduces by any of at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100%); and / or vi) improves inner / outer segment (IS / OS) junction abnormalities; and / or vii) reduces RPE disruption, including pigment epithelium detachment (PED) (e.g., reduces by any of at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100%); and / or viii) reduces cumulative incidence of retinopathy (e.g., reduces by any of at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100%); and / or ix) reduces cholesteryl ester (e.g., reduces by any of at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100%) to 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 nanodiscs comprise DMPC and MSP1D1 (e.g., SEQ ID NO: 3) or a 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 a 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 RNV and / or RAP in the eye of an individual (e.g., a human) and / or reducing the risk of developing RNV and / or RAP in the eye of an individual (e.g., a human); and / or iv) reducing lipid accumulation (e.g., reducing by any of at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100%) in the retinal pigment epithelium (RPE) and / or Bruch’s membrane; and / or v) reducing retinal neurodegeneration (e.g., reducing by any of at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100%); and / or vi) improving inner / outer segment (IS / OS) junction abnormalities; and / or vii) reducing RPE disruption, including pigment epithelium detachment (PED) (e.g., reducing by any of at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100%); and / or viii) reducing cumulative incidence of retinopathy (e.g., reducing by any of at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100%); and / or ix) reducing cholesteryl ester (e.g., reducing by any of at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100%) comprising administering to the eye of an individual an effective amount of a pharmaceutical composition comprising a plurality of nanodiscs, wherein the nanodiscs comprise DMPC and MSP1E3D1 (e.g., SEQ ID NO: 8) or a 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 a 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, a method is provided that: i) inhibits neovascularization; ii) delays the development of AMD or CNV; iii) treats RNV and / or RAP in the eye of an individual (e.g., a human) and / or reduces the risk of developing RNV and / or RAP in the eye of an individual (e.g., a human); and / or iv) reduces lipid accumulation (e.g., reduces by any of at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100%) in the retinal pigment epithelium (RPE) and / or Bruch’s membrane; and / or v) reduces retinal neurodegeneration (e.g., reduces by any of at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100%); and / or vi) improves inner / outer segment (IS / OS) junction abnormalities; and / or vii) reduces RPE disruption, including pigment epithelium detachment (PED) (e.g., reduces by any of at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100%); and / or viii) reduces cumulative incidence of retinopathy (e.g., reduces by any of at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100%); and / or (ix) reduces cholesteryl ester (e.g., reduces by any of at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100%), including administering to the eye of an individual an effective amount of a pharmaceutical composition comprising a plurality of nanodiscs, wherein an effective amount of a pharmaceutical composition comprising nanodiscs is administered to the eye of an individual, wherein the nanodiscs comprise a molar ratio of POPC and MSP1D1 (e.g., SEQ ID NO: 3) of about 65: 1, about 50: 1, or about 32.5: 1. In some embodiments, the method removes or prevents accumulation of lipids (e.g., cholesterols) in the eye of an individual. In some embodiments, the nanodiscs are administered intravitreally. In some embodiments, the nanodiscs have a diameter of 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 some embodiments, the nanodiscs do not comprise a CPP. In some embodiments, the nanodiscs do not comprise a target protein. In some embodiments, the nanodiscs do not comprise a prophylactic or therapeutic agent. In some embodiments, the nanodiscs increase the rate of cholesterol efflux by at least about 2-fold (e.g., any of at least about 2.5, 3, 4, 5, 10, 15-fold or more) compared to an untreated state or by ApoA-I or MSP.In some embodiments, the nanodisc reduces lipids 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%) or prevents accumulation of lipids in the eye of the individual.

[0087] In some embodiments, the individual has AMD and is at risk of or has RAP. In some embodiments, the individual has AMD and is at risk of or has CNV. In some embodiments, the individual has AMD and is at risk of or has RNV. In some embodiments, the individual has been identified as having one or more genetic polymorphisms that increase the risk of AMD. In some embodiments, the methods of preventing or treating ocular diseases (e.g., AMD) characterized by fat accumulation described herein further comprise determining whether the individual has a genetic polymorphism that increases the risk of AMD. In some embodiments, the individual has lesions at the Bruch’s membrane. In some embodiments, the individual has defective Abca1 and / or Abcg1 cholesterol efflux transporters in rod photoreceptors. In some embodiments, the individual has at least one of RPE rupture and hyperreflective, ellipsoid zone abnormalities, inner retinal dropouts, and hyperreflective lesions. In some embodiments, the individual has at least one of inner segment / outer segment (IS / OS) junction abnormalities and RPE disruption, including pigment epithelium detachment (PED). In some embodiments, the individual has been on a high-fat diet.

[0088] In some embodiments, provided are methods of improving visual impairment and / or reducing the likelihood of vision loss associated with an eye disease characterized by lipid accumulation in an eye of an individual (e.g., a human), comprising administering to the eye of the individual an effective amount of a pharmaceutical composition comprising a nanodisc, wherein the nanodisc comprises a MSP and a phospholipid. In some embodiments, the nanodisc comprises DMPC and MSP1D1 (e.g., SEQ ID NO: 3), such as a molar ratio of about 85: 1. In some embodiments, provided are methods of improving visual impairment and / or reducing the likelihood of vision loss associated with an eye disease characterized by lipid accumulation in an eye of an individual (e.g., a human), comprising administering to the eye of the individual an effective amount of a pharmaceutical composition comprising a plurality of nanodiscs, wherein the nanodiscs comprise DMPC and MSP1D1 (e.g., SEQ ID NO: 3) or a 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 a 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, provided are methods of improving visual impairment and / or reducing the likelihood of vision loss associated with an eye disease characterized by lipid accumulation in an eye of an individual (e.g., a human), comprising administering to the eye of the individual an effective amount of a pharmaceutical composition comprising a plurality of nanodiscs, wherein the nanodiscs comprise DMPC and MSP1E3D1 (e.g., SEQ ID NO: 8) or a 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 a 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 nanodiscs comprise POPC and MSP1D1 (e.g., SEQ ID NO: 3), such as a molar ratio of about 65: 1, about 50: 1, or about 32.5: 1. In some embodiments, the methods provided herein improve visual impairment and / or reduce the likelihood of vision loss associated with an eye disease characterized by lipid accumulation in an eye by at least any of about 5%, such as at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100%. The visual 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-center or / and center GA) or neovascular AMD (including type 1, type 2, or / and type 3 NV), or the improvement in vision can occur in a subject with atrophic AMD or neovascular AMD.

[0089] In some embodiments, a method of improving normal luminance best-corrected visual acuity (NL-BCVA) score and / or low luminance best-corrected visual acuity (LL-BCVA) score in an individual (e.g., a human) is provided, the method comprising administering to the eye of the individual an effective amount of a pharmaceutical composition comprising a nanodisc, wherein the nanodisc comprises a MSP and a phospholipid. In some embodiments, the nanodisc comprises DMPC and MSP1D1 (e.g., SEQ ID NO: 3), such as a molar ratio of about 85: 1. In some embodiments, a method of improving normal luminance best-corrected visual acuity (NL-BCVA) score and / or low luminance best-corrected visual acuity (LL-BCVA) score in an individual (e.g., a human) is provided, the method comprising administering to the eye of the individual an effective amount of a pharmaceutical composition comprising a plurality of nanodiscs, wherein the nanodiscs comprise DMPC and MSP1D1 (e.g., SEQ ID NO: 3) or a 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 a 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, a method of improving normal luminance best-corrected visual acuity (NL-BCVA) score and / or low luminance best-corrected visual acuity (LL-BCVA) score in an individual (e.g., a human) is provided, the method comprising administering to the eye of the individual an effective amount of a pharmaceutical composition comprising a plurality of nanodiscs, wherein the nanodiscs comprise DMPC and MSP1E3D1 (e.g., SEQ ID NO: 8) or a 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 a 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 a molar ratio of about 65: 1, about 50: 1, or about 32.5: 1. In some embodiments, the methods provided herein increase the NL-BCVA score and / or LL-BCVA score in an individual by at least about any of 5%, such as at least about 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 method in the art.

[0090] In some embodiments, any of the methods provided herein are: i) removing or preventing accumulation of lipids; ii) preventing or treating an eye disease characterized by accumulation of lipids; iii) reducing and / or slowing progression of the total area of GA lesions; iv) inhibiting neovascularization; vi) delaying development of AMD or CNV; vi) treating and / or reducing the risk of developing RNV and / or RAP; vii) improving visual impairment and / or reducing the likelihood of vision loss associated with an eye disease characterized by accumulation of lipids; and viii) improving the NL-BCVA score and / or LL-BCVA score mentioned herein, compared to the baseline state (or untreated state) prior to treatment, the natural progression without any treatment, treatment with a placebo, or treatment with ApoA-l or MSP protein (e.g., MSP1D1) alone. In some embodiments, the efficacy of any of the methods provided herein is compared to treatment with HDL alone.

[0091] In some embodiments, the efficacy of any of the methods provided herein can be determined by any method known in the art. For example, in some embodiments, the efficacy of any of the methods provided herein can be determined by measuring photoreceptor and RPE function, optionally by full-field scotopic and light bleaching recovery ERG diagnostics. In some embodiments, the efficacy of any of the methods provided herein can be determined by measuring anatomical changes associated with AMD progression, such as RPE rupture and hyperreflectance, ellipsoid zone abnormalities, inner retinal dropouts, and hyperreflective lesions, optionally by non-invasive imaging such as optical coherence tomography (OCT). In some embodiments, the efficacy of any of the methods provided herein can be determined by measuring visual function or cumulative incidence of retinal lesions, optionally by full-field scotopic and light bleaching recovery ERG diagnostics. In some embodiments, the efficacy of any of the methods provided herein can be determined by examining potential changes in individual lipid species in the eye, such as cholesteryl ester (CE) composition, optionally by lipidomics analysis.

[0092] In some embodiments, any of the methods provided herein are safe and result in minimal side effects, where the safety of the method can be determined by any method known in the art. In some embodiments, any of the methods provided herein do not result in significant retinal inflammation or differences in retinal integrity. In some embodiments, retinal inflammation or differences in retinal integrity can be measured by examining retinal histology. In some embodiments, any of the methods provided herein do not exacerbate expected complement signaling in individuals with dry AMD. In some embodiments, complement signaling can be determined by retinal section immunostaining.

[0093] Nanodiscs

[0094] The nanodiscs provided herein refer to a nanocomplex resembling a disc in which a lipid bilayer comprising a phospholipid is surrounded by a stabilizing belt comprising a MSP. In some embodiments, the nanodisc comprises DMPC and MSP1D1, optionally at a molar ratio (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 at 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 average diameter of the nanodisc is 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 nanodisc in the pharmaceutical composition is less than about 0.2. In some embodiments, the polydispersity index of the phospholipid content of the nanodisc 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 a therapeutic agent.

[0095] a. Membrane scaffold protein (MSP)

[0096] In some embodiments, the MSP is selected from the group consisting of apolipoprotein A-I (ApoA-I), ApoA-II, ApoC, ApoE, ApoM, and variants thereof. In some embodiments, the MSP is a variant of ApoA-I.

[0097] Apolipoproteins play an important role in maintaining structural integrity and solubility of lipoproteins, recognition by lipoprotein receptors, and regulation of certain enzymes in lipoprotein metabolism. Major classes 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). The apolipoproteins can be from any organism, including, but not limited to, humans, cows, horses, sheep, monkeys, baboons, goats, rabbits, dogs, hedgehogs, badgers, mice, rats, cats, guinea pigs, hamsters, ducks, chickens, salmon, and eels. In some embodiments, the apolipoprotein is a human apolipoprotein.

[0098] 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 native 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., an insertion, a deletion, a substitution, or any combination thereof) compared to a naturally occurring apolipoprotein (e.g., ApoA-I). In some embodiments, the MSP comprises one or more cysteine (Cys) substitutions. In some embodiments, the mutation (e.g., the Cys substitution) is at the N-terminus, at the C-terminus, and / or between an amphipathic helix of the MSP. In some embodiments, the Cys substitution is in one of the helices of the MSP. In some embodiments, the cysteine can be linked to an imaging moiety, optionally a fluorescent moiety (e.g., GFP, RFP, YFP). In some embodiments, the cysteine in the MSP can be linked to an imaging moiety, optionally a fluorescent moiety (e.g., fluorescein, rhodamine, or other common light-emitting small molecules). In some embodiments, the MSP is fused to a fluorescent protein, such as GFP, RFP, or YFP. In some embodiments, the MSP comprises a tag, such as a tag commonly used for 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, for example, a tobacco etch virus protease (TEV) recognition site (boxed in Table 1).

[0099] In some embodiments, the MSP comprises a His tag. In some embodiments, the MSP does not comprise a His tag. The notation "(-)" as used herein refers to the 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-terminal end of the MSP. In some embodiments, the MSP is derived from a naturally occurring ApoA-I, such as human ApoA-I. The TEV protease recognition site sequence is boxed in Table 1.

[0100] ApoA-I is the major protein component of HDL particles in plasma. The protein facilitates the flow of fat molecules out of cells by accepting intracellular fat for transport elsewhere, such as back to LDL particles or the liver for excretion. Human ApoA-I contains an N-terminal globular domain (GLOB) of about 43 residues followed by ten amphipathic helices (H1-H10) each consisting of 11, 22, or 24 residues. Seven of the helices are 22 amino acids in length, one helix (H10) is 24 amino acids in length, and two helices (H3, H9) are 11 amino acids in length. The helices are separated by glycines or prolines. The sequences of the different domains of human ApoA-I are indicated in Table 1. 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 DEPPQSPWDRVKDLATVYVDVLKDSGRDYVSQFEGSALGKQLN (SEQ ID NO: 19).

[0101] ApoA-1 can be from any organism. In some embodiments, the ApoA-1 is derived from an animal selected from the group consisting of a human, a cow, a horse, a sheep, a monkey, a baboon, a goat, a rabbit, a dog, a hedgehog, a badger, a mouse, a rat, a cat, a guinea pig, a hamster, a duck, a chicken, a salmon, an eel, and any combination thereof. In some embodiments, the ApoA-1 is human ApoA-1. In some embodiments, the ApoA-1 comprises (or consists of, or consists essentially of) the amino acid sequence of SEQ ID NO: 1.

[0102] In some embodiments, the MSP is a variant of ApoA-l. In some embodiments, the MSP has at least about 70% (e.g., at least about any of 80%, 85%, 90%, 95%, 99%, or more) sequence identity to SEQ ID NO: 1. In some embodiments, MSPs containing an alternative amphipathic helix polypeptide sequence can be generated by adding or removing one or more helix segments (e.g., H1, H2, etc.) or partial helix segments (e.g., removing a portion of the H1 sequence of ApoA-l to achieve H(0.5)), or adding, removing, or substituting amino acid residues based on the ApoA-l (e.g., human ApoA-l) protein. In some embodiments, the MSP can be derived from ApoA-l (e.g., human ApoA-l) by removing the GLOB domain. In some embodiments, the MSP can be derived from ApoA-l (e.g., human ApoA-l) by adding one (e.g., H4), two (e.g., H4 and H5), three (e.g., H4-H6), or more helix segments.

[0103] Exemplary MSPs and their included domains and sequences are indicated in Table 1. Other MSPs having variations relative to those in Table 1 can also be used herein. For example, by adding or removing one or more helix segments or portions thereof, and / or by adding, removing, and / or substituting one or more amino acid residues at the N-terminus, internally, and / or at the C-terminus (e.g., adding a peptide linker between helix segments), and / or by adding, removing, and / or substituting a tag sequence.

