WO2025117721A1

WO2025117721A1 - Reconstituted lipoprotein particles to rescue lipid defects in the alzheimer's brain

Info

Publication number: WO2025117721A1
Application number: PCT/US2024/057721
Authority: WO
Inventors: Li-Huei Tsai; Rebecca PINALS; Claudia Fernanda LOZANO CRUZ
Original assignee: Massachusetts Institute of Technology
Current assignee: Massachusetts Institute of Technology
Priority date: 2023-11-27
Filing date: 2024-11-27
Publication date: 2025-06-05
Anticipated expiration: 2026-05-27

Abstract

The present disclosure provides products and methods for facilitating brain lipid transport in a subject. In some aspects the products are useful for slowing the progression of or preventing the development of Alzheimer's Disease or for treating Alzheimer's disease. The products include reconstituted lipoprotein particles (rLPs). Libraries and screening of libraries to identify additional rLPs are also disclosed.

Description

RECONSTITUTED LIPOPROTEIN PARTICLES TO RESCUE LIPID DEFECTS IN THE ALZHEIMER'S BRAIN

RELATED APPLICATIONS

The application claims the benefit under 35 U.S.C. 119(e) of U.S. Provisional Application number 63/603,090 filed November 27, 2023, which is herein incorporated by reference in its entirety.

BACKGROUND

Lipid dysregulation has been noted as a hallmark of Alzheimer’s disease (AD), dating back to the original description of “adipose saccules” in glial cells over a century ago by Alois Alzheimer. Most known high-risk genes for late-onset AD are implicated in lipid metabolism, transport, and homeostasis, most notably the APOE gene, which encodes a lipid transporter protein (ApoE). (Knopman, D. S. et al. Alzheimer disease. Nat Rev Dis Primers 7, 1-21 (2021)). ApoE is the main apolipoprotein component of the lipoprotein particles (LPs) found in the brain that transport lipids between glial cells — which synthesize lipids and use intracellular lipid droplets to store excess and energy-rich lipids — and neurons — which depend on lipids for their physical remodeling and high metabolic needs. (Ralhan, I., et al, Lipid droplets in the nervous system. Journal of Cell Biology 220, e202102136 (2021) & loannou, M. S. et al. Neuron-Astrocyte Metabolic Coupling Protects against Activity-Induced Fatty Acid Toxicity. Cell 177, 1522-1535. el4 (2019)). Expression oiAPOE4 leads to the over- accumulation of lipids in glia that is typical of AD pathology, causing disruption of glial support of neurons. (Sienski, G. et al. APOE4 disrupts intracellular lipid homeostasis in human iPSC-derived glia. Science Translational Medicine 13, (2021), Victor, M. B. et al. Lipid accumulation induced by APOE4 impairs microglial surveillance of neuronal-network activity. Cell Stem Cell 29, 1197-1212. e8 (2022) & Blanchard, J. W. et al. APOE4 impairs myelination via cholesterol dysregulation in oligodendrocytes. Nature 611, 769-779 (2022)). The APOE4 variant, together with age and female chromosomal sex, composes the Alzheimer’s risk triad. (Riedel, B. C., Thompson, P. M. & Brinton, R. D. Age, APOE and Sex: Triad of Risk of Alzheimer’s Disease. J Steroid Biochem Mol Biol 160, 134-147 (2016)). SUMMARY

In aspects of the current disclosure lipid profiles have been mapped in the brain, cerebrospinal fluid (CSF), and blood plasma of female vs. male and young vs. aged mice expressing humanized APOE of the three most common variants: the AD-protective APOE2, normal form APOE3, and AD-prone APOE4. It was determined that global, sex-specific changes occur in the lipidome with age, including a net accumulation of lipids in the brain. In the CSF, cholesterol, cholesteryl esters, phosphatidylcholines, and phosphatidylethanolamines - main lipid classes composing brain lipoprotein particles - increased in APOE2 and APOE3 mice with age, but increased to a lesser extent or decreased in APOE4 mice with age. It was determined that the CSF lipidome can normally adapt to buffer age-related lipid changes in brain tissue that require increased lipid transport, yet this response breaks down in the context of AD risk factors including APOE4. Based at least in part on this analysis, reconstituted lipoprotein particles (rLPs) containing functional ApoE3 were designed and prepared. The particles may be delivered to the CSF or other tissue such as brain in order to rescue this lipid transport defect and restore glial-derived lipid support of neurons in APOE4 carriers.

In some embodiments phospholipids are present in a molar ratio of 70 : 16.67 : 8.33 : 5 : 1 POPC / SOPE / POPE / cholesterol / ApoE.

In some embodiments the phospholipid comprises a Phosphatidylinositol (PI).

In some embodiments the phospholipid comprises a combination of lipids and wherein the combination is selected from one of the following groups: PC+PE, PC+PS, PC+PE+PI, PC+PE+PS, PC+PS+PI, PC+PS+PE+PI, PC+PE+PS+Sphingolipid (SL), PC+PS+PI+SL, or PC+PE+PI+SL.

In other aspects a composition comprising a reconstituted lipoprotein particle (rLP), wherein the rLP has a discoidal shape is provided. The rLP comprises: a) at least one phospholipid and at least one sphingolipid, wherein a first phospholipid is a phosphatidylcholine (PC), and b) at least one protein, optionally a brain-associated protein.

In some embodiments the rLP comprises a combination of lipids and wherein the combination is selected from one of the following groups: PC+SM, PC+SM+PE, PC+SM+PS, PC+SM+PS+PE, PC+SM+PI, PC+SM+PS+PI, PC+SM+PE+PS, PC+SM+PS+PI, PC+SM+PS+PE+PI, or PC+SM+PE+PI. Aspects of the disclosure include a composition of a reconstituted lipoprotein particle (rLP), wherein the rLP has a discoidal shape and comprises: a) at least two distinct phospholipids, wherein a first of the two phospholipids is a phosphatidylcholine (PC) and a second of the two phospholipids is a phosphatidylethanolamine (PE) or a phosphatidylserine (PS), b) cholesterol; c) sphingolipid (SL), optionally wherein the SL is a sphingomyelin; and d) at least one protein, optionally a brain-associated protein.

In some embodiments the rLP has a diameter of about 5-30nm, 8-25nm, 10-30 nm, 10-20 nm, 10-15 nm, 10-40 nm, 15-29 nm, 15-30 nm, 15-35 nm, 15-40 nm, 20-30 nm, 20-35 nm, 20-40 nm, 30-35 nm, or 30-40 nm. In some embodiments the rLP comprises phospholipids and cholesterol. In some embodiments the rLP does not comprise cholesteryl esters.

In some embodiments the phospholipid comprises POPC (16:0-18:1 PC) and SOPE (18:0-18:1 PE). In some embodiments the phospholipid comprises POPC (16:0-18:1 PC) and POPE (16:0-18:1 PE). In some embodiments the phospholipid comprises POPC (16:0-18:1 PC), POPE (16:0-18:1 PE), and SOPE (18:0-18:1 PE). In some embodiments the phospholipids are present in a molar ratio of 70 : 16.67 : 8.33 : 5 : 1 POPC / SOPE / POPE / cholesterol / ApoE.

In some embodiments the phospholipid comprises DMPC (14:0 PC) and SOPE (18:0- 18:1 PE). In some embodiments the phospholipid comprises DMPC (14:0 PC) and POPE (16:0-18:1 PE). In some embodiments the phospholipid comprises DMPC (14:0 PC), POPE (16:0-18:1 PE), and SOPE (18:0-18:1 PE). In some embodiments the phospholipids are present in a molar ratio of 85 : 9.33 : 4.67 : 1 : 1 DMPC / SOPE / POPE / cholesterol / ApoE.

In some embodiments the cholesterol / ApoE molar ratio is 5:1 in POPC-containing formulations. In some embodiments cholesterol / ApoE molar ratio is 1:1 in DMPC- containing formulations.

In some embodiments the protein is an apolipoprotein. In some embodiments the apolipoprotein is ApoE3 (C112/R158), ApoE2 (C112/C158), Christchurch ApoE3, and/or Jacksonville ApoE3. In some embodiments the apolipoprotein is ApoA-I and/or Apo J, which may be used in combination with ApoE.

In some embodiments, ApoE is modified with post-translational modifications (PTMs) consistent with that of the brain, including higher phosphorylation, glycosylation, sulfation, oxidation, and S-nitrosylation, in comparison to ApoE found in the periphery. PTMs of ApoE can include glycation (K75), glycosylation (T8, T18, S94, T194, S197, T289, S290, S296), and phosphorylation (S129, S197, S296).

In some embodiments the protein is an apolipoprotein peptide mimic. In some embodiments the apolipoprotein mimic is EpK. Aspects of the disclosure include a method of treating Alzheimer’s Disease in a subject by administering to the subject an effective amount of a reconstituted lipoprotein particle (rLP) to treat Alzheimer’s disease in the subject, wherein the rLP has a discoidal shape and comprises at least two distinct phospholipids, wherein a first of the two phospholipids is a phosphatidylcholine (PC) and a second of the two phospholipids is a phosphatidylethanolamine (PE) or a phosphatidylserine (PS), cholesterol; a sphingolipid (SL), optionally wherein the SL is a sphingomyelin; and at least one protein, optionally a brain-associated protein.. In some embodiments the rLP has a discoidal shape and comprises: at least two distinct phospholipids, optionally selected from POPC, DMPC, SOPE, and POPE and combinations thereof, cholesterol, and at least one protein.

In some embodiments the phospholipid comprises DMPC (14:0 PC) and SOPE (18:0- 18:1 PE). In some embodiments the phospholipid comprises DMPC (14:0 PC) and POPE (16:0-18:1 PE). In some embodiments the phospholipid comprises DMPC (14:0 PC), POPE (16:0-18:1 PE), and SOPE (18:0-18:1 PE). In some embodiments the phospholipids are present in a molar ratio of 85 : 9.33 : 4.67 : 1 : 1 DMPC / SOPE / POPE / cholesterol / ApoE. In some embodiments the cholesterol / ApoE molar ratio is 5:1 in POPC-containing formulations. In some embodiments cholesterol / ApoE molar ratio is 1:1 in DMPC- containing formulations.

In some embodiments the phospholipids are present in a molar ratio of 70 : 16.67 : 8.33 : 5 : 1 POPC / SOPE / POPE / cholesterol / ApoE.