[0104] Table 1. Sequences

[0105] SEQ ID NO: Description Sequence 1 Human ApoA-I globular domain (GLOB) H1 H2 H3 H4 H5 H6 H7 H8 H9 H10 DEPPQSPWDRVKDLATVYVDVLKDSGRDYVSQFEGSALGKQLNLKLLDNWDSVTSTFSKLREQLGPVTQEFWDNLEKETEGLRQEMSKDLEEVKAKVQPYLDDFQKKWQEEMELYRQKVEPLRAELQEGARQKLHELQEKLSPLGEEMRDRARAHVDALRTHLAPYSDELRQRLAARLEALKENGGARLAEYHAKATEHLSTLSEKAKPALEDLRQGLLPVLESFKVSFLSALEEYTKKLNTQ 2 MSP1 His tag (Factor Xa protease cleavage site underlined) H1 H2 H3 H4 H5 H6 H7 H8 H9 H10 MGHHHHHH IEGR LKLLDNWDSVTSTFSKLREQLGPVTQEFWDNLEKETEGLRQEMSKDLEEVKAKVQPYLDDFQKKWQEEMELYRQKVEPLRAELQEGARQKLHELQEKLSPLGEEMRDRARAHVDALRTHLAPYSDELRQRLAARLEALKENGGARLAEYHAKATEHLSTLSEKAKPALEDLRQGLLPVLESFKVSFLSALEEYTKKLNTQ 3 MSP1 D1 His tag H (0.5) H2 H3 H4 H5 H6 H7 H8 H9 H10 MGHHHHHHHDYDIPTTENLYFQGSTFSKLREQLGPVTQEFWDNLEKETEGLRQEMSKDLEEVKAKVQPYLDDFQKKWQEEMELYRQKVEPLRAELQEGARQKLHELQEKLSPLGEEMRDRARAHVDALRTHLAPYSDELRQRLAARLEALKENGGARLAEYHAKATEHLSTLSEKAKPALEDLRQGLLPVLESFKVSFLSALEEYTKKLNTQ 4 MSP1 D2 His tag H2 H3 H4 H5 H6 H7 H8 H9 H10 MSP1 D2 His tag H2 H3 H4 H5 H6 H7 H8 H9 H10 MGHHHHHHHDYDIPTTENLYFQGPVTQEFWDNLEKETEGLRQEMSKDLEEVKAKVQPYLDDFQKKWQEEMELYRQKVEPLRAELQEGARQKLHELQEKLSPLGEEMRDRARAHVDALRTHLAPYSDELRQRLAARLEALKENGGARLAEYHAKATEHLSTLSEKAKPALEDLRQGLLPVLESFKVSFLSALEEYTKKLNTQ 5 MSP1E1 His tag (factor Xa protease cleavage site underlined) H1 H2 H3 H4 H4 H5 H6 H7 H8 H9 H10 MGHHHHHH IEGR LKLLDNWDSVTSTFSKLREQLGPVTQEFWDNLEKETEGLRQEMSKDLEEVKAKVQPYLDDFQKKWQEEMELYRQKVEPYLDDFQKKWQEEMELYRQKVEPLRAELQEGARQKLHELQEKLSPLGEEMRDRARAHVDALRTHLAPYSDELRQRLAARLEALKENGGARLAEYHAKATEHLSTLSEKAKPALEDLRQGLLPVLESFKVSFLSALEEYTKKLNTQ 6 MSP1E2 His tag (factor Xa protease cleavage site underlined) H1 H2 H3 H4 H5 H4 H5 H6 H7 H8 H9 H10 MGHHHHHH IEGR LKLLDNWDSVTSTFSKLREQLGPVTQEFWDNLEKETEGLRQEMSKDLEEVKAKVQPYLDDFQKKWQEEMELYRQKVEPLRAELQEGARQKLHELQEKLSPYLDDFQKKWQEEMELYRQKVEPLRAELQEGARQKLHELQEKLSPLGEEMRDRARAHVDALRTHLAPYSDELRQRLAARLEALKENGGARLAEYHAKATEHLSTLSEKAKPALEDLRQGLLPVLESFKVSFLSALEEYTKKLNTQ]]> 7 MSP1E3 His tag (factor Xa protease cleavage site underlined) H1 H2 H3 H4 H5 H6 H4 H5 H6 H7 H8 H9 H10 MGHHHHHHIEGRLKLLDNWDSVTSTFSKLREQLGPVTQEFWDNLEKETEGLRQEMSKDLEEVKAKVQPYLDDFQKKWQEEMELYRQKVEPLRAELQEGARQKLHELQEKLSPLGEEMRDRARAHVDALRTHLAPYLDDFQKKWQEEMELYRQKVEPLRAELQEGARQKLHELQEKLSPLGEEMRDRARAHVDALRTHLAPYSDELRQRLAARLEALKENGGARLAEYHAKATEHLSTLSEKAKPALEDLRQGLLPVLESFKVSFLSALEEYTKKLNTQ 8 MSP1E3D1 His tag H(0.5) H2 H3 H4 H5 H6 H4 H5 H6 H7 H8 H9 H10 MGHHHHHHHDYDIPTTENLYFQGSTFSKLREQLGPVTQEFWDNLEKETEGLRQEMSKDLEEVKAKVQPYLDDFQKKWQEEMELYRQKVEPLRAELQEGARQKLHELQEKLSPLGEEMRDRARAHVDALRTHLAPYLDDFQKKWQEEMELYRQKVEPLRAELQEGARQKLHELQEKLSPLGEEMRDRARAHVDALRTHLAPYSDELRQRLAARLEALKENGGARLAEYHAKATEHLSTLSEKAKPALEDLRQGLLPVLESFKVSFLSALEEYTKKLNTQ 9 ApoC-I TPDVSSALDKLKEFGNTLEDKARELISRIKQSELSAKMREWFSETFQKVKEKLKIDS 10 ApoC-II DEMPSPTFLTQVKESLSSYWESAKTAAQNLYEKTYLPAVDEKLRDLYSKSTAAMSTYTGIFTDQVLSVLKGEE 11 ApoC-III SEAEDASLLSFMQGYMKHATKTAKDALSSVQESQVAQQARGWVTDGFSSLKDYWSTVKDKFSEFWDLDPEVRPTSAVAA 12 ApoE MKVLWAALLVTFLAGCQAKVEQAVETEPEPELRQQTEWQSGQRWELALGRFWDYLRWVQTLSEQVQEELLSSQVTQELRALMDETMKELKAYKSELEEQLTPVAEETRARLSKELQAAQARLGADMEDVCGRLVQYRGEVQAMLGQSTEELRVRLASHLRKLRKRLLRDADDLQKRLAVYQAGAREGAERGLSAIRERLGPLVEQGRVRAATVGSLAGQPLQERAQAWGERLRARMEEMGSRTRDRLDEVKEQVAEVRAKLEEQAQQIRLQAEAFQARLKSWFEPLVEDMQRQWAGLVEKVQAAVGTSAAPVPSDNH 13 Apo-M MFHQIWAALLYFYGIILNSIYQCPEHSQLTTLGVDGKEFPEVHLGQWYFIAGAAPTKEELATFDPVDNIVFNMAAGSAPMQLHLRATIRMKDGLCVPRKWIYHLTEGSTDLRTEGRPDMKTELFSSSCPGGIMLNETGQGYQRFLLYNRSPHPPEKCVEEFKSLTSCLDSKAFLLTPRNQEACELSNN 14 MSP1D1(-)H(0.5)H2H3H4H5H6H7H8H9H10 GSTFSKLREQLGPVTQEFWDNLEKETEGLRQEMSKDLEEVKAKVQPYLDDFQKKWQEEMELYRQKVEPLRAELQEGARQKLHELQEKLSPLGEEMRDRARAHVDALRTHLAPYSDELRQRLAARLEALKENGGARLAEYHAKATEHLSTLSEKAKPALEDLRQGLLPVLESFKVSFLSALEEYTKKLNTQ 15 MSP1 E3D1 (-) H (0.5) H2 H3 H4 H5 H6 H4 H5 H6 H7 H8 H9 H10 GSTFSKLREQLGPVTQEFWDNLEKETEGLRQEMSKDLEEVKAKVQPYLDDFQKKWQEEMELYRQKVEPLRAELQEGARQKLHELQEKLSPLGEEMRDRARAHVDALRTHLAPYLDDFQKKWQEEMELYRQKVEPLRAELQEGARQKLHELQEKLSPLGEEMRDRARAHVDALRTHLAPYSDELRQRLAARLEALKENGGARLAEYHAKATEHLSTLSEKAKPALEDLRQGLLPVLESFKVSFLSALEEYTKKLNTQ 16 MSP1 E1D1 His tag H (0.5) H2 H3 H4 H4 H5 H6 H7 H8 H9 H10 MGHHHHHHHDYDIPTTENLYFQGSTFSKLREQLGPVTQEFWDNLEKETEGLRQEMSKDLEEVKAKVQPYLDDFQKKWQEEMELYRQKVEPYLDDFQKKWQEEMELYRQKVEPLRAELQEGARQKLHELQEKLSPLGEEMRDRARAHVDALRTHLAPYSDELRQRLAARLEALKENGGARLAEYHAKATEHLSTLSEKAKPALEDLRQGLLPVLESFKVSFLSALEEYTKKLNTQ 17 MSP1 E2D1 His tag H (0.5) H2 H3 H4 H5 H4 H5 H6 H7 H8 H9 H10 MGHHHHHHHDYDIPTTENLYFQGSTFSKLREQLGPVTQEFWDNLEKETEGLRQEMSKDLEEVKAKVQPYLDDFQKKWQEEMELYRQKVEPLRAELQEGARQKLHELQEKLSPYLDDFQKKWQEEMELYRQKVEPLRAELQEGARQKLHELQEKLSPLGEEMRDRARAHVDALRTHLAPYSDELRQRLAARLEALKENGGARLAEYHAKATEHLSTLSEKAKPALEDLRQGLLPVLESFKVSFLSALEEYTKKLNTQ 18 MSP2 N2 His tag H (0.5) H2 H3 H4 H5 H6 H7 H8 H9 H10 linker H2 H3 H4 H5 H6 H7 H8 H9 H10 MGHHHHHHHDYDIPTTENLYFQGSTFSKLREQLGPVTQEFWDNLEKETEGLRQEMSKDLEEVKAKVQPYLDDFQKKWQEEMELYRQKVEPLRAELQEGARQKLHELQEKLSPLGEEMRDRARAHVDALRTHLAPYSDELRQRLAARLEALKENGGARLAEYHAKATEHLSTLSEKAKPALEDLRQGLLPVLESFKVSFLSALEEYTKKLNTQGTPVTQEFWDNLEKETEGLRQEMSKDLEEVKAKVQPYLDDFQKKWQEEMELYRQKVEPLRAELQEGARQKLHELQEKLSPLGEEMRDRARAHVDALRTHLAPYSDELRQRLAARLEALKENGGARLAEYHAKATEHLSTLSEKAKPALEDLRQGLLPVLESFKVSFLSALEEYTKKLNTQ 19 GLOB domain DEPPQSPWDRVKDLATVYVDVLKDSGRDYVSQFEGSALGKQLN 20 H(0.5) STFSKLREQLG 21 His tag portion MGHHHHHHHDYDIPTTENLYFQG 22 His tag portion MGHHHHHHIEGR 23 Factor Xa protease cleavage site IEGR

[0106] In some embodiments, the MSP comprises an N-terminal truncation relative to ApoA-I. In some embodiments, the truncation comprises deletion of 1, 2, 3, 4, 5, 10, 15, 20, 30, 40, 50, 60, 100, or more contiguous amino acids from the N-terminus of ApoA-I. In some embodiments, the truncation comprises deletion of about 10 to 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 deletion of about 10 to 70 amino acids from the N-terminus of ApoA-I. In some embodiments, the N-terminal GLOB domain of ApoA-I is removed in the MSP. In some embodiments, the first 11 amino acids of H1 of ApoA-I are removed in the MSP, resulting in the H(0.5) helix portion of STFSKLREQLG (SEQ ID NO: 20) (see H(0.5) in Table 1). In some embodiments, both the N-terminal GLOB domain and the first 11 amino acids of H1 of ApoA-I are removed in the MSP.

[0107] In some embodiments, the MSP comprises an N-terminal amino acid sequence MGHHHHHHHDYDIPTTENLYFQG (SEQ ID NO: 21), also referred to herein after as a “His tag” or “His tag portion.” The TEV protease recognition site is indicated by the box. In some embodiments, the MSP does not comprise an 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 cleavage with TEV protease. It will be understood that cleavage at the TEV protease recognition site can 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 cleavage of MSP1D1 (SEQ ID NO: 3) with TEV protease, and MSP1E3D1(-) (SEQ ID NO: 15) is obtained by cleavage of MSP1E3D1 (SEQ ID NO: 8) with TEV protease. It will also be understood that for MSPs in which the indicated His tag (SEQ ID NO: 21) is not removed, the N-terminal methionine residue will typically be removed during heterologous expression (e.g., if expressed in bacteria such as E. coli).

[0108] In some embodiments, the MSP is MSP1. In some embodiments, the 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., any of at least about 80%, 85%, 90%, 95%, 99%, or 100%) sequence identity to MSP1 (e.g., SEQ ID NO: 2).

[0109] 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 11 amino acids are removed from the first helix labeled H1 in the parent ApoA-I, resulting in a semi-helix labeled H(0.5) and MSP1D1. In some embodiments, the MSP1D1 comprises the amino acid sequence of SEQ ID NO: 3. In some embodiments, the MSP has at least about 70% (e.g., any of at least about 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 include the N-terminal His tag portion (a variant with the His tag removed) (hereinafter referred to as MSP1D1(-)), which comprises the amino acid sequence of SEQ ID NO: 14.

[0110] In some embodiments, the MSP is MSP1D2. In some embodiments, the MSP1D2 comprises the amino acid sequence of SEQ ID NO: 4. In some embodiments, the MSP has at least about 70% (e.g., any of at least about 80%, 85%, 90%, 95%, 99%, or 100%) sequence identity to SEQ ID NO: 4.

[0111] 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, Apolipoprotein III, myoglobin, or hemoglobin. In some embodiments, the additional amphipathic helix sequence is not derived from ApoA-I, ApoA-II, ApoC-I, ApoC-II, Apo-E, Apolipoprotein III, myoglobin, or hemoglobin. In some embodiments, the additional amphipathic helix sequence is synthetic or non-naturally occurring. In some embodiments, the additional amphipathic helix sequence comprises at least one (e.g., 1, 2, 3, 4, 5, or more) alpha-helix or fragment thereof. In some embodiments, the MSP comprises an additional helix sequence, such as any of one, two, or three first helix sequences, second helix sequences, or third helix sequences. In some embodiments, the additional amphipathic helix sequence is added to the N-terminus of the apolipoprotein (e.g., ApoA-I). In some embodiments, the additional amphipathic helix sequence is added to the C-terminus of the apolipoprotein (e.g., ApoA-I). In some embodiments, the additional amphipathic helix sequence is inserted between the original helices of the apolipoprotein (e.g., ApoA-I). In some embodiments, the additional amphipathic helix sequence is added to the original MSP1 sequence (e.g., SEQ ID NO: 2) after the last Q residue of H3. In some embodiments, each additional amphipathic helix sequence has about 10 to about 30 (including, e.g., 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 20 to 25, 40 to 45, 60 to 65 amino acids in length. In some embodiments, the MSP does not comprise the GLOB domain of SEQ ID NO: 19. In some embodiments, the amphipathic helix sequence of helix 4 (H4) can be inserted into MSP1D1 to generate MSP1E1D1, as defined by SEQ ID NO: 16. In some embodiments, 2 amphipathic helices of helix 4 (H4) and helix 5 (H5) can be inserted into MSP1D1, generating MSP1E2D1 (SEQ ID NO: 17). In some embodiments, three amphipathic helices H4, H5, and H6 can be inserted into MSP1D1, generating MSP1E3D1, defined by SEQ ID NO: 8.In some embodiments, nine additional amphipathic helices are added to MSP1D1 (SEQ ID NO: 3) with GT peptide linkers to generate extended MSP-MSP2N2 (SEQ ID NO: 18).

[0112] In some embodiments, the MSP comprises additional helical sequences, such as any of one, two, or three first helical sequences, second helical sequences, or third helical sequences. In some embodiments, the additional amphipathic helical sequence is added to the N-terminus of an apolipoprotein (e.g., ApoA-I). In some embodiments, the additional amphipathic helical sequence is added to the C-terminus of an apolipoprotein (e.g., ApoA-I). In some embodiments, the additional amphipathic helical sequence is inserted between helices of an apolipoprotein (e.g., ApoA-I). In some embodiments, each additional amphipathic helical sequence has about 10 to about 30 (including, e.g., any of about 15 to about 25, about 24, about 20, about 22, about 11) amino acids. In some embodiments, the total additional amphipathic helical sequences are any of about 20 to 25, about 40 to 46, about 60 to 68, or about 100 to 200 amino acids in length. In some embodiments, the MSP is MSP1E1, MSP1E2, or MSP1E3. MSP1E1, MSP1E2, and MSP1E3 are all extended membrane scaffold proteins and are obtained by inserting one, two, or three additional 22-mer amphipathic helices into the original MSP1 sequence after the last Q residue of H3. 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., any of at least about 80%, 85%, 90%, 95%, 99%, or 100%) sequence identity to any of SEQ ID NO: 5 to SEQ ID NO: 7.

[0113] In some embodiments, the MSP comprises an additional amphipathic helix sequence and truncation relative to ApoA-I. In some embodiments, the MSP is MSP1E3D1. In some embodiments, the MSP1E3D1 comprises the amino acid sequence of SEQ ID NO: 8. In some embodiments, the MSP has at least about 70% (e.g., any of at least about 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 include the N-terminal His tag portion (a variant with the His tag removed) (hereinafter referred to as MSP1E3D1(-)) comprising the amino acid sequence of SEQ ID NO: 15.

[0114] 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 contents of which are incorporated by reference herein in their entirety.

[0115] MSP2N2 was generated by extension of His-tagged MSP1D1, addition of a GT spacer, and additional helices 2-10. Detailed composition and preparation procedures of MSP2N2 are described in Grinkova et al., Protein Engineering Design and Selection 2010 23, 843-848, the contents of which are incorporated by reference herein in their entirety.

[0116] In some embodiments, the histidine tag is removed from MSP1D1, MSP1E1D1, MSP1E2D1, MSP1E3D1, or MSP2N2 by cleavage with TEV protease to generate MSP1D1(-), MSP1E1D1(-), MSP1E2D1(-), MSP1E3D1(-), or MSP2N2(-), respectively. It will be appreciated that TEV protease cleavage at the boxed region shown in Table 1 can result in a residual glycine (G) amino acid residue on the N-terminus of the resulting MSP protein. These variants are referred to herein as “variants with the His tag removed.”

[0117] In some embodiments, the MSP is a variant of Apo-C. The Apo-C family includes three closely related proteins: ApoC-I, ApoC-II, and ApoC-III, which are made mostly by the liver and, to a lesser extent, in the intestine. In some embodiments, ApoC-I, ApoC-II, and ApoC-III comprise the amino acid sequences 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., any of at least about 80%, 85%, 90%, 95%, 99%, or 100%) sequence identity to any one of SEQ ID NO: 9-11.

[0118] 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., any of at least about 80%, 85%, 90%, 95%, 99%, or 100%) sequence identity to SEQ ID NO: 12.

[0119] 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., any of at least about 80%, 85%, 90%, 95%, 99%, or 100%) sequence identity to SEQ ID NO: 13.

[0120] b. Phospholipid

[0121] Phospholipids generally refer to a class of lipids having a hydrophilic head of a phosphate moiety linked through an alcohol or glycerol backbone and two hydrophobic tails derived from fatty acids.

[0122] In some embodiments, the phospholipid is a sphingolipid. Sphingolipids generally refer to a class of lipids containing a sphingoid base backbone, which is a group of aliphatic amino alcohols including sphingosine. In some embodiments, the sphingolipid is sphingosine-1 -phosphate.

[0123] In some embodiments, the phospholipid comprises one or more acyl chains. In some embodiments, each acyl chain is independently about 5 to about 30 carbon units in length, such as about 10 to about 22 carbon units or about 10 to about 16 carbon units. In some embodiments, the phospholipid has a glycerol backbone. In some embodiments, the phospholipid is a phosphatidylcholine (PC). PC generally refers to a class of phospholipids comprising choline as a head group, a glycerophosphate 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-phosphocholine (POPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC), dimyristoyl phosphatidylcholine (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.

[0124] In some embodiments, 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 includes two myristoyl fatty acids attached to the first and second carbons of a glycerol in ester linkage; and a choline attached to the third carbon of the glycerol by a phosphodiester linkage. In some embodiments, the phospholipid is DPPC. DPPC is a synthetic phospholipid that includes two palmitoyl fatty acids attached to the first and second carbons of a glycerol in ester linkage; and a choline attached to the third carbon of the glycerol by a phosphodiester linkage. In some embodiments, the phospholipid is 1,2-dimyristoyl-sn-glycero-3-phospho-L-serine (DMPS), such as 14:0 DMPS. In some embodiments, the phospholipid is DSPC. DSPC is a synthetic phospholipid that includes two stearoyl fatty acids attached to the first and second carbons of a glycerol in ester linkage; and a choline attached to the third carbon of the glycerol by a phosphodiester linkage.

[0125] In some embodiments, 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 includes two fatty acids, palmitic acid and oleic acid, attached to the first and second carbons of a glycerol in ester linkage, respectively; and a choline attached to the third carbon of the glycerol by a phosphodiester linkage. In some embodiments, the phospholipid is PiP2, which can 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 includes two oleoyl fatty acids attached to the first and second carbons of a glycerol in ester linkage; and a choline attached to the third carbon of the glycerol by a phosphodiester linkage. In some embodiments, the phospholipid is DEPC. DEPC is a synthetic phospholipid that includes two erucic fatty acids attached to the first and second carbons of a glycerol in ester linkage; and a choline attached to the third carbon of the glycerol by a phosphodiester linkage.

[0126] In some embodiments, the phospholipid is l,2-dimyristoyl-sn-glycero-3-phospho-L- serine (DMPS), such as 14:0 DMPS. In some embodiments, the phospholipid is DSPC. DSPC is a synthetic phospholipid that includes two stearoyl fatty acids attached to the first and second carbons of glycerol in ester linkages; and choline attached to the third carbon of glycerol by a phosphodiester linkage.

[0127] In some embodiments, the phospholipid is PiP2, which can also be referred to as PI(4,5)P2. In some embodiments, the PiP2 is 18: l PI(4,5)P2 or 18:0-20:4 PI(4,5)P2. In some embodiments, the PiP2 is 18: l PI(4,5)P2. In some embodiments, the PiP2 is 18:0-20:4 PI(4,5)P2.

[0128] In some embodiments, the phospholipid is cationic, anionic, or zwitterionic, or any combination thereof.

[0129] c. MSP to phospholipid ratio

[0130] The stoichiometry of the MSP and the phospholipid is important for the formulation, function, shape uniformity, and size distribution of the nanodisks used herein.

[0131] In some embodiments, the phospholipid-to-MSP molar ratio mentioned herein is the molar ratio of phospholipid to MSP in the pharmaceutical composition (e.g., the average or median molar ratio). In some embodiments, the phospholipid-to-MSP molar ratio mentioned herein is the molar ratio of phospholipid to MSP in the nanodisks (e.g., the average or median molar ratio).

[0132] In some embodiments, without being bound by any scientific theory, the amount of phospholipid required to saturate the nanodisks (i.e., the phospholipid-to-MSP saturation ratio N S) can depend on the identity of the MSP and the phospholipid. In some embodiments, the phospholipid can comprise a phosphatidylcholine (PC) head group and more than 12 hydrocarbon tails. In some embodiments, without being bound by any scientific theory, the phospholipid can have a tendency to condense into a bilayer structure. In some embodiments, without being bound by any scientific theory, the MSP can contact the bilayer formed by the phospholipid with the hydrophobic portion of the MSP remaining proximal to the hydrophobic tails of the phospholipid and the hydrophilic portion of the MSP remaining proximal to the solvent in the pharmaceutical composition. In some embodiments, without being bound by any scientific theory, the MSP is a variant of ApoA-l and the amino acids that form the globular domain and the first 11 amino acids of helix 1 (H1, Table 1) do not contact the lipid (Denisov et al., Denisov et al., J Am Chem Soc. 2004 Mar 24; 126(11): 3477-87).

[0133] Variants of MSPs that do not have a globular domain and / or do not have a H1 domain or portions thereof and / or do not have any amino or carboxy terminal tags and protease cleavage sites, such as MSP1D1(-), MSPE1D1(-), MSPE2D1(-), MSPE3D1(-), and MSP2N2(-) provided herein, can have the same molar ratio of phospholipid to MSP in the pharmaceutical composition required to saturate the nanodiscs as compared to their parent proteins (e.g., MSP1D1, MSPE1D1, MSPE2D1, MSPE3D1, and MSP2N2, respectively).

[0134] In some embodiments, at least about 85% (e.g., any of at least about 90%, 95%, 96%, 97%, 98%, 99%, or 100%) of the plurality of nanodiscs in the pharmaceutical composition are fully lipidated. As used herein, “fully lipidated” refers to nanodiscs in which the molar ratio of phospholipid to MSP in the pharmaceutical composition is at or slightly above the phospholipid-MSP saturation ratio N S , such as equal to or 0% to 10% higher than the phospholipid-MSP saturation ratio N S In some embodiments, the N S of an exemplary lipid and MSP combination is shown in Table 2.

[0135] In some embodiments, the nanodiscs comprise a phospholipid and an MSP, wherein at least about 85% (e.g., any of at least about 90%, 95%, 96%, 97%, 98%, 99%, or 100%) of the plurality of nanodiscs are under-lipidated. As used herein, “under-lipidated” refers to nanodiscs in which the molar ratio of phospholipid to MSP in the pharmaceutical composition is less than the phospholipid-MSP saturation ratio N S , such as less than the phospholipid-MSP saturation ratio N Sabout 95% (e.g., less than about any of 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, or 40%). In some embodiments, the N S shown in Table 2. In some embodiments, the molar ratio of phospholipid to MSP in the pharmaceutical composition is less than the phospholipid-MSP saturation ratio N S .

[0136] In some embodiments, without being bound by any scientific theory, the phospholipid-MSP saturation ratio N S 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. disclose that the number of lipids required to saturate a nanodisc in a helical amino acid MSP comprising an amphipathic helical polypeptide of length M amino acid residues that form a lipid binding scaffold (the phospholipid-MSP saturation ratio N S ) is a function of the length M and the average area occupied by a single phospholipid type. In some embodiments, N S may be calculated by Equation 1.1:

[0137] (Equation 1.1)

[0138] where M is the number of amino acid residues in the MSP helix belt surrounding the phospholipid bilayer, L is the helical pitch per MSP residue (e.g., about 0.15 nm), and r is the average radius of the MSP a-helix (e.g., about 0.55 nm). In some embodiments, A L of Equation 1.1 is a constant dependent on the phospholipid, e.g., can be the area (in nm 2 ) occupied by a single phospholipid in the bilayer. In some embodiments, without being bound by any scientific theory, the area A L may depend on the identity of the lipids used and the assembly temperature relative to the bulk bilayer phase transition temperature.

[0139] In some embodiments, M can be calculated according to Equation 1.2:

[0140] (Equation 1.2)

[0141] where r is the average radius of the MSP a-helix (e.g., about 0.55 nm or about 5.5 Å), L is the helical pitch of each MSP residue (e.g., about 0.15 nm or about 1.5 Å), A L is the average surface area of each phospholipid, and N is the number of phospholipids per one phospholipid bilayer. In some embodiments, M can be referenced from Table 2.

[0142] In some embodiments, the phospholipid-MSP saturation ratio N S may be calculated by Equation 1.1, where A L of DPPC and DMPC is about 0.52, and A L of POPC is about 0.69.

[0143] In some embodiments, the length of the helical belt (M) of the MSP variant having the H(0.5) helix sequence that contacts the lipid is the same as compared to the parent MSP protein containing H1 (see Table 1). In some embodiments, this helical belt length (M) can also be the same with or without added histidines or other tags. For example, the helical belt length M of MSP1D1, MSPE1D1, MSPE2D1, MSPE3D1, and MSP2N2 used to calculate N S may be the same as the helical belt length M of MSP1D1(-), MSPE1D1(-), MSPE2D1(-), MSPE3D1(-), and MSP2N2(-) used to calculate N S In some embodiments, the amount of lipid (DMPC, DPPC, and POPC) required to saturate MSP1D1, MSPE1D1, MSPE2D1, MSPE3D1 nanodiscs can be calculated by Equation 1.1, and the results of the calculation are summarized in Table 2.

[0144] Table 2. N of exemplary nanodisks S values

[0145] MSP Amino acid residues in MSP DMPC to MSP molar ratio (N S )]> DPPC to MSP molar ratio (N S ])]] POPC to MSP molar ratio (N S )]> MSP1D1 177 81 81 62 MSP1E1D1 199 107 107 80 MSP1E2D1 221 135 135 102 MSP1E3D1 243 167 167 126 MSP2N2 358 387 387 291

[0146] In some embodiments, without being bound by any scientific theory, the stoichiometry of MSP and phospholipid is critical for the function, shape uniformity, and size distribution of the nanodisc. In some embodiments, the molar ratio of 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 N S In some embodiments, the N Sshown in Table 2.

[0147] In some embodiments, the MSP is MSP1D1 or MSP1, and the molar ratio of phospholipid to MSP (e.g., in a nanodisc, or in a pharmaceutical composition) is 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 MSP (e.g., MSP1D1 or MSP1) (e.g., in a nanodisc, or in a pharmaceutical composition) is about 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 a nanodisc, or in a pharmaceutical composition) is about 10: 1 to about 90: 1.

[0148] In some embodiments, the MSP is MSP1D1 or MSP1, the phospholipid is POPC, and the molar ratio of POPC to MSP (e.g., in a nanodisc, or in a 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 a nanodisc, or in a 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 a nanodisc, or in a 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 a nanodisc, or in a pharmaceutical composition) is about 65: 1. In some embodiments, the MSP is MSP1D1, MSP1, or a His-tag removed variant thereof, the phospholipid is POPC, and the molar ratio of POPC to MSP (e.g., in a nanodisc, or in a pharmaceutical composition) is about 10: 1 to about 65: 1.

[0149] In some embodiments, the MSP is MSP1D1 (e.g., SEQ ID NO: 3) or a His-tag removed variant thereof (e.g., SEQ ID NO: 14), the phospholipid is POPC, and at least about 85% (e.g., any of at least about 90%, 95%, 96%, 97%, 98%, 99%, or 100%) of the plurality of nanodiscs are fully lipidated. In some embodiments, the N S about 62. In some embodiments, the MSP is MSP1D1 or a His-tag removed variant thereof, and the phospholipid is POPC, wherein the molar ratio of POPC to MSP (e.g., in the nanodisc, or in the pharmaceutical composition) is about 65: 1.

[0150] In some embodiments, the MSP is MSP1D1 (e.g., SEQ ID NO: 3) or a His-tag removed variant thereof (e.g., SEQ ID NO: 14), the phospholipid is POPC, and at least about 85% (e.g., any of at least about 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 a His-tag removed variant thereof in the pharmaceutical composition is less than a phospholipid-MSP saturation ratio N S , such as less than a phospholipid-MSP saturation ratio N Sabout 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 a His tag-removed variant thereof, the phospholipid is POPC, and the molar ratio of POPC to 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 a His tag-removed variant of MSP1D1 comprising the amino acid sequence of SEQ ID NO: 14.

[0151] In some embodiments, the MSP is MSP1D1 or MSP1, the phospholipid is DMPC, and the molar ratio of DMPC to MSP (e.g., in a nanodisc, or in a pharmaceutical composition) is about any of 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 a nanodisc, or in a pharmaceutical composition) is about 80: 1. In some embodiments, the MSP is MSP1 or a His tag-removed variant thereof, the phospholipid is DMPC, wherein the molar ratio of DMPC to MSP1 or a His tag-removed variant thereof (e.g., in a nanodisc, or in a 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 a nanodisc, or in a pharmaceutical composition) is about 85: 1. In some embodiments, the MSP is MSP1D1, MSP1, or a His tag-removed variant thereof, the phospholipid is DMPC, wherein the molar ratio of DMPC to MSP in the pharmaceutical composition is about 10: 1 to about 85: 1.

[0152] In some embodiments, the MSP is MSP1D1 (e.g., SEQ ID NO: 3) or a His-tag removed variant thereof (e.g., SEQ ID NO: 14), the phospholipid is DMPC, and at least about 85% (e.g., any of at least about 90%, 95%, 96%, 97%, 98%, 99%, or 100%) of the plurality of nanodiscs are fully lipidated. In some embodiments, the N S about 81. In some embodiments, the MSP is MSP1D1 or a His-tag removed variant thereof, and the phospholipid is DMPC, wherein the molar ratio of DMPC to MSP in the pharmaceutical composition is about 85: 1.

[0153] In some embodiments, the MSP is MSP1D1 (e.g., SEQ ID NO: 3) or a His-tag removed variant thereof (e.g., SEQ ID NO: 14), the phospholipid is DMPC, wherein at least about 85% (e.g., any of at least about 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 a His-tag removed variant thereof in the pharmaceutical composition is less than a phospholipid-MSP saturation ratio N S such as less than a phospholipid-MSP saturation ratio N Sabout 78: 1, such as any of about 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 a 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 a His-tag removed variant thereof, in the pharmaceutical composition is about 65: 1 to about 75: 1.