In some embodiments the protein is an apolipoprotein. In some embodiments the apolipoprotein is ApoE3, ApoE2, ApoE3-Christchurch (R136S), and/or ApoE3-Jacksonville (V236E). In some embodiments the apolipoprotein is ApoA-I and/or ApoJ.

In some embodiments the protein is an apolipoprotein peptide mimic. In some embodiments the apolipoprotein mimic is EpK.

In some embodiments the method further comprises determining whether the subject has or is at risk of developing Alzheimer’s Disease by identifying the subject as APOE4 positive. In some embodiments the Alzheimer’s disease risk factors include other genes implicated in lipid transport, metabolism, and homeostasis, including APOE, CEU, ABCA1, ABCA7, LRP1, SORL1, TREM2, PICALM, ECHDC3, BINI, among others. In some embodiments the Alzheimer’s disease is mild to moderate Alzheimer’s disease. In some embodiments the Alzheimer’s disease is moderate to severe Alzheimer’s disease. In some embodiments the subject is identified as APOE4 positive prior to treatment. In some embodiments the subject is homozygous for APOE4. In some embodiments the subject is a female.

In other aspects, a method for identifying a composition is provided. The method involves screening a library of reconstituted lipoprotein particles (rLPs), wherein the screening involves characterization of rLPs as having a size, morphology, stability, lipid- protein content and/or binding property, and optionally a functional property.

The details of one or more embodiments of the invention are set forth in the description below. Other features or advantages of the present invention will be apparent from the following drawings and detailed description of several embodiments, and also from the appended claims.

BRIEF DESCRIPTION OF DRAWINGS

The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present disclosure, which can be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

FIGs. 1A- 1G: Schematics and graphs depicting how aging mouse brain undergoes global lipid changes in a sex- and APOE-dependent manner, exacerbated by APOE4 CSF. 1A is a schematic of the experimental design and methods used for mapping lipid profiles in the brain and periphery of mice. 1B-D: Graphs of data depicting how mouse brain lipidome changes with aging and displays sex-dependent lipid profiles. IE: Graphs of data depicting CSF fraction of lipid species within a class that increase that demonstrate defects in AP0E4 mice to increase lipid levels in response to the aging brain. IF : Graphs of data depicting CSF cholesterol and cholesteryl esters (CEs) changing in aged vs. young mouse CSF for each APOE genotype, demonstrating increases in AP0E2 and AP0E3 CSF with age but not in AP0E4. 1G: Graph of data depicting CSF lipid fold-change with age vs. brain lipid foldchange with age, separated by APOE genotype. All mice exhibit brain lipid accumulation with age, while only the CSF of AP0E2 and AP0E3 similarly increases in lipids.

FIGs. 2A- 2K: Schematics and graphs depicting human brain accumulates lipids in AD in a sex- and APOE-dependent manner, exacerbated by dysfunctional AP0E4 CSF lipoprotein particles. 2A-B: Schematic of human MAP cohort design for lipidomics study and graphs of data depicting human MAP cohort metadata balanced across key AD pathological variables. 2C-2D: Graphs of data depicting human brain lipid probability distribution function shifts for each subgroup (control and AD, female and male, APOE3/3 and APOE3/4), lipid species changes in APOE3/4 vs. APOE3/3 human brain, and fraction of lipid species within a class that increase in APOE3/4 vs. APOE3/3 human brain. 2E-2F: Graphs of data depicting human CSF lipid probability distribution function shifts for each subgroup (control and AD, female and male, APOE3/3 and APOE3/4 lipid species changes in APOE3/4 vs. APOE3/3 human CSF, and fraction of lipid species within a class that increase in APOE3/4 vs. APOE3/3 human CSF. 2G: Graphs of data depicting human CSF lipid species abundance (z-score) changes across diagnosis and risk variables (AD, sex, APOE genotype). 2H: Graphs of data depicting human CSF top 50 lipids averaged across all individuals, with colored bars signifying lipids identified in brain LPs in previous literature and colored by lipid class (key included in panel 2G). 21: Graphs of data depicting human CSF top 30 lipid abundances separated by APOE status, sex, and AD diagnosis, demonstrating trend of decreasing LP lipids in AP0E4 carriers and/or in AD cases. 2J: Graphs of data depicting CSF lipid abundance vs. brain lipid abundance, separated by APOE genotype and AD status, demonstrating shift to more brain accumulation and lower CSF lipid content in AP0E4 carriers relative to AP0E3, and in AD relative to control patients. 2K: Schematic of global and local lipid distribution systems in the brain via CSF, both of which are disrupted in AD due to lack of brain LPs mediating lipid transport through the CSF.

FIGs. 3A- 31: Schematic and graphs of rLP synthesis, characterization, and in vitro functional screening. 3A: Schematic of rLP synthesis scheme. 3B-3C: Graphs of data depicting rLP characterization by absorbance after purification by size exclusion chromatography (SEC). SEC fractions containing both protein and lipophilic dye (here, rLP- 10e-f5) contain rLP product. Full absorbance spectra show peaks for protein at 280 nm and lipophilic dye at 550 nm, shown for formulations rLP-lOe and rLP- 101. 3D-3E: Graphs of data depicting rLP characterization by dynamic light scattering (DLS) and zeta potential, demonstrating correct size range and negative surface potential as distinct from free (not lipidated) ApoE protein. 3F-3G Images of rLPs with transmission electron microscopy (TEM), demonstrating discoidal morphology and validating diameter measurements done by DLS. 3H-3I: Schematic of cholesterol efflux assay used to test rLP function for lipid efflux in vitro. Images and graphs of data depicting human iPSC-derived astrocytes after incubation with rLPs displaying lower BODIPY-cholesterol signal, quantified for a larger panel of controls and rLP formulations.

FIGs. 4A- 4H: Schematics and graphs demonstrating that rLPs ameliorate pathological defects in an APOE4 AD mouse model. 4A-4C: Graphs of data and images depicting total amyloid clearance in the hippocampus (HPC) of rLP-treated APOE4', 5XFAD mice in comparison to saline-treated controls, quantified by integrated fluorescence intensity and surface coverage of D54D2 antibody stain. 4D-4F : Graphs of data and images depicting total amyloid clearance in the prefrontal cortex (PFC) of rLP-treated APOE4', 5XFAD mice in comparison to saline-treated controls, quantified by integrated fluorescence intensity and surface coverage of D54D2 antibody stain. 4G-4H: Graphs of data depicting lower lipid droplet load in the subiculum of rLP-treated APOE4', 5XFAD mice in comparison to saline- treated controls, quantified by integrated fluorescence intensity and lipid droplet count of BODIPY neutral lipid stain. 4I-4J: Graphs of data depicting lower levels of activated microglia in the PFC of rLP-treated AP0E4', 5XFAD mice in comparison to saline-treated controls, quantified by integrated fluorescence intensity and surface coverage of Ibal antibody stain.

FIGs. 5A-5B: Schematics disclosing the Alzheimer’s disease risk triad are provided in FIGs 5A. Schematic of lipids stored in the brain in the form of lipid droplets and lipids transported in the CSF in the form of lipoprotein particles (LPs) in FIG. 5B.

DETAILED DESCRIPTION

AP0E4 is a strong genetic risk factor for late-onset Alzheimer’s disease (AD). The ability to regulate this pathway has important implications for the treatment of disorders such as AD. Lipid dysregulation in the brain is a key feature of AD. Brain lipoproteins containing ApoE are responsible for global lipid distribution (delivery and clearance) in the brain. The strongest genetic AD risk factor, AP0E4, decreases the ability of ApoE to transport lipids.

In aspects of the disclosure an understanding of the lipids involved in various neuropathologies has been undertaken. To better understand how AD risk variables interact to impact lipid homeostasis, lipid profiles in the brain, cerebrospinal fluid (CSF), and blood plasma of female vs. male and young vs. aged mice expressing humanized APOE of the three most common variants: the AD-protective AP0E2, most common form AP0E3, and AD- prone AP0E4 were mapped. It was discovered that brain lipoprotein particle (LP)-associated lipids increase in cerebrospinal fluid (CSF) during “healthy” aging in AP0E2 and AP0E3 mice, but do not display the same increase in “AD-risked” aging in AP0E4 mice. It was further demonstrated, using postmortem human CSF samples, that brain LP-associated lipids are lower in CSF of AP0E4 carriers and also lower in AD patients compared to non- AD patients (even within APOE3I3 patient groups).

Based at least in part on these collective findings, reconstituted lipoprotein particles (rLPs) have been developed for delivery into a subject to rescue this lipid transport defect and restore glial derived lipid support of neurons. The rLPs can be delivered in a variety of ways including intrathecal delivery and intravenous delivery. Enhancing LP-mediated intercellular lipid transport reduces risk of AD by buffering lipid imbalances in the brain. This approach may also have beneficial secondary effects for ameliorating AD pathology and symptoms, including clearing amyloid beta plaques from the brain extracellular space. Thus, it was discovered, quite unexpectedly, that rLPs could be designed and used for treating diseases associated with brain deficits and diseases such as AD based on an understanding of the lipid profiles and dysregulation associated with the disease.

The rLPs disclosed herein may be used in the treatment of diseases including Alzheimer’s disease. While a number of lipid particles are used to deliver compounds such as therapeutics, diagnostics, and other agents to a subject, the rLPs disclosed herein may be used as a direct therapeutic, with or without an alternative payload or co-administered with another therapeutic agent. Additionally, the rLPs may be prepared and used with ApoE3, a non-AD- risk protein variant, to supplement this protein and lipids for AP0E4 patient carriers. This therapy would assist in correcting the AP0E4 and lipid deficiencies that arise with age and/or disease.

Another important aspect of the rLPs is the size. Lipid nanoparticles (LNPs) are typically 100 nm in diameter and cannot readily traverse in brain extracellular space. The pore size in brain extracellular matrix is less than 100 nm; typically reported -50-80 nm. Unlike larger LNPs, rLPs are sufficiently small to transport through the brain and reach various brain regions and cell types. The small size of the rLPs may also mediate better transport ability across biological barriers including the blood-brain barrier and blood-CSF barrier for delivery from the periphery to the brain.

The changes in lipids have been found to be associated not only with disease genotype and age but also sex. The data included in the Examples demonstrates that global, sexspecific changes occur in the lipidome with age. It was also determined that a drastic drop in cholesteryl esters, unique to the aging female brain and regardless of APOE status occurred. There is also an increase in triacylglycerols seen in all aging female brains. These findings suggest age- and sex-related changes in glial lipid storage that can be ameliorated by supplementing functional lipoprotein particles to restore lipid transport functions. In the CSF, the main lipid classes composing brain lipoprotein particles were increased in APOE2 and APOE3 mice with age, but increased to a lesser extent or decreased in APOE4 mice. Moreover, the specific lipid changes were sex-specific in APOE4 CSF. These results indicate that the CSF lipidome can normally adapt to buffer age-related lipid changes in brain tissue that require increased lipid transport, yet this response may break down in the context of disease and age and may be regulated differently in males and females.

Aspects of the disclosure include a method of treating AD in a subject by administering to the subject an effective amount of a reconstituted lipoprotein particle (rEP) in an effective amount to treat AD in the subject. In addition to the use of rLPs in the treatment of AD, the rLPs are beneficial in the treatment of many pathological conditions in which lipid transport and extracellular space clearance is important. The components and structure of the rLPs may be fine-tuned to accommodate multiple different disorders that involve lipid transport defects and/or aggregates accumulating in the brain extracellular space.

Thus, compositions for performing multifaceted functions based on rebalancing or substituting lipids have been developed. The compositions of the disclosure include a composition of a reconstituted lipoprotein particle (rLP). The rLP has a discoidal shape and comprises at least two distinct phospholipids, cholesterol, and at least one protein.

The rLP includes lipids as a core structural lipid. The lipids, in some embodiments have melting temperatures which transition from the gel to liquid crystalline state, which is important for disc formation, below a temperature that would cause denaturation of the protein (for instance, an ApoE protein). In some embodiments the temperature that would cause denaturation of a protein is approximately 50°C. In some embodiments the lipids have melting temperatures above the denaturation temperature of the protein, in which case the protein refolds onto the lipid disc. In some embodiments the lipids useful in the rLP has only a few or no double bounds, where double bonds are labile to oxidation and thus inherently less stable. For instance, the lipid may have fewer than 5, fewer than 4, fewer than 3, fewer than 2, or no double bounds.

The lipids useful in the rLPs are those which are brain relevant, synthetically feasible, and functional for lipid efflux/brain transport. A brain-relevant lipid is a lipid which is associated with brain tissue, such as a lipid that is found in a lipoprotein particle in a brain. The components of brain lipoprotein particles have been described in the art and others are disclosed herein as a component of the human CSF analysis. The lipids should also be synthetically feasible, meaning that they can be incorporated into a small (i.e., 10-40 nm) protein-loaded, cholesterol-carrying particle. For instance, the lipids should have properties such as the phase transition temperatures as described above and a low degree of unsaturation (i.e., low number of double bonds) to limit oxidative degradation. In some embodiments the lipids should also be functional for lipid efflux / brain transport. Eipids that are functional for lipid efflux / brain transport are known in the art and others are disclosed herein as a component of the human CSF analysis. The lipids, in some embodiments, may be sphingomyelins and phospholipids broadly, including phosphatidylcholines (PCs), phosphatidylethanolamines (PEs), phosphatidylinositols (Pls), and phosphatidylserines (PSs). In some embodiments the phospholipids include for instance PCs and PEs with varying carbon chain lengths and degree of unsaturation. The rLPs optimally include at least one phosphatidylcholine (PC) and at least one phosphatidylethanolamine (PE). ,

PCs are a class of phospholipids that incorporate choline as a headgroup. This phospholipid is composed of a choline head group and glycerophosphoric acid, with a variety of fatty acids, which may be saturated or unsaturated. More than one tail group may be present in the phosphatidylcholine in some cases, and the tail groups may be the same or different. Non-limiting examples of phosphatidylcholines that could be used include one or more of a mixture of lauric, myristic, palmitic, stearic, oleic, arachidic, and/or behenic diglycerides linked to a choline ester head group. In some embodiments the PC phospholipids are selected from DMPC and POPC (16:0-18:1 PC).

POPC is l-palmitoyl-2-oleoylphosphatidylcholine:

DMPC is l,2-Dimyristoyl-sn-glycero-3-phosphocholine and has the following structure:

PEs are glycerophospholipids, comprised of a glycerol backbone, two fatty acids, and a phosphoethanolamine molecule. Exemplary phosphatidylethanolamines include but are not limited to dilauroyl phosphatidylethanolamine (DLPE), dierucoyl phosphatidylethanolamine (DEPE), dioleoyl phosphatidylethanolamine (DOPE), distearoyl phosphatidylethanolamine (DSPE), dipalmitoyl phosphatidylethanolamine (DPPE) dimyristoyl phosphatidylethanolamine (DMPE), l-(lZ-octadecenyl)-2-arachidonoyl-sn-glycero-3- phosphoethanolamine (C18(plasm)-20:4 PE), l-(lZ-octadecenyl)-2-oleoyl-sn-glycero-3- phosphoethanolamine (C18(plasm)-18:O PE), SOPE (18:0-18:1 PE), and POPE (16:0-18:1 PE). In some embodiments the PE phospholipids are selected from SOPE and POPE.

SOPE is l-Stearoyl-2-Oleoyl-sn-glycero-3-PE:

POPE is l-Palmitoyl-2-Oleoyl-sn-glycero-3-PE:

Phosphatidylserines (PS) are glycerophospholipids having two fatty acids attached in ester linkage to the first and second carbon of glycerol and serine attached through a phosphodiester linkage to the third carbon of the glycerol. PS is a major acidic phospholipid class in the human cerebral cortex. PSs typically have melting temperatures between PCs and PEs and are highly abundant in brain LPs, following PCs and PEs.

Phosphatidylinositols (Pls) is a lipid which contains a phosphate group, two fatty acid chains, and one inositol sugar molecule. PI is an amphiphilic lipid.

The rLP in some aspects includes at least two distinct phospholipids selected from the group of PC, PS, PE and in some embodiments Pls and/or sphingolipids (SL). The term distinct indicates that the at least two phospholipids are different from one another. In some embodiments the at least two phospholipids are in different classes from one another. Classes of phospholipids include but are not limited to PC, PE, PS, and PI. In some embodiments the at least two distinct phospholipids are PC+PE, PC+PS, PS+PE, PC+PI, PS+PI, PC+PE+PS, PC+PS+PI, PS+PE+PI, PC+PE+PI, PC+PE+PS+SL, PC+PS+PI+SL, PS+PE+PI+SL, or PC+PE+PI+SL.

Sphingolipids (SLs) are lipids comprising a sphingoid base. They are aliphatic amino alcohols having a polar head group and two hydrophobic tails. One form of SL including a phosphocholine bonded to the terminal oxygen atom is a sphingomyelin (SM).

Glyco sphingolipids are another form of SL. Sphingomyelins are found in high abundance in native brain LPs.

In some aspects the rLP includes at least one PC and at least one SM in addition to a protein such as a brain- associated protein. Such rLPs do not require, but optionally could include cholesterol. In some embodiments the rLPs may comprise a lipid combination such as one of the following examples: PC+SM, PC+SM+PE, PC+SM+PS, PC+SM+PS+PE, PC+SM+PI, PC+SM+PS+PI, PC+SM+PE+PS, PC+SM+PS+PI, PC+SM+PS+PE+PI, or PC+SM+PE+PI.

The ratios of lipids to each other and to the protein play a role in the function of the rLPs. These parameters were empirically tested to develop optimized formulations for stability and lipid efflux ability.

The rLPs also include cholesterol or modified cholesterols such as monocholesteryl succinate. Ideally the rLP is designed to be below the cholesterol crystallization point (approximately 30 wt%). An advantage of including in the rLPs instead of cholesteryl esters is that cholesterol enables the production of a discoidal shape rather than spherical. Nascent, discoidal LPs can serve as better lipid acceptors to facilitate efflux of accumulated lipids that arise in the aging/ AD brain. Monocholesteryl succinate is useful for preventing maturation of rLPs from cholesterol to cholesteryl ester, and thus for making more “permanent” discs that could not be remodeled into spheres in vivo.

In some embodiments the lipids and cholesterol are separate entities and are present in the rLP, arranged through non-covalent interactions. In some embodiments one or more of the lipids and cholesterol are linked to one another through a covalent bonds. Lor instance, one or more of the phospholipids may be conjugated to a cholesterol by attaching the cholesterol to a single aliphatic chain using ester and carbonate ester bonds. In some embodiments the cholesterol may be conjugated to a sphingolipid or sphingomyelin (SM). SM has a hydroxyl group that enables its conjugation with cholesterol.

In some embodiments the rLPs have one of two main morphologies (or combinations of both: discoidal (e.g., similar to nascent LPs as secreted from astrocytes) and spherical (e.g., similar to mature LPs in which the cholesterol has been esterified, such as the major population found in CSF). Discoidal rLPs may be, for instance, synthesized by a thin-film rehydration approach. Preferably the rLP has a discoidal shape.

The rLPs include at least one protein, which is in some embodiments a brain- associated protein. A brain-associated protein, as used herein, refers to protein that is present in brain tissue and which is involved in lipid transport or efflux. In some embodiments the brain-associated protein is an apolipoprotein. The apolipoprotein may be any type. For instance, ApoE3 which is the “normal” apolipoprotein variant of ApoE and not associated with AD risk may be used. Other ApoE protein variants e.g. ApoE2 or Christchurch ApoE3 may also be used. Besides ApoE, other Apo proteins may be used and include ApoJ (clusterin) and ApoA-I, which are also found on brain lipid particles. Peptide mimics for apolipoproteins can also be used. A peptide mimic, EpK peptide version of ApoE has been synthesized. Peptides are highly tunable and relatively simple to synthesize. An advantage of incorporating apolipoproteins in rLPs derives from their receptor- and lipid-binding abilities. For instance, ApoE on the surface of LPs mediates cell-surface receptor binding and lipid loading/unloading. In other embodiments the rLPs comprise disease-risk forms of ApoE, such as ApoE4 (R112/R158). In some embodiments the protein is ApoE4 in combination with ApoA-I and/or ApoJ. Such constructs are useful for research purposes.

Some exemplary LPs, including controls, are shown in the following Table 1. The last column discloses features and properties of the designed LPs.

Table 1

The methods may involve the introduction of functional, brain in vivo-like ApoE3- rLPs to ameliorate lipid transport defects present in the AP0E4 brain and drive system toward the age-adaptive, neuro supportive CSF present in AP0E3 and AP0E2 carriers.