[0154] In some embodiments, the MSP is MSP1D1 (e.g., SEQ ID NO: 3) or a His-tag removed variant thereof (e.g., SEQ ID NO: 14), and the phospholipid is DMPC, wherein the molar ratio of phospholipid to MSP in the pharmaceutical composition is about 72: 1 to about 78: 1, such as about 75: 1.

[0155] In some embodiments, the MSP is MSP1D1 (e.g., SEQ ID NO: 3) or a His-tag removed variant thereof (e.g., SEQ ID NO: 14), and the phospholipid is DMPC, wherein the molar ratio of phospholipid to MSP in the pharmaceutical composition is about 40: 1 to about 60: 1, such as about 46: 1 to about 55: 1, or about 51: 1.

[0156] In some embodiments, the MSP is MSP1D1 or MSP1, the phospholipid is DPPC, and the molar ratio of DPPC to MSP (e.g., in the nanodiscs, 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 nanodiscs, or in the pharmaceutical composition) is about 90:1. In some embodiments, the MSP is MSP1D1, MSP1, or a His-tag removed variant thereof, the phospholipid is DPPC, wherein the molar ratio of DPPC to MSP (e.g., in the nanodiscs, or in the pharmaceutical composition) is about 10:1 to about 90:1.

[0157] In some embodiments, the MSP is MSP1D1 (e.g., SEQ ID NO: 3) or a His-tag removed variant thereof (e.g., SEQ ID NO: 14), the phospholipid is DPPC, wherein at least about 85% (e.g., any of at least about 90%, 95%, 96%, 97%, 98%, 99%, or 100%) of the plurality of nanodiscs in the pharmaceutical composition are completely lipidated. In some embodiments, the N S In some embodiments, the MSP is MSP1 or a His-tag removed variant thereof, the phospholipid is DPPC, wherein the molar ratio of DPPC to MSP in the pharmaceutical composition is about 90:1.

[0158] In some embodiments, the MSP is MSP1D1 (e.g., SEQ ID NO: 3) or a His-tag removed variant thereof (e.g., SEQ ID NO: 14), the phospholipid is DPPC, wherein at least about 85% (e.g., any of at least about 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 a His-tag removed variant thereof in the pharmaceutical composition is less than a phospholipid-MSP saturation ratio N S , such as less than a phospholipid-MSP saturation ratio N Sabout 95% (e.g., less than about any of 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, or 40%). In some embodiments, the molar ratio of DPPC to MSP1D1, or a 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 a His-tag removed variant thereof, in the pharmaceutical composition is about 10: 1 to about 80: 1, such as about any of 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 a His-tag removed variant thereof, the phospholipid is DPPC, and the molar ratio of DPPC to MSP in the pharmaceutical composition is less than about 75: 1.

[0159] In some embodiments, the MSP is MSP1E3, and the molar ratio of phospholipid to MSP1E3 (e.g., in the nanodiscs or in the pharmaceutical composition) is 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 nanodiscs, or in the pharmaceutical composition) is about 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.

[0160] In some embodiments, the MSP is MSP1E3, MSP1E3D1, or a His-tag removed variant thereof, and the molar ratio of phospholipid to MSP (e.g., in the nanodiscs, or in the pharmaceutical composition) is 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.

[0161] In some embodiments, the MSP is MSP1E3, the phospholipid is POPC, and the molar ratio of POPC to MSP1E3 (e.g., in a nanodisc, or in a pharmaceutical composition) is about 100:1 to about 250:1 (including, e.g., 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 a nanodisc, or in a pharmaceutical composition) is about 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 a nanodisc, or in a pharmaceutical composition) is about 130:1. In some embodiments, the MSP is MSP1E3 or a variant thereof that has a His tag removed, the phospholipid is POPC, wherein the molar ratio of POPC to MSP (e.g., in a nanodisc, or in a pharmaceutical composition) is about 130:1.

[0162] In some embodiments, the MSP is MSP1E3, the phospholipid is DMPC, and the molar ratio of DMPC to MSP1E3 (e.g., in a nanodisc, or in a pharmaceutical composition) is about 100:1 to about 200:1 (including, e.g., 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 a nanodisc, or in a pharmaceutical composition) is about 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 a nanodisc, or in a pharmaceutical composition) is about 150:1. In some embodiments, the MSP is MSP1E3 or a variant thereof that has a His tag removed, the phospholipid is DMPC, wherein the molar ratio of DMPC to MSP (e.g., in a nanodisc, or in a pharmaceutical composition) is about 150:1.

[0163] In some embodiments, the MSP is MSP1E3, the phospholipid is DPPC, and the molar ratio of DPPC to MSP1E3 (e.g., in the nanodiscs, or in the pharmaceutical composition) is about 120: 1 to about 220: 1, including for example 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 nanodiscs, 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 nanodiscs, or in the pharmaceutical composition) is about 170: 1. In some embodiments, the MSP is MSP1E3 or a His-tag removed variant thereof, the phospholipid is DPPC, wherein the molar ratio of DPPC to MSP (e.g., in the nanodiscs, or in the pharmaceutical composition) is about 170: 1.

[0164] In some embodiments, the MSP is MSP1E3D1 (e.g., SEQ ID NO: 8) or a 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 completely lipidated. In some embodiments, the MSP is MSP1E3D1 or a His-tag removed variant thereof, the phospholipid is DMPC, wherein the molar ratio of phospholipid to MSP in the pharmaceutical composition is less than a phospholipid-MSP saturation ratio N S about 167.

[0165] In some embodiments, the MSP is MSP1E3D1 (e.g., SEQ ID NO: 8) or a 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 a His-tag removed variant thereof, the phospholipid is DMPC, wherein the molar ratio of phospholipid to MSP in the pharmaceutical composition is less than a phospholipid-MSP saturation ratio N S about 167. Sabout 60%, 55%, 50%, 45%, or 40%). In some embodiments, the molar ratio of DMPC to MSP1E3D1, or a 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 any of about 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 a His tag removed variant thereof, in the pharmaceutical composition is about 50: 1 to about 160: 1, such as about any of 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 a His tag removed variant thereof, the phospholipid is DMPC, and the molar ratio of DMPC to MSP in the pharmaceutical composition is about 150: 1.

[0166] In some embodiments, the MSP is MSP1E3D1 (e.g., SEQ ID NO: 8) or a His tag removed variant thereof (e.g., SEQ ID NO: 15), the phospholipid is DMPC, wherein the molar ratio of DMPC to 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.

[0167] In some embodiments, the MSP is MSP1E3D1, the phospholipid is DMPC, and the molar ratio of 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 a His tag removed variant of MSP1E3D1 comprising the amino acid sequence of SEQ ID NO: 15.

[0168] In some embodiments, the MSP is MSP1E3D1 (e.g., SEQ ID NO: 8) or a His-tag removed variant thereof (e.g., SEQ ID NO: 15), the phospholipid is DPPC, and at least about 85% (e.g., any of at least about 90%, 95%, 96%, 97%, 98%, 99%, or 100%) of the plurality of nanodiscs in the pharmaceutical composition are fully lipidated. In some embodiments, the MSP is MSP1E3D1 or a His-tag removed variant thereof, the phospholipid is DPPC, and the molar ratio of DPPC to MSP in the pharmaceutical composition is about 170: 1. S In some embodiments, the MSP is MSP1E3D1 (e.g., SEQ ID NO: 8) or a His-tag removed variant thereof (e.g., SEQ ID NO: 15), the phospholipid is DPPC, and at least about 85% (e.g., any of at least about 90%, 95%, 96%, 97%, 98%, 99%, or 100%) of the plurality of nanodiscs in the pharmaceutical composition are fully lipidated. In some embodiments, the MSP is MSP1E3D1 or a His-tag removed variant thereof, the phospholipid is DPPC, and the molar ratio of DPPC to MSP in the pharmaceutical composition is about 170: 1.

[0169] In some embodiments, the MSP is MSP1E3D1 (e.g., SEQ ID NO: 8) or a His-tag removed variant thereof (e.g., SEQ ID NO: 15), the phospholipid is DPPC, and at least about 85% (e.g., any of at least about 90%, 95%, 96%, 97%, 98%, 99%, or 100%) of the plurality of nanodiscs in the pharmaceutical composition are fully lipidated. In some embodiments, the MSP is MSP1E3D1 or a His-tag removed variant thereof, the phospholipid is DPPC, and the molar ratio of DPPC to MSP in the pharmaceutical composition is about 170: 1. S In some embodiments, the MSP is MSP1E3D1 (e.g., SEQ ID NO: 8) or a His-tag removed variant thereof (e.g., SEQ ID NO: 15), the phospholipid is DPPC, and at least about 85% (e.g., any of at least about 90%, 95%, 96%, 97%, 98%, 99%, or 100%) of the plurality of nanodiscs in the pharmaceutical composition are fully lipidated. In some embodiments, the MSP is MSP1E3D1 or a His-tag removed variant thereof, the phospholipid is DPPC, and the molar ratio of DPPC to MSP in the pharmaceutical composition is about 170: 1. Sabout 95% (e.g., less than about any of 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, or 40%). In some embodiments, the molar ratio of DPPC to MSP1E3D1, or a 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 a His tag-removed variant thereof, in the pharmaceutical composition is about 70: 1 to about 160: 1, such as about any of 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 a His tag-removed variant thereof, and the phospholipid is DPPC, wherein the molar ratio of DPPC to MSP in the pharmaceutical composition is less than about 158: 1. In some embodiments, the MSP is MSP1E3D1. In some embodiments, the MSP is a His tag-removed variant of MSP1E3D1 comprising the amino acid sequence of SEQ ID NO: 15.

[0170] In some embodiments, the MSP is MSP1E3D1 (e.g., SEQ ID NO: 8) or a His tag-removed variant thereof (e.g., SEQ ID NO: 15), and 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 plurality of nanodiscs are fully lipidated. S about 126.

[0171] In some embodiments, the MSP is MSP1E3D1 (e.g., SEQ ID NO: 8) or a variant thereof with its His tag removed (e.g., SEQ ID NO: 15), and the phospholipid is POPC, wherein at least about 85% (e.g., at least about 90%, 95%, 96%, 97%, 98%, 99%, or 100%) of the plurality of nanodiscs are under-esterified. In some embodiments, the MSP is MSP1E3D1 or a variant thereof with its His tag removed, and the phospholipid is POPC, wherein the molar ratio of POPC to MSP in the pharmaceutical composition is less than a phospholipid-MSP saturation ratio N of about 126:1. S Such as a phospholipid-MSP saturation ratio of less than approximately 126:1 N S The content is approximately 95% (e.g., less than any one of about 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, or 40%). In some embodiments, the molar ratio of POPC in the pharmaceutical composition to MSP1E3D1 or its His-labeled variant is less than or equal to about 120:1, such as less than or equal to any one or less of about 118:1, 115:1, 110:1, 105:1, 100:1, 95:1, 90:1, 85:1, 80:1, 75:1, 70:1, or 65:1. In some embodiments, the molar ratio of POPC to MSP1E3D1 or a His-tagged variant thereof in the pharmaceutical composition is from about 60:1 to about 120:1, such as from about 70:1 to about 120:1, from about 70:1 to about 110:1, from about 70:1 to about 100:1, from about 80:1 to about 110:1, from about 80:1 to about 100:1, from about 80:1, from about 90:1, from about 100:1, or from about 110:1. In some embodiments, the MSP is MSP1E3D1 or a His-tagged variant thereof, and the phospholipid is POPC, wherein the molar ratio of POPC to MSP in the pharmaceutical composition is less than about 118:1. In some embodiments, the MSP is MSP1E3D1. In some embodiments, the MSP is a His-tagged variant of MSP1E3D1 comprising the amino acid sequence of SEQ ID NO: 15.

[0172] In some embodiments, the MSP is MSP1E1D1 (e.g., SEQ ID NO: 16) or a variant thereof with its His tag removed, and the phospholipid is DMPC, wherein at least about 85% (e.g., at least about 90%, 95%, 96%, 97%, 98%, 99%, or 100%) of the plurality of nanodiscs in the pharmaceutical composition are fully esterified. In some embodiments, N S It is approximately 107.

[0173] In some embodiments, the MSP is MSP1E1D1 (e.g., SEQ ID NO: 16) or a His tag- removed variant thereof, the phospholipid is DMPC, wherein at least about 85% (e.g., any of at least about 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 a His tag-removed variant thereof, the phospholipid is DMPC, wherein the molar ratio of phospholipid to MSP in the pharmaceutical composition is less than a phospholipid-MSP saturation ratio N S , such as less than a phospholipid-MSP saturation ratio N S of about 95% (e.g., less than any of about 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, or 40%). In some embodiments, the molar ratio of DMPC to MSP1D1E1 or a 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 any of about 100: 1, 95: 1, 90: 1, 85: 1, 80: 1, 75: 1, 70: 1, 65: 1, 60: 1, 55: 1, 50: 1, 45: 1, or less. In some embodiments, the molar ratio of DMPC to MSP1E1D1 or a 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 a 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.

[0174] In some embodiments, the MSP is MSP1E1D1 (e.g., SEQ ID NO: 16) or a His tag- removed variant thereof, the phospholipid is DPPC, wherein at least about 85% (e.g., any of at least about 90%, 95%, 96%, 97%, 98%, 99%, or 100%) of the plurality of nanodiscs in the pharmaceutical composition are fully lipidated. In some embodiments, N S is about 107.

[0175] In some embodiments, the MSP is MSP1E1D1 (e.g., SEQ ID NO: 16) or a His tag- removed variant thereof, the phospholipid is DPPC, wherein at least about 85% (e.g., any of at least about 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 a His tag-removed variant thereof, the phospholipid is DPPC, wherein the molar ratio of phospholipid to MSP in the pharmaceutical composition is less than a phospholipid-MSP saturation ratio N S , such as less than a phospholipid-MSP saturation ratio N S of about 95% (e.g., less than any of about 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, or 40%). In some embodiments, the molar ratio of DPPC to MSP1D1E1 or a 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 any of about 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 a 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 a 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.

[0176] In some embodiments, the MSP is MSP1E1D1 (e.g., SEQ ID NO: 16) or a His tag- removed variant thereof, the phospholipid is POPC, wherein at least about 85% (e.g., any of at least about 90%, 95%, 96%, 97%, 98%, 99%, or 100%) of the plurality of nanodiscs in the pharmaceutical composition are fully lipidated. In some embodiments, N S is about 80.

[0177] In some embodiments, the MSP is MSP1E1D1 (e.g., SEQ ID NO: 16) or a His tag- removed variant thereof, the phospholipid is POPC, and at least about 85% (e.g., any of at least about 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 a His tag-removed variant thereof, the phospholipid is POPC, wherein the molar ratio of phospholipid to MSP in the pharmaceutical composition is less than a phospholipid-MSP saturation ratio N S , such as less than a phospholipid-MSP saturation ratio N S of about 95% (e.g., less than any of about 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, or 40%). In some embodiments, the molar ratio of POPC to MSP1E1D1 or a 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 any of about 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 a His tag-removed variant thereof in the pharmaceutical composition is about 10: 1 to about 75: 1, such as any of about 20: 1 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 a 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.

[0178] In some embodiments, the MSP is MSP1E2D1 (e.g., SEQ ID NO: 17) or a His tag- removed variant thereof, the phospholipid is DMPC, wherein at least about 85% (e.g., any of at least about 90%, 95%, 96%, 97%, 98%, 99%, or 100%) of the plurality of nanodiscs in the pharmaceutical composition are fully lipidated. In some embodiments, N S is about 135.

[0179] In some embodiments, the MSP is MSP1E2D1 (e.g., SEQ ID NO: 17) or a His-tag removed variant thereof, the phospholipid is DMPC, wherein at least about 85% (e.g., any of at least about 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 a His-tag removed variant thereof, the phospholipid is DMPC, wherein the molar ratio of phospholipid to MSP in the pharmaceutical composition is less than a phospholipid-MSP saturation ratio N S , such as less than a phospholipid-MSP saturation ratio N S of about 95% (e.g., less than any of about 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, or 40%). In some embodiments, the molar ratio of DMPC to MSP1E2D1 or a 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 any of about 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 a 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.

[0180] In some embodiments, the MSP is MSP1E2D1 (e.g., SEQ ID NO: 17) or a His-tag removed variant thereof, the phospholipid is DPPC, wherein at least about 85% (e.g., any of at least about 90%, 95%, 96%, 97%, 98%, 99%, or 100%) of the plurality of nanodiscs in the pharmaceutical composition are fully lipidated. In some embodiments, N S is about 135.