In some embodiments, the rLPs are ultrasmall rLPs. For instance, an ultrasmall rLP has an average diameter about 5-30nm, 8-25nm, 10-30 nm, 10-20 nm, 10-15 nm, 10-40 nm, 15-29 nm, 15-30 nm, 15-35 nm, 15-40 nm, 20-30 nm, 20-35 nm, 20-40 nm, 30-35 nm, or 30- 40 nm. The small size is particularly suited for brain transport through dense brain ECM.

The novel rLP formulations offer a non-genetically modifying potential therapeutic avenue for treating A /Y9E4- related lipid defects in the context of the Alzheimer’s brain. The extensive lipidomics dataset disclosed herein may be used as a roadmap for patient stratification (e.g., by chromosomal sex) to match the rLP formulation to the observed lipid defects. The effectiveness of the rLPs has been demonstrated in vivo. Lor instance, it is shown herein that rLPs successfully clear amyloid that accumulates in the extracellular space. Additionally, reductions in lipid droplet accumulation were observed in the treated group (quantified by BODIPY neutral lipid staining) and a trend toward reduced microglia activation (quantified by Ibal staining).

The rLPs disclosed herein may be used in the treatment of diseases including Alzheimer’s disease. The rLPs are functional in the absence of any therapeutic agent. Although most lipid particles are used to deliver drugs to a patient, the rLPs have therapeutic activity on their own. The rLPs mediate lipid transport such as delivery and clearance within the brain and to/from the brain. Lor instance, the rLPs can mediate processes such as cholesterol efflux, fatty acid transport, myelin debris clearance, remyelination after injury, and protein transport. In addition to the treatment of AD, the rLPs may be used to treat other neurodegenerative diseases and brain disorders. For example, the rLPs may be used in clearance of AB, tau, alpha- synuclein, and abnormal extracellular matrix deposits. Accordingly, rLPs may be useful in the treatment of diseases such as: Frontotemporal dementia, Vascular dementia, Cerebral amyloid angiopathy, Cerebral small vessel disease, Progressive supranuclear palsy, Dementia with lewy bodies, Parkinson’s disease, Multiple sclerosis, Huntington’s disease, Amyotrophic lateral sclerosis, Prion diseases (e.g., Creutzfeld- Jakob disease), Niemann-Pick disease, Traumatic brain injury, and Brain cancers (e.g., glioblastoma).

While a number of lipid particles are used to deliver compounds such as therapeutics, diagnostics and other agents to a subject, the rLPs disclosed herein may be used as a direct therapeutic, with or without an alternative payload or co-administered with another therapeutic agent. Thus, the rLPs may be used as a therapeutic or diagnostic agent carriers. The particles are particularly useful for facilitating the delivery of therapeutics to the brain. For example, delivery of ApoE3 to AP0E4 patients can provide an important therapeutic benefit. Additional therapeutics for treating brain disorders can also be included in the rLP for delivery to the brain. For diagnostics, imaging contrast agent may be encapsuled in the rLP. For example, MRLbased contrast agents that display poor water solubility but are intended for brain imaging applications may be delivered to the brain in rLPs.

The rLPs may be administered using any means known in the art, including inhalation, intraperitoneally, intravenously, intramuscularly, subcutaneously, intrathecally, intraventricularly, orally, enterally, parenterally, intranasally, or dermally. In some embodiments the rLPs are delivered by intracisternal, intracerebroventricular, or intravenous routes.

In some embodiments the subjects are identified as having or at risk of developing myelin degeneration based on genotype, whether they are AP0E4 positive, and successfully treated with the compounds described herein. If the subject is AP0E4 positive, those subjects are at risk of developing disorders such as CAA or Alzheimer’s disease.

In some embodiments, the methods are directed to treating or managing diseases or disorders in which abnormal lipid regulation occurs in the brain, such as those diseases associated with learning and/or memory or Alzheimer’s disease. In a non-limiting example, the rLPs disclosed herein are administered to a patient diagnosed as having or at risk for developing Alzheimer's disease, cerebral amyloid angiopathy (CAA), mild cognitive impairment, moderate cognitive impairment, and combinations thereof.

The subject may have been diagnosed with the disease, such as Alzheimer’s disease. In some embodiments the subject can be treated following diagnosis, at varying stage of the disease, or as a prophylactic measure in instances where genetic traits, family history, or other factors put the patient at risk for the neurodegenerative disease or disorder. Successful dosage amounts and schedules may be established and monitored by metrics indicative of effective treatment, for example the extent of inhibition, delay, prevention or reduction of symptoms such as cognitive decline, loss of myelination in the brain, and neurodegeneration which are detected following the initiation of treatment.

In some embodiments the subject is determined to be AP0E4 positive. A number of genetic factors in early- and late-onset familial Alzheimer's disease have been documented. The AP0E4 variant is strongly associated with late-onset/sporadic Alzheimer's disease, with a reported allele frequency of 50%-65% in patients with Alzheimer's disease, which is approximately three times that in the general population and for other neurologic disorders. In addition to Alzheimer's disease, the AP0E4 gene variant has been implicated in other amyloid-forming disorders, including CAA.

Thus, in some embodiments the methods disclosed herein are useful for treating Alzheimer’s disease. The methods of treatment may alleviate the pathological symptoms of Alzheimer's disease, including and not limited to amyloid-P accumulation or aggregation, brain cell aging, and synapse loss. As used herein, the inhibiting accumulation and/or aggregation encompasses clearing of amyloid- P by glymphatic flow, inhibiting aggregation by suppressing the production or synthesis of amyloid-P, and/or inhibiting accumulation by degrading already produced amyloid-P .

The deposition of extracellular amyloid plaques in the brain is a hallmark pathologic finding in Alzheimer’s disease. These amyloid plaques are primarily composed of Abeta peptides generated by the sequential cleavage of amyloid precursor protein ("APP") via P and y-secretase activity. Techniques and tools have been developed to visualize the presence of plaques in patients. For example, position emission tomography ("PET") scans using imaging agents, such ¹⁸F-florbetapir, that detect amyloid-beta can be used to detect the presence of amyloid in the brain.

A “subject” herein is typically a human. In certain embodiments, a subject is a nonhuman mammal. Exemplary non-human mammals include laboratory, domestic, pet, sport, and stock animals, e.g., non-human primates, mice, cats, dogs, horses, and cows. In one embodiment, such eligible subject or patient is one that is experiencing or has experienced one or more signs, symptoms, or other indicators of an amyloid disease or has been diagnosed with a disease, whether, for example, newly diagnosed, previously diagnosed or at risk for developing a disease such as Alzheimer's disease. Diagnosis of disease may be made based on clinical history, clinical examination, and established imaging modalities. A "patient" or "subject" herein includes any single human subject eligible for treatment who is experiencing or has experienced one or more signs, symptoms, or other indicators of disease. Intended to be included as a subject are any subjects involved in clinical research trials, or subjects involved in epidemiological studies, or subjects once used as controls.

The methods of treatment provided herein can be applied to subjects suffering from Alzheimer's disease. The subject may, in some embodiments have mild to moderate Alzheimer's disease. In other embodiments the subject may have moderate to severe Alzheimer's disease. The severity of the disease can be assessed using a number of diagnostic criteria known in the art, such as biomarkers. For instance, mild Alzheimer's disease or Stage 1 disease may be an asymptomatic patient characterized by PET or CSF positive for amyloid P, a Stage 2 disease may show downstream neurodegeneration biomarkers such as tau, FDG- PET, or structural MRI, and Stage 3 disease may present as amyloidosis plus neuronal injury and cognitive/behavioral decline.

In some aspects, the methods provided herein are methods of reducing or slowing decline due to Alzheimer's disease in patients suffering from early, mild, or mild to moderate Alzheimer's disease. In some embodiments, the decline is one or more of: clinical decline, cognitive decline, and functional decline. In some embodiments, the decline is a decline in cognitive capacity or cognitive decline. In some embodiments, the decline comprises a decline in functional capacity or functional decline. Various tests and scales have been developed to measure cognitive capacity (including memory) and/or function. In various embodiments, one or more test is used to measure clinical, functional, or cognitive decline. A standard measurement of cognitive capacity is the Alzheimer's Disease Assessment Scale Cognitive (ADAS-Cog) test, for example, the 12-item ADAS-Cog or ADAS-Cogl2, or the 13-item ADAS-Cog or ADAS-Cog- 13. Thus, in some embodiments, the reduction or slowing in decline in cognitive capacity (or cognitive decline) in patients being treated with the rLPs of the disclosure is determined using the ADAS-Cogl2 test. An increase in ADAS-Cogl2 score is indicative of worsening in a patient's condition. In some embodiments, the reduction or slowing in cognitive decline in patients being treated with the rLPs of the disclosure is determined by a Clinical Dementia Rating Scale/Sum of Boxes (CDR-SB) score. In some embodiments, reduction or slowing in functional decline (or decline in functional ability) in patients being treated with the rLPs of the disclosure is determined using the Instrumental Activities of Daily Living (iADL) scale. In some embodiments, decline of one or more types is assessed and one or more of the foregoing tests or scales is used to measure reduction or slowing in decline.

Amyloid-positive subjects or patients may have brain amyloid load consistent with that seen in patients diagnosed with Alzheimer's disease. A subject suffering from mild cognitive impairment or Alzheimer's disease or having preclinical Alzheimer's disease, prodromal Alzheimer's disease, early or mild Alzheimer's disease, are typically subjects with an MMSE score of 20 or above (e.g., 20-30, 20-26, 24-30, 21-26, 22-26, 22-28, 23-26, 24-26, or 25-26) or with a Clinical Dementia Rating-Global Score (CDR-GS) of 0.5 or 1.0, and subjects with a Free and Cued Selective Reminding Test-Immediate Recall (FCSRT-IR) Cueing Index of 0.67 or above and a total free recall score of 27 or greater.

Several Alzheimer's disease -risk genes are expressed in cells that constitute the brain and may directly influence the accumulation and clearance of Ap. In particular, Apolipoprotein E (APOE) protein is highly expressed in astrocytes and microglia of the brain. In humans, there are three genetic polymorphisms of APOE, e2, e3, and e4. The e4 isoform of APOE (APOE4) is the most significant known genetic risk factor for CAA and sporadic Alzheimer's disease. In some embodiments, subjects are carriers of at least one APOE4 allele ("APOE4 carriers”).