[0181] In some embodiments, the MSP is MSP1E2D1 (e.g., SEQ ID NO: 17) or a His-tag removed variant thereof, the phospholipid is DPPC, wherein at least about 85% (e.g., any of at least about 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 a His-tag removed variant thereof, the phospholipid is DPPC, wherein the molar ratio of phospholipid to MSP in the pharmaceutical composition is less than a phospholipid-MSP saturation ratio N S , such as less than a phospholipid-MSP saturation ratio N S of about 95% (e.g., less than any of about 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, or 40%). In some embodiments, the molar ratio of DPPC to MSP1E2D1 or a 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 any of about 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 a 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.

[0182] In some embodiments, the MSP is MSP1E2D1 (e.g., SEQ ID NO: 17) or a His-tag removed variant thereof, the phospholipid is POPC, wherein at least about 85% (e.g., any of at least about 90%, 95%, 96%, 97%, 98%, 99%, or 100%) of the plurality of nanodiscs in the pharmaceutical composition are fully lipidated. In some embodiments, N S is about 102.

[0183] In some embodiments, the MSP is MSP1E2D1 (e.g., SEQ ID NO: 17) or a His tag- removed variant thereof, the phospholipid is POPC, and at least about 85% (e.g., any of at least about 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 a His tag-removed variant thereof, the phospholipid is POPC, wherein the molar ratio of phospholipid to MSP in the pharmaceutical composition is less than a phospholipid-MSP saturation ratio N S , such as less than a phospholipid-MSP saturation ratio N S of about 102: 1 (e.g., any of less than about 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, or 40%). In some embodiments, the molar ratio of POPC to MSP1E2D1 or a His tag-removed variant thereof in the pharmaceutical composition is less than about 96: 1, such as any of less than about 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 a 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.

[0184] In some embodiments, the MSP is MSP2N2 (e.g., SEQ ID NO: 18) or a His tag- removed variant thereof, the phospholipid is DMPC, wherein at least about 85% (e.g., any of at least about 90%, 95%, 96%, 97%, 98%, 99%, or 100%) of the plurality of nanodiscs in the pharmaceutical composition are fully lipidated. In some embodiments, N S is about 387.

[0185] In some embodiments, the MSP is MSP2N2 (e.g., SEQ ID NO: 18) or a His-tag removed variant thereof, the phospholipid is DMPC, and at least about 85% (e.g., any of at least about 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 (e.g., SEQ ID NO: 18) or a His-tag removed variant thereof, the phospholipid is DMPC, wherein the molar ratio of phospholipid to MSP in the pharmaceutical composition is less than the phospholipid-MSP saturation ratio N S of about 387: 1, such as less than about 95% (e.g., any of less than about 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, or 40%) of the phospholipid-MSP saturation ratio N S of about 387: 1. In some embodiments, the molar ratio of DMPC to MSP2N2 or a 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 any of about 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 a 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.

[0186] In some embodiments, the MSP is MSP2N2 (e.g., SEQ ID NO: 18) or a His-tag removed variant thereof, the phospholipid is DPPC, and at least about 85% (e.g., any of at least about 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.

[0187] In some embodiments, the MSP is MSP2N2 (e.g., SEQ ID NO: 18) or a His-tag removed variant thereof, the phospholipid is DPPC, and at least about 85% (e.g., any of at least about 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 a His-tag removed variant thereof, the phospholipid is DPPC, and the molar ratio of phospholipid to MSP in the pharmaceutical composition is less than the phospholipid-MSP saturation ratio N S , such as less than the phospholipid-MSP saturation ratio N S of about 95% (e.g., less than any of about 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, or 40%). In some embodiments, the molar ratio of DPPC to MSP2N2 or a 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 any of about 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 a 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.

[0188] In some embodiments, the MSP is MSP2N2 (e.g., SEQ ID NO: 18) or a His-tag removed variant thereof, the phospholipid is POPC, and at least about 85% (e.g., any of at least about 90%, 95%, 96%, 97%, 98%, 99%, or 100%) of the plurality of nanodiscs in the pharmaceutical composition are fully lipidated. In some embodiments, N S is about 291.

[0189] In some embodiments, the MSP is MSP2N2 (e.g., SEQ ID NO: 18) or a His-tag removed variant thereof, the phospholipid is POPC, wherein at least about 85% (e.g., any of at least about 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 a His-tag removed variant thereof, the phospholipid is POPC, wherein the molar ratio of phospholipid to MSP in the pharmaceutical composition is less than the phospholipid-MSP saturation ratio N S , such as less than the phospholipid-MSP saturation ratio N S of about 95% (e.g., any of less than about 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, or 40%). In some embodiments, the molar ratio of POPC to MSP2N2 or a His-tag removed variant thereof in the pharmaceutical composition is less than or equal to about 280: 1, such as any of less than or equal to about 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 a 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.

[0190] d. Properties

[0191] 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.

[0192] In some embodiments, the nanodiscs increase the rate of cholesterol efflux at least about 2-fold (e.g., at least about any one of 2.5-fold, 3-fold, 4-fold, 5-fold, 10-fold, 15-fold or more) compared to an untreated state (e.g., prior to administration of the nanodiscs) or the rate of cholesterol efflux achieved by ApoA-I or MSP (e.g., MSP1D1). In some embodiments, the nanodiscs increase the rate of cholesterol efflux in retinal pigment epithelial cells or macrophages (e.g., by at least about any one of 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 10-fold or more). In some embodiments, the nanodiscs decrease lipid in the eye of the individual by at least about 5% (e.g., by at least about any one of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%) or prevent accumulation of lipid in the eye of the individual.

[0193] In some embodiments, the nanodisc pharmaceutical compositions described herein increase the rate of cholesterol efflux at least about 20% (e.g., at least about any one 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 the rate of cholesterol efflux achieved by ApoA-I or high density lipoprotein (HDL) alone. In some embodiments, the under-lipidated nanodisc pharmaceutical compositions described herein increase the rate of cholesterol efflux at least about 20% (e.g., at least about any one 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 (molar ratio of phospholipid to MSP in the pharmaceutical composition is about the phospholipid-MSP saturation ratio N S ) containing the same MSP and phospholipid.

[0194] In some embodiments, the nanodiscs do 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 translocation sequence (MTS).

[0195] In some embodiments, the nanodiscs do not comprise a target protein. In some embodiments, the nanodiscs do not comprise a prophylactic or therapeutic agent.

[0196] 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 nanodisc can be tuned by the length of the MSP. In some embodiments, the diameter of the nanodisc allows the nanodisc to penetrate into or through an ocular muscle. In some embodiments, the diameter of the nanodisc allows the nanodisc to reach the back of the eye without a CPP. In some embodiments, the diameter of the nanodisc can be tuned by the length of the MSP band.

[0197] In some embodiments, the average diameter of the nanodisc 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 nanodisc 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 nanodisc can be tuned by the length of the MSP.

[0198] 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.

[0199] 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.

[0200] 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 at about 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% by weight. In some embodiments, the nanodisc comprises phospholipid at about 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% by weight. In some embodiments, the nanodisc comprises MSP and phospholipid at about any of about 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 by weight. In some embodiments, about any of about 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 nanodisc is MSP.

[0201] In some embodiments, the polydispersity index (a measure of 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 can be determined by combining multi-angle light scattering with size exclusion chromatography (SEC-MALS). In some embodiments, at least about any of 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 any of 60%, such as at least about any of 70%, 75%, 80%, 85%, 90%, 95%, or 99% of the nanodiscs retain a size of about 2 nm to about 20 nm after being frozen and thawed one or more times, such as after being frozen at -80 °C and thawed with or without a cryoprotectant. In some embodiments, at least about any of 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 substantially unchanged size (e.g., the size of the nanodiscs in the pharmaceutical composition changes by less than about any of 20%, 15%, 10%, 5%, 2%, or 1%) after being frozen and thawed one or more times, such as after being frozen at -80 °C and thawed with or without a cryoprotectant.

[0202] In some embodiments, the polydispersity index (a measure of 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 any of 60%, such as at least about any of 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% of the nanodiscs have a molar ratio of phospholipid to MSP that is less than the phospholipid-MSP saturation ratio N S In some embodiments, at least about any of 60%, such as at least about any of 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% of the nanodiscs have a molar ratio of phospholipid to MSP that is less than the phospholipid-MSP saturation ratio N Sabout 95% (e.g., less than about any of 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, or 40%). In some embodiments, the molar ratio between the phospholipid and the MSP remains substantially unchanged (e.g., the size of the nanodiscs in the pharmaceutical composition changes by less than about any of 20%, 15%, 10%, 5%, 2%, or 1%) for at least about 60%, such as at least about any of 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% of the nanodiscs after being frozen and thawed one or more times, such as being frozen at -80 °C and thawed with or without a cryoprotectant.

[0203] In some embodiments, the molar ratio between the phospholipid and the MSP remains substantially unchanged (e.g., the size of the nanodiscs in the pharmaceutical composition changes by less than about any of 20%, 15%, 10%, 5%, 2%, or 1%) in the pharmaceutical composition after being stored at room temperature for at least about 1 hour, such as at least about any of 2 hours, 5 hours, 10 hours, 12 hours, 15 hours, 20 hours, or 24 hours, or 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 15 days, 30 days, 40 days, 50 days, 100 days. In some embodiments, the molar ratio between the phospholipid and the MSP remains substantially unchanged (e.g., the size of the nanodiscs in the pharmaceutical composition changes by less than about any of 20%, 15%, 10%, 5%, 2%, or 1%) after being stored at -80 °C for at least about 1 month, such as at least about any of 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 15 months, 30 months, 40 months, 50 months, 100 months.

[0204] e. Methods of preparation

[0205] Nanodiscs can be formed by the interaction between the MSP and the phospholipid. Methods of making nanodiscs are known in the art, and a description of an exemplary method 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 2002 2 (8), 853-856. DOI: 10.1021 / nl025623k, the contents of which are incorporated by reference herein in their entirety. See also Example 1 and Examples 8-10 for exemplary methods.

[0206] In some embodiments, 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) is provided, the method comprising: (1) incubating MSP and a phospholipid to obtain a preparation mixture, wherein prior to incubation, the phospholipid is solubilized by a detergent or an organic compound; and (2) removing the detergent or organic compound from the preparation mixture, thereby obtaining the nanoparticle. In some embodiments, the molar ratio of phospholipid to 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 phospholipid to MSP in the pharmaceutical composition. In some embodiments, the method further comprises purifying the obtained nanodiscs (one or more times), e.g., by SEC. 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.

[0207] In some embodiments, the nanodiscs can be synthesized by incubating solubilized phospholipids with MSPs. In some embodiments, the phospholipids are solubilized by a detergent or organic compound. In some embodiments, the solubilized phospholipids are prepared by adding a solution of a detergent or organic compound to a phospholipid film. Exemplary detergents or organic compounds include, but are not limited to, alkyl glucosides such as n-dodecyl-P-D-maltoside (DDM), octyl-P-glucoside (OG), Triton X-100, 3-[(3-cholamidopropyl)dimethylammonio]-l-propanesulfonate (CHAPS), 2,2-didecylpropane-l,3-bis-P-D-maltopyranoside (LMNG), cholate salts, and any combination thereof. In some embodiments, the phospholipids are solubilized by a cholate salt, such as sodium cholate. In some embodiments, the MSPs are 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 MSPs in the solution are 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 MSPs in the solution are about 0.5 mg / mL to about 10 mg / mL, such as about any of 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.

[0208] In some embodiments, the method comprises removing the detergent or organic compound from the preparation mixture, thereby obtaining the nanodiscs. In some embodiments, after the nanodiscs are formed, the detergent or organic compound (e.g., cholate salt) 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 the nanodiscs can be determined by tools known in the art, such as size exclusion chromatography (SEC) and electron microscopy. ® XAD-2 ® beads). In some embodiments, the formation and size of the nanodiscs can be determined by tools known in the art, such as size exclusion chromatography (SEC) and electron microscopy.

[0209] 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 a cryoprotectant.

[0210] In some embodiments, the method further comprises purifying the prepared nanodisks, such as isolating nanodisks having different phospholipid to MSP ratios. In some embodiments, the purifying comprises using SEC. In some embodiments, the method comprises more than one purification cycle until a desired distribution of phospholipid to MSP ratios is achieved (e.g., full saturation, or less than the phospholipid-polymer saturation ratio N S ].

[0211] In some embodiments, the method further comprises measuring the actual phospholipid to MSP ratio in the nanodisk pharmaceutical composition. In some embodiments, the actual phospholipid to MSP ratio in the nanodisk pharmaceutical composition can be determined by measuring the fatty acid content using gas chromatography (GC) analysis.

[0212] In some embodiments, the molar ratio of phospholipid to MSP in the preparation mixture (e.g., the mixture of step (1) of the preparation method described above) is the same as the molar ratio of phospholipid to MSP in the obtained nanodisk pharmaceutical composition. In some embodiments, the molar ratio of phospholipid to MSP in the preparation mixture (e.g., the mixture of step (1) of the preparation method described above) is less than the molar ratio of phospholipid to MSP in the obtained nanodisk pharmaceutical composition (e.g., less than about any of 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, or 40%). In some embodiments, the molar ratio of phospholipid to 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 nanodisks in the preparation state are under-lipidated, such as having a molar ratio of phospholipid to MSP that is less than the phospholipid-MSP saturation ratio N S In some embodiments, the molar ratio of phospholipid to 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 nanodisks in the preparation state are fully lipidated.

[0213] In some embodiments, provided herein is a pharmaceutical composition comprising nanodisks prepared according to any of the preparation methods detailed herein.

[0214] 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.

[0215] Routes of administration and dosages

[0216] The nanodisks described herein, or pharmaceutical compositions thereof, can be administered by any suitable route of administration. In some embodiments, the nanodisks, or pharmaceutical compositions thereof, are administered via topical ocular administration.

[0217] In some embodiments, suitable routes of administration include, but are not limited to, intravitreal, topical, periocular injection, intraocular or periocular implant, intravitreal implant, and suprachoroidal implant or particulate or polymeric compositions, or any release system such as emulsion, solid non-biodegradable or biodegradable implant or tablet, or a micro pump. In some embodiments, the nanodisks, or pharmaceutical compositions thereof, are administered directly to the eye by injection into ocular tissue, such as periocular, conjunctival, subtenon, intracameral, intravitreal, intraocular, subretinal, subconjunctival, retrobulbar, suprachoroidal, or intracanalicular injection. In some embodiments, the nanodisks, 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 implant comprising porous, non-porous, or gelatinous material. In some embodiments, the nanodisks, or pharmaceutical compositions thereof, are administered by topical ocular drops or ointment. In some embodiments, the nanodisks, or pharmaceutical compositions thereof, are administered in the fornices by a slow release device, or implanted near (transsclerally) or in (intrascierally) the sclera or suprachoroidally or intraocularly. Intracameral injection can be through the cornea of the eye into the anterior chamber to allow the agent to reach the trabecular meshwork. Intracanalicular injection can be into the venous collector channel of the draining Schlemm’s canal or into the Schlemm’s canal. In some embodiments, the nanodisks, or pharmaceutical compositions thereof, are administered intravitreally.

[0218] In some embodiments, the pharmaceutical composition comprising nanodisks (e.g., a plurality of nanodisks) can be in the form of an isotonic sterile saline solution (sodium phosphate dibasic or monobasic, sodium chloride, potassium chloride, calcium chloride, or magnesium chloride, etc. or mixtures of such salts), a sterile aqueous dispersion (e.g., water-oil emulsion), or a dry, especially lyophilized composition, which, on the addition of sterile water or physiological saline, as the case can be, allows the constitution of an injectable solution.

[0219] In some embodiments, the pharmaceutical composition comprising nanodisks (e.g., a plurality of nanodisks) and a pharmaceutically acceptable excipient is administered as an ophthalmic eye drop. Suitable pharmaceutically acceptable excipients include, but are not limited to, an ophthalmically acceptable preservative, solvent, surfactant, viscosity enhancer, penetration enhancer, buffer, isotonic agent, stabilizer, pH adjuster, 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 a viscosity builder such as hydroxymethylcellulose, hydroxyethylcellulose, hydroxypropylmethylcellulose, methylcellulose, carboxymethylcellulose, polyvinylpyrrolidone, hyaluronic acid, and the like.

[0220] In some embodiments, the pharmaceutical composition comprising nanodisks (e.g., a plurality of nanodisks) 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 petrolatum, or white petrolatum.

[0221] In some embodiments, the pharmaceutical composition is an anhydrous dosage form comprising nanodisks. Such anhydrous dosage forms can be prepared using anhydrous or low moisture content ingredients and low moisture or low humidity conditions.

[0222] 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 buffered saline (PBS). In some embodiments, the isotonic buffer does not comprise tris.

[0223] 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, methyl paraben, polyvinyl alcohol, and phenethyl alcohol.

[0224] In some embodiments, the excipient can be selected by one of ordinary skill in the art. Exemplary excipients include, for example, those described in Handbook of Pharmaceutical Excipients, Rowe et al. (eds.) 6thEd. (2009), the contents of which are incorporated herein by reference in their entirety.

[0225] In some embodiments, the physician can determine that the dose they consider appropriate according to the prophylactic or therapeutic treatment and according to the age, weight, condition, and other factors specific to the individual to be treated.

[0226] In some embodiments, the frequency and dose can also vary according to factors specific to each individual, depending on the particular therapy (e.g., therapeutic or prophylactic), the route of administration, and the age, body, weight, response, and past medical history of the individual. In some embodiments, an effective dose can be extrapolated from dose-response curves derived from in vitro or animal model test systems.

[0227] In some embodiments, the dosage of the nanodisc or pharmaceutical composition thereof when administered to remove or prevent the accumulation of lipids (e.g., cholesterol) in the eye, or to prevent or treat an eye disease characterized by the accumulation of lipids (e.g., cholesterol) in the individual is from about 50 pg / kg of the individual’s weight to about 50 mg / kg of the individual’s weight (including, for example, any of from about 50 pg / kg of the individual’s weight to about 30 mg / kg of the individual’s weight, from about 70 pg / kg of the individual’s weight to about 20 mg / kg of the individual’s weight, from about 100 pg / kg of the individual’s weight to about 10 mg / kg of the individual’s weight, from about 100 pg / kg of the individual’s weight to about 500 pg / kg of the individual’s weight, or from about 200 pg / kg of the individual’s weight to about 500 pg / kg of the individual’s weight). In some embodiments, the dosage of the nanodisc or pharmaceutical composition thereof when administered to remove or prevent the accumulation of lipids (e.g., cholesterol) in the eye, or to prevent or treat an eye disease characterized by the accumulation of lipids (e.g., cholesterol) in the individual is from about 0.1 pg / kg of the individual’s weight to about 20 pg / kg of the individual’s weight (including, for example, any of from about 0.5 pg / kg of the individual’s weight to about 18 mg / kg of the individual’s weight, from about 1 pg / kg of the individual’s weight to about 15 mg / kg of the individual’s weight, from about 1 pg / kg of the individual’s weight to about 10 mg / kg of the individual’s weight, from about 1.5 pg / kg of the individual’s weight to about 9 pg / kg of the individual’s weight, or from about 2 pg / kg of the individual’s weight to about 9 pg / kg of the individual’s weight). In some embodiments, the dosage of the nanodisc or pharmaceutical composition thereof for use in the methods described herein is from about 1 pg to about 50 mg (including, for example, any of from about 1 pg to about 30 mg, from about 1 pg to about 20 mg, from about 1 pg to about 10 mg, from about 10 pg to about 20 mg, about 10 pg, or about 15 mg). In some embodiments, the dosage of the nanodisc or pharmaceutical composition thereof for use in the methods described herein is from about 1 pg to about 1000 pg (including, for example, any of from about 5 pg to about 800 mg, from about 5 pg to about 600 mg, from about 8 pg to about 600 mg, or from about 10 pg to about 600 mg).

[0228] In some embodiments, a dose of the nanodisc or pharmaceutical composition thereof can be administered according to a suitable schedule, e.g., 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, administration of the nanodisc or pharmaceutical composition thereof can be repeated once about every three days. In some embodiments, administration of the nanodisc or pharmaceutical composition thereof can be repeated once about every 2 to 4 weeks. In some embodiments, administration of the nanodisc or pharmaceutical composition thereof can be repeated once about every 3 weeks. In some embodiments, administration of the nanodisc or pharmaceutical composition thereof can be repeated, such as 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 apart.

[0229] In some cases, it can be necessary to use a dose of the nanodisc or pharmaceutical composition thereof outside the ranges disclosed herein, 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 individual response.

[0230] Enumerated embodiments

[0231] Embodiment 1. A method of removing or preventing accumulation of lipids in an eye of an individual, the method 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.

[0232] Embodiment 2. A method of preventing or treating an eye disease characterized by accumulation of lipids in an individual, the method comprising administering to the eye of the individual an effective amount of a pharmaceutical composition comprising a nanodisc, wherein the nanodisc comprises a MSP and a phospholipid.

[0233] Embodiment 3. The method of embodiment 1 or 2, wherein the MSP comprises an apolipoprotein variant selected from the group consisting of apolipoprotein A-I (ApoA-I), ApoA-II, ApoC, ApoE, and ApoM.

[0234] Embodiment 4. The method of any one of embodiments 1 to 3, wherein the MSP is a variant of ApoA-I.

[0235] Embodiment 5. The method of embodiment 4, wherein the MSP comprises one or more amphipathic helix sequences relative to ApoA-I.

[0236] Embodiment 6. The method of embodiment 5, wherein the one or more amphipathic helix sequences are derived from ApoA-I.

[0237] Embodiment 7. The method of embodiments 5 or 6, wherein the MSP is MSP1E1, MSP1E2, or MSP1E3.

[0238] Embodiment 8. The method of any one of embodiments 4-7, wherein the MSP comprises a truncation relative to the N-terminus of ApoA-I.

[0239] Embodiment 9. The method of embodiment 8, wherein the MSP is MSP1, MSP1D1, MSP1D2, or MSP1E3D1.

[0240] Embodiment 10. The method of any one of embodiments 1-9, wherein the phospholipid is phosphatidylcholine (PC).

[0241] Embodiment 11. The method of embodiment 10, wherein the phospholipid is selected from the group consisting of dimyristoyl phosphatidylcholine (DMPC), palmitoyloleoyl phosphatidylcholine (POPC), dipalmitoyl phosphatidylcholine (DPPC), distearoyl phosphatidylcholine (DSPC), and combinations thereof.

[0242] Embodiment 12. The method of any one of embodiments 10 or 11, wherein the phospholipid is DMPC, DPPC, or POPC.

[0243] 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 about 10: 1 to about 90: 1.

[0244] Embodiment 14. The method of embodiment 13, wherein the phospholipid is POPC, and wherein the molar ratio of POPC to MSP is about 10: 1 to about 65: 1.

[0245] 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.

[0246] 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.

[0247] Embodiment 17. The method of embodiment 13, wherein the phospholipid is DMPC, and wherein the molar ratio of DMPC to MSP is about 10: 1 to about 85: 1.

[0248] 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.

[0249] 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.

[0250] 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.

[0251] 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 about 100: 1 to about 200: 1.

[0252] 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.

[0253] 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.

[0254] 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.

[0255] Embodiment 25. The method of any one of embodiments 1-24, wherein the nanodisc has a diameter of about 5 nm to about 20 nm.

[0256] Embodiment 26. The method of any one of embodiments 1-25, wherein the lipid that is removed or prevented from accumulating in the eye is cholesterol.

[0257] Embodiment 27. The method of any one of embodiments 1-26, wherein the nanodisc does not comprise a cell-penetrating peptide (CPP).

[0258] Embodiment 28. The method of embodiment 27, wherein the CPP is selected from the group consisting of a penetratin, polyarginine (R8), LL-37, a transport protein, Pep-1, and a membrane translocating sequence (MTS).

[0259] Embodiment 29. The method of any one of embodiments 1-28, wherein the nanodisc does not comprise a target protein.

[0260] Embodiment 30. The method of any one of embodiments 1 to 29, wherein the nanodisc does not comprise a prophylactic or a therapeutic agent.

[0261] Embodiment 31. The method of any one of embodiments 26 to 30, wherein the nanodisc increases the rate of cholesterol efflux at least about 2-fold compared to an untreated state or by ApoA-I or MSP.

[0262] Embodiment 32. The method of any one of embodiments 1 to 31, wherein the pharmaceutical composition is administered via intravitreal injection.

[0263] Embodiment 33. The method of any one of embodiments 2 to 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, vision loss, hemianopsia, visual agnosia, strabismus, retinal neurovascular disorders, lipid keratopathy, corneal lipidosis, and combinations thereof.

[0264] Embodiment 34. The method of embodiment 33, wherein the eye disease characterized by lipid accumulation is AMD.

[0265] Embodiment 35. The method of embodiment 34, wherein the AMD is dry AMD.

[0266] Embodiment 36. The method of embodiment 35, wherein the dry AMD is geographic atrophy (GA).

[0267] Embodiment 37. The method of any one of embodiments 1 to 36, wherein the nanodisc reduces lipid in the eye of the individual by at least about 5% or prevents lipid accumulation in the eye of the individual.

[0268] Examples

[0269] Example 1: Preparation of nanodisks

[0270] Nanodiscs are assembled from phospholipids, 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 contents of which are incorporated by reference herein in their entirety

[0271] In short, a frozen solution containing MSP in 20 mM glycerol (Tris) (pH 7.4), 0.1 M NaCl, 0.5 mM EDTA, and 0.01% NaN3 was thawed and filtered through a 0.22 micron filter. The concentration of MSP was determined spectrophotometrically using a molar extinction coefficient ε280 = 21,000 M. -1 cm -1 Measurement.

[0272] Phospholipid stock solutions were prepared in chloroform at 50 mM to 100 mM for long-term storage in 4 ml glass vials with Teflon-lined screw caps at -20 °C. The concentration of the stock solution was determined by phosphate analysis. The required amount of chloroform-lipid stock solution was aliquoted into disposable glass culture tubes, and the solvent was dried using a mild nitrogen stream in a fume hood. The tubes were rotated at an angle to obtain a thin film on the lower wall. Residual solvent was removed by placing the tubes in a vacuum desiccator under high vacuum for at least 4 hours.

[0273] Add a buffer containing detergent and sodium cholate to the tube to achieve a cholate to lipid ratio of 2:1. Vortex the tube, heat it in hot tap water, and sonicate it in an ultrasonic bath until the solution is clear and no lipid remains on the tube walls. Add MSP to the solution and incubate for at least 15 minutes.

[0274] To remove bile salts and initiate the self-assembly of nanodisks, 0.5 g to 0.8 g of Amberlite XAD 2 beads (Sigma) were added per ml of the reconstruction mixture. The suspension was placed on a fixed-orbit shaker and incubated overnight. The optimal lipid-to-MSP ratios and incubation temperatures for various lipids, as well as the optimized incubation temperatures and minimum incubation times, are shown in Table B-1.

[0275] Table B-1

[0276] Phospholipid Optimal ratio (e.g., preparation ratio) of MSP1D1 Optimal ratio (e.g., preparation ratio) of MSP1E3 Incubation temperature Minimum incubation time DPPC 90:1 170:1 37℃ 2 hours DMPC 80:1 150:1 25℃ 2 hours POPC 65:1 130:1 4℃ 4 hours

[0277] Samples were extracted from Amberlite XAD 2 beads, filtered through a 0.22-micron filter, and fractionated in MSP standard buffer on a Superdex 200 increase 10 / 300 GL column (GE Healthcare) at a flow rate of 0.75 ml / min. Samples were filtered prior to injection, and fractions were collected every minute. Figure 1 An exemplary chromatogram of the reaction (MSP1D1+DMPC) is shown.

[0278] Example 2A: Nanodisk preparation with different lipid to MSP ratios

[0279] Nanodiscs were constructed using the same method as in Example 1. However, MSP1D1 was added to the sodium cholate lipid mixture at lipid-to-MSP ratios of 10:1, 32.5:1, 50:1, and 65:1 for POPC and 20:1, 30:1, 40:1, 50:1, 60:1, and 85:1 for DMPC. No lipids were added as a control (lipid-free).

[0280] like Figure 2A As shown, a POPC to MSP1D1 ratio of 65:1 produces fully esterified nanodisks (10 nm in diameter). Figure 2B As shown, fully esterified nanodiscs were formed when the molar ratio of DMPC to MSP1D1 was 85:1.

[0281] Example 3: Exemplary nanodisks alter the ability of cholesterol to egress from macrophages in vitro

[0282] Using the methods of Examples 1 and 2, nanodisks containing different lipid concentrations of DMPC (lipid A) or POPC (lipid B) were formed with MSP1D1. J774 cells derived from the mouse macrophage cell line were plated and treated with 74 kBq per millimeter. 3 H-cholesterol was radiolabeled. ABCA1 was upregulated by incubation with 0.3 mM 8-(4-chlorophenylthio)-cAMP for 6 h. Subsequently, nanodiscs and effluent medium were added and incubated for 4 h.

[0283] All steps were performed in the presence of the acyl-CoA cholesterol acyltransferase inhibitor CP113,818 (2 μg / mL). Liquid scintillation counting was used to quantify the efflux of radioactive cholesterol from cells. The amount of radioactive cholesterol incorporated into cellular lipids was calculated by isopropanol extraction from control wells not exposed to nanodisks / culture medium. The percentage of cholesterol efflux was calculated using the following formula:

[0284]

[0285] All experiments were performed in duplicate and in accordance with publicly available 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 full text of which is incorporated herein by reference).

[0286] likeFigure 3 As shown, POPC-containing nanodiscs with lipid-to-MSP1D1 ratios of 50:1 and 32.5:1 exhibited a higher percentage of cholesterol efflux than fully esterified POPC-containing nanodiscs with a lipid-to-MSP1D1 ratio of 65:1. Compared to DMPC-containing nanodiscs with a lipid-to-MSP1D1 ratio of 85:1, POPC-containing nanodiscs with lipid-to-MSP1D1 ratios ranging from 32.5:1 to 65:1 all showed better cholesterol efflux properties. Furthermore, all tested nanodiscs exhibited at least approximately twice the cholesterol efflux achieved through the MSP1D1 protein itself.

[0287] Example 4: Exemplary nanodisks remove cholesterol from J774 murine macrophages in vitro

[0288] Nanodiscs with a DMPC to MSP1D1 ratio of 85:1 were formed using the methods of Examples 1 and 2. The method of Example 3 was repeated to measure cholesterol efflux achieved by these exemplary nanodiscs compared to naturally occurring ApoA-I. Figure 4 As shown, the percentage of cholesterol efflux achieved using the exemplary nanodiscs is more than twice that achieved using naturally occurring ApoA-I.

[0289] Example 5: Exemplary nanodisks remove cholesterol from human retinal pigment epithelial cells in vitro

[0290] 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 cholesterol efflux measurement method of Example 3 was performed on human retinal pigment epithelial (RPE) cells to measure cholesterol efflux at 2, 4, and 6 hours of incubation. Passive efflux (without added protein or nanodiscs) and MSP1D1 protein alone were used for comparison.

[0291] Figures 5A-5C It shows that after 2 hours of incubation ( Figure 5A ), 4 hours ( Figure 5B ) and 6 hours ( Figure 5CThe percentage of cholesterol efflux from RPE cells was then measured. These results collectively demonstrate that nanodiscs with a POPC / MSP1D1 ratio of 65:1 and nanodiscs with a DMPC / MSP1D1 ratio of 85:1 continuously promoted cholesterol efflux from RPE cells for at least 6 hours, which was significantly greater than cholesterol efflux achieved by MSP1D1 protein alone. Furthermore, during the 2- to 4-hour incubation window, nanodiscs with a DMPC / MSP1D1 ratio of 85:1 appeared to efflux cholesterol faster than nanodiscs with a POPC / MSP1D1 ratio of 65:1, but the rate of efflux accelerated at 6 hours for nanodiscs with a POPC / MSP1D1 ratio of 65:1.

[0292] Example 6: Exemplary nanodisks deliver in vivo in the mouse eye

[0293] To determine the in vivo penetration and persistence of exemplary nanodisks in the eye, nanodisks comprising MSP1D1 and DMPC in a molar ratio of 1:85 were prepared, wherein MSP1D1 was linked to a green fluorescent label via cysteine ​​residues engineered into the MSP1D1 protein.

[0294] Nanodisks were prepared the day before the procedure and stored at 4°C.