Alleviating a neurodegenerative disease such as Alzheimer’s Disease, includes delaying the development or progression of the disease or reducing disease severity. Alleviating the disease does not necessarily require curative results. As used therein, "delaying" the development of a disease means to defer, hinder, slow, retard, stabilize, and/or postpone progression of the disease. This delay can be of varying lengths of time, depending on the history of the disease and/or individuals being treated. A method that "delays" or alleviates the development of a disease, or delays the onset of the disease, is a method that reduces probability of developing one or more symptoms of the disease in a given time frame and/or reduces extent of the symptoms in a given time frame, when compared to not using the method. Such comparisons are typically based on clinical studies, using a number of subjects sufficient to give a statistically significant result. "Development" or "progression" of a disease means initial manifestations and/or ensuing progression of the disease. Development of the disease can be detectable and assessed using standard clinical techniques as well known in the art. However, development also refers to progression that may be undetectable. For purpose of this disclosure, development or progression refers to the biological course of the symptoms. "Development" includes occurrence, recurrence, and onset. As used herein "onset" or "occurrence" of a neurodegenerative disease includes initial onset and/or recurrence.

The rLP is administered to the brain of the patient, either directly or indirectly by administration to other regions of the body. The rLP may be administered directly by intrathecal, intraventricular, or intranasal administration. The rLP may be administered indirectly to the brain by administration through any route that delivers a rLP to a body of a subject. In some embodiments the rLP is administered as an immediate release formulation. In some embodiments the rLP is administered as a sustained release formulation.

The rLPs may be administered directly to a subject in the absence of any additional carriers of formulation agents. In other embodiments the rLPs may be combined with pharmaceutically acceptable carriers or excipients for administration to a subject.

Pharmaceutically acceptable excipients (carriers) including buffers, which are well known in the art. See, e.g., Remington: The Science and Practice of Pharmacy 20th Ed. (2000) Lippincott Williams and Wilkins, Ed. K. E. Hoover. The pharmaceutical compositions to be used in the present methods can comprise pharmaceutically acceptable carriers, excipients, or stabilizers in the form of lyophilized formulations or aqueous solutions. (Remington: The Science and Practice of Pharmacy 20^th Ed. (2000) Lippincott Williams and Wilkins, Ed. K. E. Hoover). Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations used, and may comprise buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrans; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counter-ions such as sodium; metal complexes (e.g., Zn-protein complexes); and/or non-ionic surfactants such as TWEEN™ (polysorbate), PLURONICS™ (poloxamers) or polyethylene glycol (PEG).

In one embodiment, the pharmaceutical formulations include those suitable for oral, parenteral (including subcutaneous, intradermal, intramuscular, intravenous, and intraarticular), inhalation (including fine particle dusts or mists which may be generated by means of various types of metered doses, pressurized aerosols, nebulizers or insufflators), rectal and topical (including dermal, transdermal, transmucosal, buccal, sublingual, and intraocular) administration, although the most suitable route may depend upon, for example, the condition and disorder of the recipient. Pharmaceutically acceptable excipients and salts are further described herein. In some embodiments the pharmaceutically acceptable salt is a hydrochloride.

The formulations may conveniently be presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy. All methods include the step of bringing the active ingredient into association with the carrier which constitutes one or more accessory ingredients. In general, the formulations are prepared by uniformly and intimately bringing into association the active ingredient with liquid carriers or finely divided solid carriers or both and then, if necessary, shaping the product into the desired formulation.

Exemplary compositions for oral administration include suspensions which can contain, for example, microcrystalline cellulose for imparting bulk, alginic acid or sodium alginate as a suspending agent, methylcellulose as a viscosity enhancer, and sweeteners or flavoring agents such as those known in the art; and immediate release tablets which can contain, for example, microcrystalline cellulose, dicalcium phosphate, starch, magnesium stearate and/or lactose and/or other excipients, binders, extenders, disintegrants, diluents and lubricants such as those known in the art. The rLP can also be delivered through the oral cavity by sublingual and/or buccal administration. Molded tablets, compressed tablets or freeze-dried tablets are exemplary forms which may be used. Exemplary compositions include those formulating the present rLPs with fast dissolving diluents such as mannitol, lactose, sucrose and/or cyclodextrins. Also included in such formulations may be high molecular weight excipients such as celluloses (avicel) or polyethylene glycols (PEG). Such formulations can also include an excipient to aid mucosal adhesion such as hydroxy propyl cellulose (HPC), hydroxy propyl methyl cellulose (HPMC), sodium carboxy methyl cellulose (SCMC), maleic anhydride copolymer (e.g., Gantrez), and agents to control release such as polyacrylic copolymer (e.g. Carbopol 934). Lubricants, glidants, flavors, coloring agents and stabilizers may also be added for ease of fabrication and use. Formulations for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents, thickening agents. The formulations may be presented in unit-dose or multi-dose containers, for example sealed ampoules and vials, and may be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example saline or water-for-injection, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets of the kind previously described. Exemplary compositions for parenteral administration include injectable solutions or suspensions which can contain, for example, suitable non-toxic, parenterally acceptable diluents or solvents, such as mannitol, 1,3- butanediol, water, Ringer's solution, an isotonic sodium chloride solution, or other suitable dispersing or wetting and suspending agents, including synthetic mono- or diglycerides, and fatty acids, including oleic acid, or Cremapnor. In one embodiment, compositions for parenteral administration comprise up to 15% Cremaphor and up to 85% alcohol. In one embodiment, compositions for parenteral administration comprise up to 50% Cremaphor and up to 50% alcohol. In one embodiment, compositions for parenteral administration comprise up to 15% Cremaphor and up to 85% ethanol. In one embodiment, compositions for parenteral administration comprise up to 50% Cremaphor and up to 50% ethanol. An aqueous carrier may be, for example, an isotonic buffer solution at a pH of from about 3.0 to about 8.0, preferably at a pH of from about 3.5 to about 7.4, for example from 3.5 to 6.0, for example from 3.5 to about 5.0. Useful buffers include sodium citrate-citric acid and sodium phosphate-phosphoric acid, and sodium acetate/acetic acid buffers. Excipients that can be included are, for instance, non-toxic auxiliary substances, such as wetting or emulsifying agents, preservatives, and pH buffering agents and the like, for example sodium acetate or sorbitan monolaurate.

Preferred unit dosage formulations are those containing an effective dose, as disclosed herein. It should be understood that in addition to the ingredients particularly mentioned above, the formulations of this invention may include other agents conventional in the art having regard to the type of formulation in question, for example those suitable for oral administration may include flavoring agents. The rLPs are also suitably administered as sustained-release systems. Suitable examples of sustained-release systems of the disclosure include suitable polymeric materials, for example semi-permeable polymer matrices in the form of shaped articles, e.g., films, or microcapsules; suitable hydrophobic materials, for example as an emulsion in an acceptable oil; or ion exchange resins; and sparingly soluble derivatives of the rLP of the disclosure, for example, a sparingly soluble salt. Sustained-release systems may be administered orally; rectally; parenterally; intracistemally; intravaginally; intraperitoneally; topically, for example as a powder, ointment, gel, drop or transdermal patch; bucally; or as an oral or nasal spray.

Preparations for administration can be suitably formulated to give controlled release of rLPs of the disclosure. For example, the pharmaceutical compositions may be in the form of particles comprising one or more of biodegradable polymers, polysaccharide jellifying and/or bioadhesive polymers, amphiphilic polymers, agents capable of modifying the interface properties of the particles of the rLPs. These compositions exhibit certain biocompatibility features which allow a controlled release of the active substance.

The rLPs may be delivered by way of a pump or by continuous subcutaneous infusions, for example, using a mini-pump. An intravenous bag solution may also be employed. The key factor in selecting an appropriate dose is the result obtained, as measured by improvements in one or more symptoms of neurodegenerative disorders of interest, or by other criteria for measuring control or prevention of one or more symptoms of neurodegenerative disorders of interest, as are deemed appropriate by the practitioner. In another aspect of the disclosure, rLPs are delivered by way of an implanted pump.

Implantable drug infusion devices are used to provide patients with a constant and long term dosage or infusion of a drug or any other therapeutic agent. Essentially such device may be categorized as either active or passive. The rLPs may be formulated as a depot preparation. Such a long acting depot formulation can be administered by implantation, for example subcutaneously or intramuscularly; or by intramuscular injection. Thus, for example, the rLPs can be formulated with suitable polymeric or hydrophobic materials, for example as an emulsion in an acceptable oil; or ion exchange resins; or as a sparingly soluble derivatives, for example, as a sparingly soluble salt.

A therapeutically effective amount of the rLPs may be administered as a single pulse dose, as a bolus dose, or as pulse doses administered over time. Thus, in pulse doses, a bolus administration of a rLP of the disclosure is provided, followed by a time period wherein no rLP of the disclosure is administered to the subject, followed by a second bolus administration. In specific, non-limiting examples, pulse doses of a rLP of the disclosure are administered during the course of a day, during the course of a week, or during the course of a month.

In some embodiments the daily dose administered to the patient is between 1 and 50 mg and the dose is administered once daily. Therapeutically effective amount of the rLPs will be dependent on the molecule utilized, the subject being treated, the severity and type of the affliction, and the manner and route of administration. For example, a therapeutically effective amount of the rLPs may vary from about 0.001 mg/Kg to about 2000 mg/Kg body weight. In one embodiment, a therapeutically effective amount of the rLPs may vary from about 0.01 mg/Kg to about 1 mg/Kg body weight. In one embodiment, a therapeutically effective amount of the rLPs may vary from about 0.001 mg/Kg to about 0.9 mg/Kg body weight, about 0.8 mg/Kg body weight, about 0.001 mg/Kg to 0.7 mg/Kg body weight, about 0.001 mg/Kg to 0.6 mg/Kg body weight, about 0.001 mg/Kg to 0.5 mg/Kg body weight, about 0.001 mg/Kg to 0.4 mg/Kg body weight, about 0.001 mg/Kg to 0.3 mg/Kg body weight, about 0.001 mg/Kg to 0.2 mg/Kg body weight, about 0.001 mg/Kg to 0.1 mg/Kg body weight, about 0.001 mg/Kg to 0.09 mg/Kg body weight, about 0.001 mg/Kg to 0.08 mg/Kg body weight, about 0.001 mg/Kg to 0.07 mg/Kg body weight, about 0.001 mg/Kg to 0.06 mg/Kg body weight, about 0.001 mg/Kg to 0.05 mg/Kg body weight, about 0.001 mg/Kg to 0.04 mg/Kg body weight, about 0.001 mg/Kg to 0.03 mg/Kg body weight, about 0.001 mg/Kg to 0.02 mg/Kg body weight. 0.01 mg/Kg body weight, about 0.001 mg/Kg to 0.009 mg/Kg body weight, about 0.001 mg/Kg to 0.008 mg/Kg body weight, about 0.001 mg/Kg to 0.007 mg/Kg body, about 0.001 mg/Kg to 0.006 mg/Kg body, about 0.001 mg/Kg to 0.005 mg/Kg body weight, about 0.001 mg/Kg to 0.004 mg/Kg body weight, about 0.001 mg/Kg to 0.003 mg/Kg body weight, and about 0.001 mg/Kg to about 0.002 mg/Kg body weight. In one embodiment, a therapeutically effective amount of the rLP may vary from about 0.001 mg/Kg to about 20 mg/Kg body weight.

In some embodiments, a therapeutically effective amount of the rLP is selected from the group consisting of about 0.01 mg/m², about 0.02 mg/m², about 0.03 mg/m², about 0.04 mg/m², about 0.05 mg/m², about 0.06 mg/m², about 0.07 mg/m², about 0.08 mg/m², about 0.09 mg/m², and about 0.1 mg/m². In some embodiments, a therapeutically effective amount of the rLP is selected from the group consisting of about 0.1 mg/m², about 0.2 mg/m², about 0.3 mg/m², about 0.4 mg/m², about 0.5 mg/m², about 0.6 mg/m², about 0.7 mg/m², about 0.8 mg/m², about 0.9 mg/m², about 1 mg/m², about 1.1 mg/m², about 1.2 mg/m², about 1.3 mg/m², about 1.4 mg/m², about 1.5 mg/m², about 1.6 mg/m², about 1.7 mg/m², about 1.8 mg/m², about 1.9 mg/m², about 2 mg/m², about 2.1 mg/m², about 2.2 mg/m², about 2.3 mg/m², about 2.4 mg/m², about 2.5 mg/m², about 2.6 mg/m², about 2.7 mg/m², about 2.8 mg/m², about 2.9 mg/m², and about 3 mg/m².

The practice of the present invention will employ, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry and immunology, which are within the skill of the art.

EXAMPLES

The following Examples provide exemplification of embodiments and aspects of the disclosure.

A library of rLPs with different particle morphologies and lipid/protein compositions are developed herein based on the mapping of lipid profiles in the brain, CSF, and blood plasma of different subjects under different conditions/classifications. The library of rLPs is a useful tool for identifying and optimizing new compositions for treating and preventing AD.

Characterization of library members may include size (dynamic light scattering; to measure hydrodynamic diameter), morphology (transmission electron microscopy; to show discoidal shape and confirm diameter), stability (zeta potential), lipid and protein content (small-molecule assays and mass spectrometry), purity (native polyacrylamide gel electrophoresis) and binding (lipid uptake and immunoblotting). Functional testing may include in vitro testing in cells or tissues, such as brain cells or human iPSC-derived brain cell cultures and in vivo testing via intracisternal injection in mice.

A tissue lipidomics analysis was performed. Briefly, the data demonstrated a few conclusions. The aged brain undergoes global, sex-specific changes in the lipidome. Increase in lipids overall with age, including TGs (esp. unsaturated), DGs, cholesterol, SMs, antioxidants, has been observed. A distinct signature of aged females reveals even higher net increase in lipids, including TGs, but lower CEs and DGs was also observed. In AP0E2 and AP0E3, the CSF lipidome is age-adaptive to buffer lipid changes that arise in aged brain tissue; absent in risk-prone AP0E4. Sex differences in lipidome reflect storage defects/bioenergetic shifts that can be ameliorated vs. exacerbated as a function of APOE status. Decreased CEs in AP0E4 aged female CSF were also observed. In view of the knowledge gained from the lipidomics analysis, additional work was performed to create rLPs which could be used as stand-alone therapeutics and/or carriers for therapeutics in the treatment of neurological diseases of lipid transport and extracellular space clearance is important. This additional work is described in the Examples.

Example 1: Assessment of AD Risk Triad-Dependent Lipid Changes in APOE Mouse Models

Lipid changes were studied in mice with humanized homozygous APOE2, APOE3, and APOE4 at ages 3- and 12-months. Both male and female animals were studied (Fig. 1A). Blood plasma was collected by facial bleed, cerebrospinal fluid (CSF) was collected via capillary needle puncture of the cistema magna connected to a manual syringe, then mice were perfused with PBS and the brain tissue was harvested. Brain tissue, CSF, and blood plasma were flash-frozen and processed for mass spectrometry-based lipidomics including lipid extraction. Principal component analysis of brain tissue revealed that age was the most distinguishing feature separating out the lipid fingerprints of the mouse brains (Fig. IB). Further analysis of brain tissue revealed increases in various phospholipid and triacylglycerol (TG) species in 12-month compared to 3-month-old animals (Fig. 1C). TGs and cholesteryl esters (CEs) are the primary lipid species that compose lipid droplets in the brain. When examining aged females vs. males, females display enrichment of TGs and depletion of CEs (Fig. ID). Fisher’s test is applied to determine statistical significance of the fraction of lipid species within a class that increase in females compared to males. These findings demonstrate that the brain accumulates lipids with age, though lipid species vary in a sex-dependent manner.

In CSF, lipid classes representative of lipoprotein particle (LP) lipid classes are shown, including PCs, PEs, cholesteryl esters (CEs), TGs, and sphingomyelins (SMs), and Fisher’s test is applied to determine statistical significance. The results demonstrate that LP- associated lipids do not increase as strongly in AP0E4 mice, with sex-dependent changes, in comparison to AP0E3 mice (Fig. IE). Similarly, cholesterol and CE species increase in both AP0E2 and AP0E3 CSF with age, especially with higher degrees of unsaturation (more double bonds in carbon chains), while not displaying any significant changes in AP0E4 CSF with age, indicating a deficiency in LPs. Significantly changing lipid species are colored with darker opacity (Fig. IF). Brain and CSF findings are summarized in Fig. 1G, wherein CSF lipid log2fold-change vs. brain lipid log2fold-change reveals brain accumulation with age in all samples, with compensatory CSF lipid increases with age only in “age-adaptive” APOE2 and APOE3 CSF and not in AD-risked APOE4 CSF. Taken together, these results suggest a defect in APOE4 CSF lipid transport capabilities.

Example 2: Evaluation of Lipid Changes driven by AD Risk Triad in Postmortem Human Samples

A study was designed to characterize lipid levels in postmortem samples from human donors of the Memory and Aging Project (MAP) study at Rush University (Fig. 2A). Groups were balanced for National Institute on Aging/Reagan Institute of the Alzheimer Association Consensus Recommendations for the Postmortem Diagnosis of AD (NIA/Reagan Diagnosis of AD), overall cognitive diagnostic category (Cogdx), Consortium to Establish a Registry for Alzheimer’s Disease (CERAD) score, Braak stage, and postmortem interval (Fig. 2B). The study included equal numbers of female and male individuals, homozygous APOE3/3 and heterozygous APOE3/4 carriers, and AD and non- AD patients. Lipidomics were performed on postmortem brain tissue sampled from the prefrontal cortex (PFC), ventricular cerebrospinal fluid (CSF), and blood serum. Brain tissue mass was normalized then homogenized, biofluid volumes were normalized, and then all tissues were processed for mass spectrometry-based lipidomics including lipid extraction and spiking with internal lipid standard mixture. From these lipidomics experiments, probability distribution functions of baseline lipid z-scores (normalized abundances) were noted to be higher in the brains of female donors and increase in males with AD to the level of female donors (Fig. 2C). In addition, APOE3/4 individuals (grouped males and females) were determined to show significant increases in brain lipids compared to APOE3/3 individuals, including in cholesterol and cholesteryl esters (Fig. 2D). In CSF, lipid z-scores were noted to have decreased in AD patients compared to non- AD patients (Fig. 2E), with APOE3/4 individuals displaying lower lipid levels compared to APOE3/3 (Fig. 2F). Full heatmapping of normalized lipid abundance illustrates broad CSF lipid decreases in AD patients compared to healthy controls (Fig. 2G). Absolute abundances of the top 50 CSF lipids averaged across all individuals are aligned to previous literature (DeMattos et al. Neurochemistry International, 2001) and inform rLP design choices (Fig. 2H). A correlation of decreasing LP lipids in APOE4 carriers was also noted (Fig. 21). Importantly, CSF lipid z-score compared to brain lipid z-score indicates a shift to more brain accumulation and lower CSF lipid content in the progression of APOE3/3 control, APOE3/4 control, APOE3/3 AD, then APOE3/4 AD (Fig. 2J). This result indicates that AD patients and AP0E4 carriers exhibit heightened brain lipid accumulation, without the compensatory CSF lipoprotein particle transport mechanisms to offload this increased lipid burden that exists in healthy controls and AP0E3 carriers. These results underscore both the global and local lipid transport functions that ApoE carries out in the brain tissue (Fig. 2K), impacted negatively by the poor lipid binding and transport abilities of the ApoE4 variant.

Example 3: Manufacturing and Characterization of Reconstituted Lipoprotein Particles (rLPs) rLPs were synthesized and loaded with ApoE3 by (i) thin-film rehydration; (ii) solubilization and reconstitution; and (iii) downstream purification to remove free ApoE and larger lipid structures (Fig. 3A). Briefly, (i) individual lipids were dissolved in chloroform to known concentrations, mixed together in known molar ratios, and the chloroform was completely removed in a high-throughput manner that limits lipid oxidation by using vacuum centrifugation; (ii) 50 mM sodium cholate was added to the thin lipid film and sonicated to solubilize the lipids, ApoE was added with gentle mixing by pipetting, more sodium cholate was added to be above the critical micelle concentration (~20 mM), and the mixture was incubated for 12 hours to aid in lipid/protein assembly at a temperature near but slightly above the melting temperature of the lipid with the maximum melting temperature in the mixture; (iii) sodium cholate was slowly removed by dialysis against phosphate-buffered saline for 24 hours to drive disc formation (membrane molecular weight cutoff 7-10 kDa), the sample was centrifuged to remove precipitates and preliminarily assess colloidal stability, any remaining free ApoE or large lipid aggregates was removed by size-exclusion chromatography (Superdex™ 200 resin), then the fraction containing rLPs was spin-filtered to concentrate the final product. Concentration of protein was measured by absorbance at 280 nm (Fig. 3B). During size exclusion chromatography purification, fractions containing product were determined by co-elution of protein and a lipophilic dye incorporated into the lipid discs (Vybrant™ Dil Cell-Labeling Solution, ThermoFisher Scientific, Waltham, MA) (Fig. 3C). Hydrodynamic diameter of two exemplary formulations (rLP-lOe and rLP-101) was compared to free ApoE3 by dynamic light scattering and reported as number distribution (Fig. 3D). This experiment reveals diameters of 5.28 nm, 23.88 nm, and 27.77 nm for free ApoE, rLP-101, and rLP-lOe, respectively. The results demonstrate that rLPs are synthesized with target size characteristics and are larger than free ApoE, as expected upon lipidation. Additionally, zeta-potential (Fig. 3E) and TEM (Fig. 3F-G) of rLP-lOe and rLP-101 were measured and show that rLPs are colloidally stable (more negative surface potential) and possess discoidal morphology, respectively.