[0295] Six-week-old C57Bl / 6J mice were anesthetized via intraperitoneal injection of a mixture of 87.6 mg / kg ketamine and 10 mg / kg toluenethiazide, and pain was managed with 0.5% promecaine hydrochloride eye drops. The prepared nanodiscs were administered intravitreally at a dose of 7 μg / eye in a volume of 1.14 μl. A subset of mice were enlarged in the left eye with a 1% tropicamide ophthalmic solution, followed by the application of a 2.5% Gonak hydroxypropyl methylcellulose palliative solution, and non-invasive fundus imaging was performed at various time points using a Phoenix Micron III imaging system. Figures 6A-6B At the time points shown, mice were euthanized and their eyes removed. The eyes were fixed in 10% neutral buffered formalin at room temperature (RT) for 2 hours, then transferred to fractionated sucrose (10%, then 30%) overnight. The fixed, cryopreserved eyes were mounted at the optimal cutting temperature (OCT) and slides were prepared by cutting sections (20 μM) on a Lieca 1850 cryostat. Cover slips were applied using a Vectasheild anti-fading mounting agent with Dapi to counterstain cell nuclei and avoid additional processing steps associated with other counterstaining methods. The prepared slides were imaged on an Olympus FV1000 confocal microscope using a system-optimized z-step for each corresponding objective. This allowed direct visualization of fluorescently labeled nanodisks after successful cholesterol removal from the retina.

[0296] likeFigure 6A and Figure 6B As shown, the nanodiscs were successfully delivered into the mouse eyes via intravitreal injection, and the nanodiscs were able to enter from the vitreous humor, reach the retinal space, enter the RPE, and enter the extraocular muscles by 6 hours. By 24 hours, the disks appeared to have left the eye. These results demonstrate that the exemplary nanodiscs have excellent in vivo permeability and do not remain in the eye for extended periods (>24 hours).

[0297] Example 7: Exemplary nanodisks are safe and efficacious in the mouse eye in vivo

[0298] To determine the in vivo safety and efficacy of nanodisks in the eye, exemplary nanodisks comprising MSP1D1 and DMPC in a molar ratio of 1:85 were prepared, wherein MSP1D1 was linked to a green fluorescent label via cysteine ​​residues engineered into the MSP1D1 protein.

[0299] Fluorescently labeled exemplary nanodisks were delivered intravitreally into the eyes of anesthetized mice, and the diffusion path and rate of the nanodisks in the eye were visualized by imaging post-mortem retinal tissue over a 24-hour timeframe following injection. Figure 7A ).like Figure 7B As shown, the fluorescence signal was detectable within 15 minutes of injection and diffused rapidly across the retina via a vitreous-to-RPE (retinal pigment epithelium) gradient, indicating that the nanodiscs can be delivered into the eye and efficiently transported to the target tissue via the retina. These results collectively demonstrate that the exemplary nanodiscs can be distributed into the RPE.

[0300] Next, a “wet” AMD mouse model with laser-induced Bruch’s membrane injury to induce choroidal neovascularization (CNV) was used. Exemplary nanodiscs were injected intravitreally in conjunction with laser injury or 3 days after laser injury (“+d3”) to examine the size of the resulting CNV area. Figure 8A Importantly, the mean CNV area was similar in control and exemplary nanodisc-treated mice, indicating that exemplary nanodiscs did not exacerbate the prominent features of AMD in mice. Figure 8B and Figure 8C These studies are crucial because they demonstrate that exemplary nanodisks can be effectively delivered to the retina without exacerbating the characteristics of neovascular AMD in mice, unlike currently approved complement inhibitors for the treatment of geographic atrophy (GA).

[0301] To determine whether exemplary nanodisks could reduce lipid load in the eye, transgenic mouse species were used, allowing for the specific loss of the Abca1 and Abcg1 cholesterol efflux transporters in rod photoreceptors (Abca1 / g1-rod / -rod), resulting in lipid accumulation in the RPE and Bruch's membrane, as well as retinal neurodegeneration similar to that observed in patients with early to mid-stage dry AMD. To accelerate lipid accumulation, mice were fed a high-fat diet throughout the study. Figure 9A As shown, for the first mouse synchronous group (Study 1), under high-fat diet induction and exemplary nanodiscs ( Figure 9B and Figure 9C Intravitreal delivery or blank injection of the "disc" in the text Figure 9B and Figure 9C Before administering the "mediator" (as described in the text), baseline (T0) optical coherence tomography (OCT) fundus images of the mouse retina were collected. A second intravitreal injection was administered 3 weeks later. Six weeks after exposure to a high-fat diet, follow-up OCT fundus imaging (T1) and blood sample collection were performed, followed by electroretinal spectroscopy (ERG) visual function testing and tissue harvesting. First, full-field scotopic vision and mild bleaching recovery ERG diagnostics, measuring photoreceptor and RPE function, showed no difference between mice injected with the mediator and those injected with the exemplary nanodisc, demonstrating the safety of the exemplary nanodisc for retinal electrical signal transduction. Figure 9B and Figure 9C Further confirmation of safety was provided by examination of retinal histology, in which the differences in retinal inflammation or retinal integrity between mouse groups were identical. Figure 9D Furthermore, immunostaining of retinal sections revealed that complement 3 (C3) staining was detectable in the mouse retina, but similar expression was observed in mice treated with the mediated substance and mice treated with exemplary nanodiscs, indicating that exemplary nanodiscs do not exacerbate the expected complement signaling in this dry AMD model. Figure 9E RPE rupture and hyperreflectivity, ellipsoidal region abnormalities, inner retinal deposition, and hyperreflective lesions are the main anatomical changes associated with AMD progression, identified using non-invasive imaging. Therefore, we examined follow-up (T1) optical coherence tomography (OCT) images for potential structural changes induced by genetic mutations and a high-fat diet. As expected, after 6 weeks of a high-fat diet absent from baseline T0 images, a high incidence of various inner segment / outer segment (IS / OS) junction abnormalities and RPE disruption (including pigment epithelial detachment (PED)) was found in images of mice injected with the vector. Figure 9F However, these abnormalities were rarely detected or absent in mouse eyes treated with exemplary nanodisks, indicating improved characterization of AMD on OCT imaging.

[0302] To replicate this study and to further emphasize the high-fat diet-induced phenotype, a second cohort of mice on a 6-week high-fat diet was tested (Study 2), but the exemplary nanodiscs or mediators were not injected until 2 and 4 weeks after diet induction. Figure 10A At 6 weeks, visual function was reassessed, and no difference was found in full-field dark vision and bright bleach recovery response between the medium-treated group and the exemplary nanodisk-treated group, confirming the safety of the nanodisk. Figure 10B and Figure 10C In analyzing follow-up OCT imaging, more lesions were observed in the IS / OS junction and RPE in mice injected with the mediator, but far fewer lesions were observed in mice injected with the exemplary nanodisc. Furthermore, a greater number of lesions were observed in both groups 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 the cumulative incidence of retinopathy in mediator-treated and exemplary nanodisc-treated mice via OCT imaging across the two studies). Nevertheless, exemplary nanodisc treatment resulted in a nearly four-fold reduction in retinopathy in both studies compared to the mediator.

[0303] Table B-2 Cumulative incidence of retinopathy

[0304] Vehicle Nanodisks Lesions per mouse (vehicle vs. nanodisks) Study 1 (Example 1) Figure 9A ) 11 1 3.3 vs 0.33 Study 2 (Part 1) Figure 10A ) 30 11 10 vs 2.75 Combined 44 12 6.7 vs 1.7

[0305] Finally, lipidomics analysis was performed on retinal samples to examine potential changes in individual lipid species in mouse eyes following symptomatic nanodisk treatment. A reduction in cholesterol ester (CE) composition (a major component of AMD causing drusen) was found in the retinas of nanodisk-treated mice, indicating that this treatment reduces cholesterol load in diseased mice characteristic of AMD. Furthermore, this reduction in cholesterol esters (a major lipid species found in drusen), along with the improvement in OCT lesions, provides strong support for the mechanism of action and scientific prospects of the nanodisks presented in this paper.

[0306] Example 8: Preparation of illustrative under-lipidated nanodisks

[0307] Under-esterified nanodiscs were prepared from phospholipids, MSP, and organic compounds according to the scheme provided below.

[0308] Materials

[0309] MSP1D1(-) refers to MSP1D1 with the N-terminal histidine affinity tag removed, which was used in the preparation described herein. MSP1D1(-) contains the sequence of SEQ ID NO: 14. MSP1D1 also refers to the form without the N-terminal histidine affinity tag removed, containing SEQ ID NO: 3. Concentration was measured by UV-Vis spectroscopy, which showed an extinction coefficient of 18450 M at 280 nm. -1 (0.84 mg / ml).

[0310] Phospholipids (e.g., DMPC, DPPC) are dissolved in chloroform and their concentration is measured by determining total phosphorus (see, for example, Chen, Toribara and Warner (1956) Anal. Chem. 28: 1756-1758 (2016), the contents of which are incorporated herein by reference in their entirety).

[0311] Other reagents used in 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.

[0312] Preparation of DMPC / MSP1D1(-) nanodisks

[0313] All operations were performed at room temperature (20°C to 22°C).

[0314] A DMPC / chloroform stock solution was added to a glass tube to prepare a final DMPC to MSP1D1(-) ratio of 30:1. The DMPC was dried under a nitrogen stream while the glass tube was rotated to form a film on the side of the tube. The tube containing the DMPC was then placed in a desiccator and a vacuum was applied for at least 4 hours, e.g., overnight.

[0315] The DMPC was then hydrated in water. To determine the required amount of water, the desired final sodium cholate concentration was first determined. The final cholate to lipid ratio was determined to be greater than 2. An exemplary concentration was 17 mM cholate. The amounts of water and 200 mM sodium cholate were determined such that the final sodium cholate concentration was 17 mM. The amount of protein was then determined such that the final protein concentration was approximately 3.5 mg / mL. At this point, only water was added to the dried DMPC.

[0316] The water / DMPC mixture was vortexed until the solution turned milky white. Then, 200 mM sodium cholate was added to the hydrated DMPC to obtain a final cholate concentration of 17 mM in the assembly mixture. The sodium cholate / DMPC mixture was completely dissolved by placing the tube in an ultrasonic water bath for 5 min, followed by gentle heating at approximately 40 °C, repeated twice. MSP1D1(-) was then added to the sodium cholate / DMPC mixture and thoroughly mixed to obtain a preparative mixture with a protein concentration of approximately 3.5 mg / mL. The preparative mixture was incubated at room temperature for 10 to 20 minutes.

[0317] Assembling of nanodisks is initiated by adding Amberlite XAD-2 resin, in which Amberlite absorbs the bile salt detergent. The amount of Amberlite XAD-2 resin added should be at least 50% of the volume of the assembly mixture; for example, for a 5 ml preparation mixture, Amberlite resin is added to the preparation mixture until the volume is 7.5 ml. The assembly mixture containing Amberlite resin is gently agitated by placing it on a platform shaker or tube rotator at room temperature for at least four hours. The supernatant containing the assembled nanodisks is recovered from the Amberlite XAD resin using a pipette and placed in a new tube. The Amberlite XAD resin is washed twice with PBS, and the wash solution containing the remaining assembled nanodisks is added to the sample tube. Large nanodisks are removed from the sample by passing them through a 0.2 μm filter. Depending on the nanodisk sample volume, this can also be done using a centrifuge filter (Amicon Ultra Free) or a syringe equipped with a 0.2 μm filter.

[0318] Similarly, another under-esterified nanodisc containing DMPC and MSP1D1(-) was fabricated, wherein the molar ratio of DMPC to MSP1D1(-) in the mixture was approximately 55:1. Similarly, a third under-esterified nanodisc containing DMPC and MSP1D1(-) was fabricated, wherein the molar ratio of DMPC to MSP1D1(-) in the mixture was approximately 40:1.

[0319] Under-lipolated nanodisks comprising DMPC and MSP1E3D1(-) were prepared similarly using different molar ratios of lipids to MSP in the preparation mixture. The molar ratios of lipids to MSP used in the various DMPC / MSP1E3D1(-) compositions are also detailed in Example 12. MSP1E3D1(-) refers to MSP1E3D1 with the N-terminal histidine affinity tag removed, which was used in the preparation herein. MSP1E3D1(-) contains the sequence of SEQ ID NO: 15. MSP1E3D1 also refers to the form without the N-terminal histidine affinity tag removed, containing SEQ ID NO: 8. To prepare the under-lipolated nanodisks, the final ratio of DMPC to MSP1E3D1(-) in the preparation mixture was approximately 50:1.

[0320] Similarly, another under-esterified nanodisc containing DMPC and MSP1E3D1(-) was prepared, wherein the molar ratio of DMPC to MSP1E3D1(-) in the mixture was approximately 90:1.

[0321] Nanodisks containing other MSPs (or other phospholipid / MSP ratios) were prepared using a similar scheme detailed in this article.

[0322] Example 9: Purification of illustrative under-lipidated nanodisks

[0323] The filtered sample containing DMPC / MSP1D1(-) nanodiscs or DMPC / MSP1E3D1(-) nanodiscs from Example 8 was further purified by size exclusion chromatography (SEC). The control nanodiscs were purified similarly.

[0324] For the column: Use Superdex S-200 increase 10 / 300 (approximately 25 ml bed volume). The buffer is PBS. The flow rate is 0.75 ml / min. The injection volume is 0.5 mL.

[0325] The chromatogram of the preparative SEC of DMPC / MSP1D1(-) nanodiscs (preparative mixture ratio of approximately 30:1) is shown in Figure 11A In the middle. For example Figure 11A As shown, two peaks were separated. The region on the right (dark gray) corresponds to a more under-lipidized nanodisc (lower lipid-to-protein ratio) containing approximately 51 lipids per MSP1D1(-). The peak located at 20 min (see...) Figure 11A The small image at the top shows the remaining lipid-free protein.

[0326] Chromatograms of preparative SEC of DMPC / MSP1E3D1(-) nanodiscs (preparative mixture ratio approximately 50:1) are shown. Figure 11B In the middle. For example Figure 11BAs shown, a single prominent peak was collected, which contains an under-esterified nanodisc with a molar ratio of DMPC to MSP1E3D1(-) of approximately 62:1.

[0327] Example 10: Measurement of phospholipid to MSP ratio

[0328] This assay measures the phospholipid-to-MSP ratio by first extracting the phospholipids, then saponifying and methylating the fatty acids for rapid gas chromatography (GC) analysis. The amount of fatty acid / MSP measured is twice the amount of the starting phospholipids. Since the phospholipid DPPC is used as a standard, two fatty acids per phospholipid have been taken into account.

[0329] The ratio of phospholipids to MSP in the nanodiscs was determined according to the procedure detailed below.

[0330] The MSP concentration of the nanodiscs described herein was measured by UV-Vis spectroscopy. Nanodisc samples were added to 2 mL GC vials to ensure approximately 1 μg of phospholipids. Each nanodisc sample was run in triplicate. The nanodisc samples were diluted to 100 μL with PBS. 10 μL of a known concentration of standard (DPPC) was added to each nanodisc sample. Total phospholipids were extracted using the modified Bligh and Dyer techniques described in conditions E and E' of the phase diagram in Figure 7 of Int. J. Mol. Sci. 2017, 18(4), 708.

[0331] Phospholipids were extracted and dried under a nitrogen flow. Fatty acids were simultaneously saponified and methylated 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 JLipid Res. 2010; 51(3):635-40). The mixture was incubated at 100 °C for 1 hour. In other batches, the mixture was incubated overnight at 45 °C.

[0332] Then, 0.5 mL of water and 0.5 mL of hexane were added to the mixture. The mixture was vortexed, and the top hexane layer was recovered for GC analysis. A standard curve of DMPC in chloroform at a known concentration was prepared by serial dilution, with 10 μL of DMPC standard added to each dilution, and saponification was performed as described in Section 176. J Lipid Res. 2010; 51(3):635-40.

[0333] The DMPC content was determined by measuring the area ratio of C14 fatty acid methyl ester (DMPC) to C16 fatty acid methyl ester (DPPC) and comparing it with a standard curve.

[0334] The lipid to MSP ratio of the nanodiscs was determined by measuring protein and DMPC concentrations using UV-Vis spectroscopy.

[0335] Using DMPC / MSP1D1(-) nanodisks from Example 8 (preparation mixture ratio of approximately 30:1), exemplary results generated by GC method are shown in... Figure 12 In. Figure 12 In this context, DMPCfame is methyl myristate, and DPPCfame is methyl palmitate, which are methylated fatty acids derived from DMPC and DPPC, respectively.