Lipid efflux function of rLPs was tested using an in vitro cholesterol / cholesteryl ester efflux assay. A schematic of the mechanisms of action of the assay is shown in Fig. 3H. Briefly, AP0E4 astrocytes, derived from human induced pluripotent stem cells (iPSCs), were plated at 15k/well on a Matrigel-coated glass-bottom 96- well plate and allowed to recover for approximately four days to reach a near-confluent monolayer, with every-other day media changes. Astrocytes were then loaded with BODIPY-tagged cholesterol (diluted to 1 pg/mL in phenol-red-free, serum-free astrocyte media) for 1 hour, washed, then incubated with either controls or rLP formulations (diluted to 10 g/mL in the same media) for 24 hours (Fig. 3H).The cells were then washed and imaged on a confocal microscope to visualize BODIPY- cholesterol remaining in the cells. Lipid droplets were quantified using an automated image analysis workflow in FIJI and fold-change in lipid droplet number is plotted normalized to the control (media alone, no cholesterol acceptor).

A panel of rLPs such as those shown in Table 1 were prepared and tested in the cholesterol efflux assay. Tested formulations include formulations rLP-lOe, rLP-101, and controls of free ApoE3, ApoA-I, and HDL that are considered good cholesterol acceptors. The formulations corresponding to the list along the x-axis in Fig. 31 are described in Table 2. The data depicting human iPSC-derived astrocytes after incubation with rLPs display lower BODIPY-cholesterol signal. The results demonstrate that rLPs facilitate cholesterol efflux. These results are further supported by BODIPY-cholesterol fluorescence in the supernatant of rLP-treated wells.

Table 2

Example 4: Determination of the Effect of Reconstituted Lipoprotein Particles (rLPs) on Alzheimer’s Disease (AD)

A mouse model of AD including a combination of humanized APOE4 and familial AD genes is used to analyze the effects of rLPs in vivo. Both male and female 12-month-old AP0E4 5XFAD mice were treated with rLPs by intracistemal injection, allowed to recover, and sacrificed after 24 hours. The mouse brains were prepared using standard techniques including perfusion, fixation, sectioning, and immunohistochemical staining. Total amyloid was quantified by D54D2 antibody immunofluorescence of tissue samples taken from the hippocampus (HPC) or prefrontal cortex (PFC). Total amyloid integrated fluorescence intensity and total amyloid coverage were quantified using an automated image analysis workflow in FIJI. Total amyloid fluorescence and coverage were reduced in the rLP-treated group relative to the saline control group in both the hippocampus (Fig. 4A-C) and the PFC (Fig. 4D-F). In the same model, lipid droplets were assessed by BODIPY staining of tissue samples taken from the subiculum. Lipid droplet integrated fluorescence intensity and lipid droplet count were reduced in the rLP-treated group relative to the saline control group (Fig. 4G-H). Thus, the in vitro lipid efflux assay appears to be predictive of in vivo function for lipid droplet reduction. Further, microglia activation may also be reduced in the rLP-treated group, as suggested by decreased total Ibal stain intensity and surface coverage (Fig. 41- J). Taken together, rLPs may represent a promising therapeutic route to impact the AD pathological hallmarks of amyloid aggregation, lipid droplet accumulation, and glia activation in the brain.

ADDITIONAL EMBODIMENTS

Additional embodiments disclosed herein include: Embodiment 1. A method of treating Alzheimer’s Disease in a subject comprising administering to the subject an effective amount of a reconstituted lipoprotein particle (rLP) in an effective amount to treat Alzheimer’ s disease in the subject, wherein the rLP has a discoidal shape and comprises: a) at least two distinct phospholipids, optionally selected from POPC, DMPC, SOPE, and POPE and combinations thereof, b) cholesterol and c) at least one protein.

Embodiment 2. The method of Embodiment 1, further comprising determining whether the subject has or is at risk of developing Alzheimer’s Disease by identifying the subject as AP0E4 positive prior to treatment.

Embodiment 3. The method of Embodiment 1 or Embodiment 2, wherein the subject is a female.

4. The method of any one of Embodiments 1-3, wherein the Alzheimer's disease is mild to moderate Alzheimer's disease.

Embodiment 5. The method of any one of Embodiments 1-3, wherein the Alzheimer's disease is moderate to severe Alzheimer's disease.

Embodiment 6. The method of any one of Embodiments 1-5, wherein the subject is homozygous for APOE4.

Embodiment 7. The method of any one of Embodiments 1-6, wherein a population of the rLP has a diameter of 5-30nm, 8-25nm, 10-30 nm, 10-20 nm, 10-15 nm, 10-40 nm, 15-29 nm, 15-30 nm, 15-35 nm, 15-40 nm, 20-30 nm, 20-35 nm, 20- 40 nm, 30-35 nm, or 30-40 nm.

Embodiment 8. The method of any one of Embodiments 1-6, wherein a population of the rLP has a diameter of 10-40 nm. Embodiment 9. The method of any one of Embodiments 1-8, wherein the rLP does not comprise cholesteryl esters.

Embodiment 10. The method of any one of Embodiments 1-9, wherein the phospholipid comprises POPC (16:0-18:1 PC) and SOPE (18:0-18:1 PE).

Embodiment 11. The method of any one of Embodiments 1-9, wherein the phospholipid comprises POPC (16:0-18:1 PC) and POPE (16:0-18:1 PE).

Embodiment 12. The method of any one of Embodiments 1-9, wherein the phospholipid comprises SOPE (18:0-18:1 PE) and POPE (16:0-18:1 PE).

Embodiment 13. The method of any one of Embodiments 1-9, wherein the phospholipid comprises POPC (16:0-18:1 PC), POPE (16:0-18:1 PE), and SOPE (18:0-18:1 PE).

Embodiment 14. The method of any one of Embodiments 1-13, wherein the phospholipids are present in a molar ratio of 70 : 16.67 : 8.33 : 5 : 1 POPC / SOPE / POPE / cholesterol / ApoE.

Embodiment 15. The method of any one of Embodiments 1-14, wherein the protein is an apolipoprotein.

Embodiment 16. The method of Embodiment 15, wherein the apolipoprotein is ApoE3, ApoE2, and/or Christchurch ApoE3.

Embodiment 17. The method of Embodiment 15, wherein the apolipoprotein is ApoA-I and/or ApoJ.

Embodiment 18. The method of any one of Embodiments 1-14, wherein the protein is an apolipoprotein peptide mimic. Embodiment 19. The method of Embodiment 18, wherein the apolipoprotein mimic is EpK.

Embodiment 20. A composition comprising a reconstituted lipoprotein particle (rLP), wherein the rLP has a discoidal shape and comprises: a) at least two distinct phospholipids, optionally selected from POPC, DMPC, SOPE, and POPE and combinations thereof, b) cholesterol, and c) at least one protein.

Embodiment 21. The composition of Embodiment 20, wherein a population of the rLP has a diameter of 5-30nm, 8-25nm, 10-30 nm, 10-20 nm, 10-15 nm, 10-40 nm, 15-29 nm, 15-30 nm, 15-35 nm, 15-40 nm, 20-30 nm, 20-35 nm, 20-40 nm, SO- 35 nm, or 30-40 nm.

Embodiment 22. The composition of Embodiment 20, wherein a population of the rLP has a diameter of 10-40 nm.

Embodiment 23. The composition of any one of Embodiments 20-22, wherein the rLP does not comprise cholesteryl esters.

Embodiment 24. The composition of any one of Embodiments 20-23, wherein the phospholipid comprises POPC (16:0-18:1 PC) and SOPE (18:0-18:1 PE).

Embodiment 25. The composition of any one of Embodiments 20-23, wherein the phospholipid comprises POPC (16:0-18:1 PC) and POPE (16:0-18:1 PE).

Embodiment 26. The composition of any one of Embodiments 20-23, wherein the phospholipid comprises SOPE (18:0-18:1 PE) and POPE (16:0-18:1 PE).

Embodiment 27. The composition of any one of Embodiments 20-23, wherein the phospholipid comprises POPC (16:0-18:1 PC), POPE (16:0-18:1 PE), and SOPE (18:0-18:1 PE). Embodiment 28. The composition of any one of Embodiments 20-27, wherein the phospholipids are present in a molar ratio of 70 : 16.67 : 8.33 : 5 : 1 POPC / SOPE / POPE / cholesterol / ApoE.

Embodiment 29. The composition of any one of Embodiments 20-28, wherein the protein is an apolipoprotein.

Embodiment 30. The composition of Embodiment 29, wherein the apolipoprotein is ApoE3, ApoE2, and/or Christchurch ApoE3.

Embodiment 31. The composition of Embodiment 29, wherein the apolipoprotein is ApoA-I and/or ApoJ.

Embodiment 32. The composition of any one of Embodiments 20-27, wherein the protein is an apolipoprotein peptide mimic.

Embodiment 33. The composition of Embodiment 32, wherein the apolipoprotein mimic is EpK.

Embodiment 33. A method for identifying a composition, comprising screening a library of reconstituted lipoprotein particles (rLPs), wherein the screening involves characterization of rLPs as having a size, morphology, stability, lipid-protein content and/or binding property, and optionally a functional property.

Embodiment 35. The method of Embodiment 34, wherein the characterization of the rLPs comprises identifying rLPs having the following properties: a) a diameter of about 5-30nm, 8-25nm, 10-30 nm, 10-20 nm, 10-15 nm, 10- 40 nm, 15-29 nm, 15-30 nm, 15-35 nm, 15-40 nm, 20-30 nm, 20-35 nm, 20-40 nm, 30-35 nm, or 30-40 nm, b) a discoidal shape, c) colloidal stability, d) at least two distinct lipids and at least one protein, and/or e) lipid uptake ability.