[0336] Based on the measurement results, such as Figure 11A As detailed in the paper, starting with a preparation mixture of DMPC and MSP1D1(-) at a molar ratio of approximately 30:1, under-esterified nanodiscs with a DMPC:MSP1D1(-) molar ratio of approximately 72:1 (hereinafter referred to as "DMPC:MSP1D1(-)=72:1 nanodiscs") and under-esterified nanodiscs with a DMPC:MSP1D1(-) molar ratio of approximately 51:1 (hereinafter referred to as "DMPC:MSP1D1(-)=51:1 nanodiscs") were obtained, with the yield of the DMPC:MSP1D1(-)=51:1 nanodiscs being optimized. Therefore, this condition was further used to prepare DMPC:MSP1D1(-)=51:1 nanodiscs.

[0337] Starting with a mixture of DMPC and MSP1D1(-) at a molar ratio of approximately 55:1, under-esterified nanodisks with a DMPC:MSP1D1(-) molar ratio of approximately 75:1 were obtained, hereinafter referred to as "DMPC:MSP1D1(-)=75:1 nanodisks". This condition was further used to prepare DMPC:MSP1D1(-)=75:1 nanodisks.

[0338] Starting with a mixture of DMPC and MSP1D1(-) at a molar ratio of approximately 40:1, under-esterified nanodiscs with a DMPC:MSP1D1(-) molar ratio of approximately 51:1 were obtained, hereinafter referred to as "DMPC:MSP1D1(-)=51:1 nanodiscs". This condition was further used to prepare DMPC:MSP1D1(-)=51:1 nanodiscs.

[0339] Starting with a preparation mixture of DMPC and MSP1E3D1(-) at a molar ratio of approximately 50:1, under-esterified nanodiscs with a molar ratio of DMPC to MSP1E3D1(-) of approximately 62:1 were obtained, hereinafter referred to as "DMPC:MSP1E3D1(-)=62:1 nanodiscs".

[0340] Starting with a preparation mixture of DMPC and MSP1E3D1(-) at a molar ratio of approximately 60:1, under-esterified nanodiscs with a molar ratio of DMPC to MSP1E3D1(-) of approximately 63:1 were obtained, hereinafter referred to as "DMPC:MSP1E3D1(-)=63:1 nanodiscs".

[0341] Starting with a preparation mixture of DMPC and MSP1E3D1(-) at a molar ratio of approximately 90:1, under-esterified nanodiscs with a molar ratio of DMPC to MSP1E3D1(-) of approximately 93:1 were obtained, hereinafter referred to as "DMPC:MSP1E3D1(-)=93:1 nanodiscs".

[0342] For a control, starting with a preparation mixture of DMPC and MSP1D1 with a molar ratio of approximately 85:1, nanodiscs with a molar ratio of DMPC to MSP1D1 of approximately 83:1 (fully esterified) were obtained, hereinafter referred to as "DMPC:MSP1D1=83:1 nanodiscs".

[0343] For another control, starting with a preparation mixture of DMPC and MSP1D1(-) at a molar ratio of approximately 80:1, nanodiscs (fully esterified) with a molar ratio of DMPC to MSP1D1(-) of approximately 81:1 were obtained, hereinafter referred to as "DMPC:MSP1D1(-)=81:1 nanodiscs".

[0344] Example 11: Correspondence between phospholipid / MSP1D1(-) ratio in preparation mix and final nanodisk composition

[0345] The correspondence between the phospholipid / MSP1D1(-) ratio in the preparation mixture and the final nanodiscs was examined. Exemplary compositions comprising multiple DMPC / MSP1D1(-) nanodiscs were synthesized and purified according to the procedures described in Examples 8 and 9, and the ratios were measured as in Example 10. For nanodiscs starting with DMPC / MSP1D1(-) preparation mixture ratios of 80:1, 55:1, and 40:1, the fractions collected from the first SEC were repurified using a second SEC (repurification). For nanodiscs starting with a DMPC / MSP1D1(-) preparation mixture ratio of 40:1, a third SEC purification was performed (repurification 2). For nanodiscs starting with a DMPC / MSP1D1(-) preparation mixture ratio of 30:1, only one SEC step was performed. After the final purification step, the lipid-to-protein ratio was measured in the final nanodiscs. The results were shown in… Figures 13A to 13D In the middle. For example Figure 13A As shown, nanodiscs were generated by an initial DMPC / MSP1D1(-) ratio of 80:1 in the mixture, where the DMPC / MSP1D1(-) ratio was approximately 81:1. Figure 13BAs shown, nanodiscs were generated by an initial DMPC / MSP1D1(-) ratio of 55:1 in the mixture, where the DMPC / MSP1D1(-) ratio was approximately 75:1. Figure 13C As shown, nanodiscs were generated by an initial DMPC / MSP1D1(-) ratio of 40:1 in the mixture, where the DMPC / MSP1D1(-) ratio was approximately 51:1. Figure 13D As shown, a 30:1 initial DMPC / MSP1D1(-) ratio in the preparation mixture yields nanodiscs, where the DMPC / MSP1D1(-) ratio is approximately 51:1. Furthermore, DMPC and MSP1D1(-) at a 30:1 (preparation ratio) are adequately separated in a single purification step. These results collectively demonstrate that low phospholipid / MSP ratios (e.g., an initial DMPC / MSP1D1(-) ratio of less than 81:1 in the preparation mixture (the phospholipid-MSP saturation ratio N of DMPC and MSP1D1) are suitable for nanodiscs. S It can produce surprisingly stable, under-esterified compositions.

[0346] Example 12: Correspondence between phospholipid / MSP1E3D1(-) ratio in preparation mix and final nanodisk composition Figures 14A to 14C

[0347] The correspondence between the phospholipid / MSP1E3D1(-) ratio in the prepared mixture and the final nanodisc composition was examined. Exemplary nanodiscs containing multiple DMPC / MSP1E3D1(-) nanodiscs were synthesized and purified according to the procedures described in Examples 8 and 9, and the ratio was measured as in Example 10. Only one SEC step was performed. After the final purification step, the lipid-to-protein ratio was measured in the final nanodiscs. The results were shown... Figure 14A In the middle. For example Figure 14B As shown, nanodiscs are generated by an initial DMPC / MSP1E3D1(-) ratio of 90:1 in the preparation mixture, where the DMPC / MSP1D1(-) ratio is approximately 93, such as 93±3. Figure 14C As shown, nanodiscs are generated by an initial DMPC / MSP1E3D1(-) ratio of 60:1 in the preparation mixture, where the DMPC / MSP1D1(-) ratio is approximately 63:1, such as 63 ± 4:1. Example 13: Cholesterol efflux assay As shown, a starting DMPC / MSP1D1(-) ratio of 50:1 in the preparation mixture yields nanodiscs, where the DMPC / MSP1D1(-) ratio is approximately 62:1, such as 62 ± 0.5:1. These results collectively demonstrate that low phospholipid / MSP ratios (e.g., starting DMPC / MSP1E3D1(-) ratios less than 167:1 in the preparation mixture) (the phospholipid-MSP saturation ratio N of DMPC and MSP1E3D1) are favorable for nanodisc production. S It can produce surprisingly stable, under-esterified nanodiscs.

[0348] Figure 15A

[0349] Perform this assay as described in the product information for the Cholesterol Effusion Assay Kit (Cell-Based) (ab196985).

[0350] In summary, J774 mouse macrophages were cultured and placed in 96-well tissue culture plates. Cells were labeled with fluorescent cholesterol, washed, and incubated overnight. The cells were then treated for 4 hours with a culture medium containing the test nanodiscs generated as described in Examples 1 through 3. The culture medium containing the test nanodiscs was recovered, and cholesterol fluorescence (F) was measured in the medium. 培养基 Cells were lysed, and the residual fluorescence (F) in the cells was measured. 细胞 Cholesterol efflux is defined as:

[0351] % outflow = F 培养基 / (F 培养基 +F 细胞 )

[0352] Figure 15B and Figure 15A Effluent assays performed on purified exemplary nanodiscs as described in Examples 8 through 10 are shown. The phospholipid to MSP ratio is the ratio measured from the final nanodiscs. In addition to the fully lipidated nanodisc control, MSP1D1(-) (protein only, no lipids), Apo-AI (protein only, no lipids), and high-density lipoprotein (HDL) were also used as controls. A positive control from a cholesterol efflux assay kit (ab196985) was included. PBS was used as a negative control (not shown).

[0353] like Figure 13B As shown, when 2.5 μg of sample was incubated with cells labeled with fluorescent cholesterol, the composition with nanodiscs having a high lipid content (e.g., nanodiscs with insufficient lipidation of DMPC:MSP1D1(-) = 75:1) was compared with that of other nanodiscs. Figure 13A Nanodiscs shown in the image) and fully esterified DMPC:MSP1D1(-)=81:1 nanodiscs (e.g., Figure 13D Compared to the nanodiscs shown, the nanodiscs with insufficient esterification (DMPC:MSP1D1(-)=51:1) (e.g., Figure 15A The nanodiscs shown exhibit a much larger cholesterol outflow rate. Figure 15AFurther, it was shown that removing the N-terminal his-tag portion of the MSP protein did not affect cholesterol efflux (comparing nanodiscs with fully lipotropic DMPC:MSP1D1(-) = 81:1 and nanodiscs with fully lipotropic DMPC:MSP1D1 = 83:1). The phospholipid-MSP saturation ratio of nanodiscs containing DMPC and MSP1E3D1 (or MSP1E3D1(-)) was [not specified]. S The value is 167 (see Table 2). Therefore, the nanodiscs with DMPC:MSP1E3D1(-)=62:1 and DMPC:MSP1E3D1(-)=94:1 are highly under-esterified. Figure 15B As shown, nanodiscs with a DMPC:MSP1E3D1(-) ratio of 62:1 also exhibited slightly better cholesterol efflux rates compared to nanodiscs with a DMPC:MSP1E3D1(-) ratio of 94:1 (which has a higher lipid content). Compared to lipid-free MSP1D1(-) protein controls, Apo-AI controls, and HDL controls, nanodiscs with insufficient lipidation (DMPC:MSP1D1(-) ratio of 51:1, 62:1, and 94:1) all showed significantly higher cholesterol efflux rates. Example 14: Stability of under-lipidated nanodisk composition to freeze-thaw cycles The results showed that cholesterol efflux capacity increased significantly with increasing nanodisc dosage (10 μg vs. 2.5 μg). Surprisingly, 2.5 μg of under-esterified DMPC:MSP1D1(-)=51:1 nanodiscs even showed stronger cholesterol efflux capacity compared to 10 μg of fully esterified DMPC:MSP1D1(-)=81:1 nanodiscs, further demonstrating the technical superiority (e.g., cholesterol efflux capacity) of the nanodiscs presented in this paper, especially the under-esterified nanodiscs.

[0354] Figure 11A

[0355] The stability of the nanodisks prepared in Examples 8 to 10 was tested. One exemplary DMPC / MSP1D1(-) nanodisk composition tested is... Figure 16 The right-hand region of the chromatographic peak corresponds to the under-esterified DMPC:MSP1D1(-)=51:1 nanodisc. The sample was frozen at -80°C with or without 10% glycerol as a cryoprotectant. The frozen sample was then removed from -80°C storage and rapidly thawed. The sample was subjected to SEC and compared with the chromatogram obtained before freezing. The results are shown in... Figure 17The results show that the chromatograms of the freeze-thawed samples (without glycerol) are almost identical to those of the pre-frozen samples. The chromatograms of samples frozen with or without cryoprotectant after freeze-thaw cycles are also almost identical (data not shown). These results demonstrate the surprisingly high stability of the prepared, under-esterified nanodiscs when subjected to freeze-thaw cycles.

[0356] Freeze-thaw cycles on under-esterified nanodiscs did not affect cholesterol efflux efficiency. Figure 17 The image shows a comparison of cholesterol efflux from under-lipidized DMPC:MSP1D1(-)=51:1 nanodiscs before and after freeze-thaw cycles. ​ As shown, the cholesterol efflux efficiency of the under-lipidated nanodiscs remained high after freeze-thaw cycles (second column), and was superior to that of the fully lipidated nanodisc control with high DMPC content (nanodiscs with DMPC:MSP1D1=83:1), as well as higher than that of lipid-free HDL and blank control PBS solutions.

Claims

1. A method of removing or preventing accumulation of lipids in an eye of an individual, the method comprising administering to the eye of the individual an effective amount of a pharmaceutical composition comprising a plurality of nanodiscs, wherein the nanodiscs comprise a membrane skeleton protein (MSP) and a phospholipid.

2. A method of preventing or treating an eye disease characterized by accumulation of lipids in an individual, the method comprising administering to the eye of the individual an effective amount of a pharmaceutical composition comprising a plurality of nanodiscs, wherein the nanodiscs comprise a 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 variants thereof.

4. The method of any one of claims 1 to 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 a truncation relative to the N-terminus of 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 variants thereof that are free of the N-terminal His-tag portion (His-tag free variants).

8. The method of claim 7, wherein the MSP is: (i) a His-tag free variant of MSP1D1 comprising the amino acid sequence of SEQ ID NO: 14; or (ii) a His-tag free variant of MSP1E3D1 comprising the amino acid sequence of SEQ ID NO:

15.

9. The method of any one of claims 1 to 8, wherein the phospholipid comprises DMPC, DPPC, DMPS, DSPC, POPC, POPC, PiP2, or combinations thereof.

10. The method of any one of claims 1 to 9, wherein the phospholipid is phosphatidylcholine (PC).

11. The method of claim 10, wherein the phospholipid is selected from the group consisting of DMPC, POPC, DPPC, DSPC, and combinations thereof.

12. The method of any one of claims 1 to 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 to 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 N S .

15. The method of claim 14, wherein the molar ratio of the phospholipid to the MSP in the pharmaceutical composition is less than the phospholipid-MSP saturation ratio N S about 95%.

16. The method of any one of claims 1 to 15, wherein the MSP is MSP1D1, MSP1, or a His-tag free variant thereof, and wherein the molar ratio of the phospholipid to the MSP in the pharmaceutical composition is about 10: 1 to about 90:

1.

17. The method of claim 16, wherein the MSP is MSP1D1, MSP1, or a 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 10: 1 to about 65:

1.

18. The method of claim 17, wherein the MSP is MSP1D1 or a 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 MSPl Dl or a His-tag removed variant thereof, the phospholipid is POPC, and the N S 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.

22. The method of claim 16, wherein the MSP is MSP1D1, MSP1, or a 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 about 10: 1 to about 85:

1.

23. The method of claim 22, wherein the MSP is MSP1D1 or a 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 MSPl Dl or a His-tag removed variant thereof, the phospholipid is DMPC, and the N S 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 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 a 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 a 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 about 10: 1 to about 90:

1.

31. The method of claim 30, wherein the MSP is MSP1 or a 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 MSPl Dl or a His-tag removed variant thereof, the phospholipid is DPPC, and the N S is about 81.

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 to 15, wherein the MSP is MSP1E3D1, MSP1E3, or a His tag-removed variant thereof, and wherein the molar ratio of the phospholipid to the MSP in the pharmaceutical composition is about 50: 1 to about 200:

1.

35. The method of claim 34, wherein the MSP is MSP1E3 or a 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 MSPl E3D1 or a His-tag removed variant thereof, the phospholipid is POPC, and the N S 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 MSPl E3D1 or a His-tag removed variant thereof, the phospholipid is DMPC, and the N S 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 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 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 a 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.

44. The method of claim 34, wherein the MSP is MSP1E3 or a 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 MSPl E3D1 or a His-tag removed variant thereof, the phospholipid is DPPC, and the N S 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 to 15, wherein the MSP is MSPl El Dl or a His tag- removed variant thereof, the phospholipid is DMPC, and the N S 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 about 60: 1 to about 65:

1.

50. The method of any one of claims 1 to 49, wherein the nanodiscs in the pharmaceutical composition have a diameter of about 2 nm to about 20 nm.

51. The method of any one of claims 1 to 50, wherein the lipid used to remove or prevent 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 claim 51 or 52, wherein the nanodisc increases the rate of cholesterol efflux at least about 2-fold compared to an untreated state or by ApoA-I or the MSP.

54. The method of any one of embodiments 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 (AMD), photoreceptor neurodegeneration, optic nerve atrophy, vision loss, hemianopsia, visual agnosia, strabismus, retinal neurovascular disorders, lipid keratopathy, corneal lipidosis, and combinations 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 in the eye of the individual by at least about 5% or prevents lipid accumulation in the eye of the individual.

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