Embodiment 36. The method of Embodiment 34 or 35, wherein the functional property of the rLPs is determined by in vitro testing in iPSC-derived brain cell cocultures and/or in vivo testing via intracistemal injection in test subject, such as a mouse.

EQUIVALENTS AND SCOPE

All of the features disclosed in this specification may be combined in any combination. Each feature disclosed in this specification may be replaced by an alternative feature serving the same, equivalent, or similar purpose. Thus, unless expressly stated otherwise, each feature disclosed is only an example of a generic series of equivalent or similar features. From the above description, one skilled in the art can easily ascertain the essential characteristics of the present disclosure, and without departing from the spirit and scope thereof, can make various changes and modifications of the present disclosure to adapt it to various usages and conditions. Thus, other embodiments are also within the claims.

Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the present disclosure described herein. The scope of the present disclosure is not intended to be limited to the above description, but rather is as set forth in the appended claims. In the claims articles such as “a,” “an,” and “the” may mean one or more than one unless indicated to the contrary or otherwise evident from the context. Claims or descriptions that include “or” between one or more members of a group are considered satisfied if one, more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process unless indicated to the contrary or otherwise evident from the context. The present disclosure includes embodiments in which exactly one member of the group is present in, employed in, or otherwise relevant to a given product or process. The present disclosure includes embodiments in which more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process.

Furthermore, the present disclosure encompasses all variations, combinations, and permutations in which one or more limitations, elements, clauses, and descriptive terms from one or more of the listed claims is introduced into another claim. For example, any claim that is dependent on another claim can be modified to include one or more limitations found in any other claim that is dependent on the same base claim. Where elements are presented as lists, e.g., in Markush group format, each subgroup of the elements is also disclosed, and any element(s) can be removed from the group. It should it be understood that, in general, where the present disclosure, or aspects of the present disclosure, is/are referred to as comprising particular elements and/or features, certain embodiments of the present disclosure or aspects of the present disclosure consist, or consist essentially of, such elements and/or features. For purposes of simplicity, those embodiments have not been specifically set forth in haec verba herein. It is also noted that the terms “comprising” and “containing” are intended to be open and permits the inclusion of additional elements or steps. Where ranges are given, endpoints are included. Furthermore, unless otherwise indicated or otherwise evident from the context and understanding of one of ordinary skill in the art, values that are expressed as ranges can assume any specific value or sub-range within the stated ranges in different embodiments of the present disclosure, to the tenth of the unit of the lower limit of the range, unless the context clearly dictates otherwise.

This application refers to various issued patents, published patent applications, journal articles, and other publications, all of which are incorporated herein by reference. If there is a conflict between any of the incorporated references and the instant specification, the specification shall control. In addition, any particular embodiment of the present disclosure that falls within the prior art may be explicitly excluded from any one or more of the claims. Because such embodiments are deemed to be known to one of ordinary skill in the art, they may be excluded even if the exclusion is not set forth explicitly herein. Any particular embodiment of the present disclosure can be excluded from any claim, for any reason, whether or not related to the existence of prior art.

Those skilled in the art will recognize or be able to ascertain using no more than routine experimentation many equivalents to the specific embodiments described herein. The scope of the present embodiments described herein is not intended to be limited to the above Description, but rather is as set forth in the appended claims. Those of ordinary skill in the art will appreciate that various changes and modifications to this description may be made without departing from the spirit or scope of the present disclosure, as defined in the following claims.

Claims

CLAIMS What is claimed is:

1. A method of treating Alzheimer’s Disease in a subject comprising administering to the subject an effective amount of a reconstituted lipoprotein particle (rLP) in an effective amount to treat Alzheimer’s disease in the subject, wherein the rLP has a discoidal shape and comprises: a) at least two distinct phospholipids, wherein a first of the two phospholipids is a phosphatidylcholine (PC) and a second of the two phospholipids is a phosphatidylethanolamine (PE) or a phosphatidylserine (PS), b) cholesterol; c) sphingolipid (SL), optionally wherein the SL is a sphingomyelin; and d) at least one protein, optionally a brain-associated protein.

2. The method of claim 1, further comprising determining whether the subject has or is at risk of developing Alzheimer’s Disease by identifying the subject as APOE4 positive prior to treatment.

3. The method of claim 1, wherein the subject is a female.

4. The method of claim 1, wherein the Alzheimer's disease is mild to moderate Alzheimer's disease.

5. The method of claim 1, wherein the Alzheimer's disease is moderate to severe Alzheimer's disease.

6. The method of claim 1, wherein the subject is homozygous for APOE4.

7. The method of any one of claims 1-6, wherein a population of the rLP has a diameter of 5-30nm, 8-25nm, 10-30 nm, 10-20 nm, 10-15 nm, 10-40 nm, 15-29 nm, 15-30 nm, 15-35 nm, 15-40 nm, 20-30 nm, 20-35 nm, 20-40 nm, 30-35 nm, or 30-40 nm.

8. The method of any one of claims 1-6, wherein a population of the rLP has a diameter of 10-40 nm.

9. The method of any one of claims 1-6, wherein the rLP does not comprise cholesteryl esters.

10. The method of any one of claims 1-6, wherein the phospholipid comprises POPC (16:0-18:1 PC) and SOPE (18:0-18:1 PE).

11. The method of any one of claims 1-6, wherein the phospholipid comprises POPC (16:0-18:1 PC) and POPE (16:0-18:1 PE).

12. The method of any one of claims 1-6, wherein the phospholipid comprises DMPC, SOPE (18:0-18:1 PE) and POPE (16:0-18:1 PE).

13. The method of any one of claims 1-6, wherein the phospholipid comprises POPC (16:0-18:1 PC), POPE (16:0-18:1 PE), and SOPE (18:0-18:1 PE).

14. The method of any one of claims 1-6, wherein the phospholipids are present in a molar ratio of 70 : 16.67 : 8.33 : 5 : 1 POPC / SOPE / POPE / cholesterol / ApoE.

15. The method of any one of claims 1-6, wherein the protein is an apolipoprotein.

16. The method of claim 15, wherein the apolipoprotein is ApoE3, ApoE2, and/or Christchurch ApoE3.

17. The method of claim 15, wherein the apolipoprotein is ApoA-I and/or Apo J.

18. The method of any one of claims 1-6, wherein the protein is an apolipoprotein peptide mimic.

19. The method of claim 18, wherein the apolipoprotein mimic is EpK.

20. A composition comprising a reconstituted lipoprotein particle (rLP), wherein the rLP has a discoidal shape and comprises: a) at least two distinct phospholipids, wherein a first of the two phospholipids is a phosphatidylcholine (PC) and a second of the two phospholipids is a phosphatidylethanolamine (PE) or a phosphatidylserine (PS), b) cholesterol; c) sphingolipid (SL), optionally wherein the SL is a sphingomyelin; and d) at least one protein, optionally a brain-associated protein.

21. The composition of claim 20, wherein a population of the rLP has a diameter of 5- 30nm, 8-25nm, 10-30 nm, 10-20 nm, 10-15 nm, 10-40 nm, 15-29 nm, 15-30 nm, 15-35 nm, 15-40 nm, 20-30 nm, 20-35 nm, 20-40 nm, 30-35 nm, or 30-40 nm.

22. The composition of claim 20, wherein a population of the rLP has a diameter of 10-40 nm.

23. The composition of claim 20, wherein the rLP does not comprise cholesteryl esters.

24. The composition of any one of claims 20-23, wherein the phospholipid comprises POPC (16:0-18:1 PC) and SOPE (18:0-18:1 PE).

25. The composition of any one of claims 20-23, wherein the phospholipid comprises POPC (16:0-18:1 PC) and POPE (16:0-18:1 PE).

26. The composition of any one of claims 20-23, wherein the phospholipid comprises DMPC, SOPE (18:0-18:1 PE) and POPE (16:0-18:1 PE).

27. The composition of any one of claims 20-23, wherein the phospholipid comprises POPC (16:0-18:1 PC), POPE (16:0-18:1 PE), and SOPE (18:0-18:1 PE).

28. The composition of any one of claims 20-23, wherein the phospholipids are present in a molar ratio of 70 : 16.67 : 8.33 : 5 : 1 POPC / SOPE / POPE / cholesterol / ApoE.

29. The composition of any one of claims 20-23, wherein the protein is an apolipoprotein.

30. The composition of claim 28, wherein the apolipoprotein is ApoE3, ApoE2, and/or Christchurch ApoE3.

31. The composition of claim 28, wherein the apolipoprotein is ApoA-I and/or ApoJ.

32. The composition of any one of claims 20-23, wherein the protein is an apolipoprotein peptide mimic.

33. The composition of claim 32, wherein the apolipoprotein mimic is EpK.

34. The composition of any one of claims 20-23, wherein the phospholipid comprises a Phosphatidylinositol (PI).

35. The composition of any one of claims 20-23, wherein the phospholipid comprises a combination of lipids and wherein the combination is selected from one of the following groups: PC+PE, PC+PS, PC+PE+PI, PC+PE+PS, PC+PS+PI, PC+PS+PE+PI, PC+PE+PS+Sphingolipid (SL), PC+PS+PI+SL, or PC+PE+PI+SL.

36. A composition comprising a reconstituted lipoprotein particle (rLP), wherein the rLP has a discoidal shape and comprises: a) at least one phospholipid and at least one sphingolipid, wherein a first phospholipid is a phosphatidylcholine (PC), and b) at least one protein, optionally a brain-associated protein.

37. The composition of claim 36, wherein a population of the rLP has a diameter of 5- 30nm, 8-25nm, 10-30 nm, 10-20 nm, 10-15 nm, 10-40 nm, 15-29 nm, 15-30 nm, 15-35 nm, 15-40 nm, 20-30 nm, 20-35 nm, 20-40 nm, 30-35 nm, or 30-40 nm.

38. The composition of any one of claims 20-23, wherein the rLP comprises a combination of lipids and wherein the combination is selected from one of the following groups: PC+SM, PC+SM+PE, PC+SM+PS, PC+SM+PS+PE, PC+SM+PI, PC+SM+PS+PI, PC+SM+PE+PS, PC+SM+PS+PI, PC+SM+PS+PE+PI, or PC+SM+PE+PI.