Therapeutic lipid processing compositions and methods for treating age-related macular degeneration

Abstract

Described herein are compositions and methods for treating age-related macular degeneration (AMD). In particular, described herein are polypeptides comprising helical structures that have ATP binding cassette transporter membrane stabilization and agonist activity, transporter binding activity, and / or can result in cholesterol outflow and methods of using these peptides in the treatment of AMD.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This patent application claims priority to U.S. Provisional Patent Application No. 63 / 341,990, filed on May 13, 2022, entitled “THERAPEUTIC LIPID PROCESSING COMPOSITIONS AND METHODS FOR TREATING AGE-RELATED MACULAR DEGENERATION,” which is incorporated herein by reference in its entirety. Incorporated by Reference

[0002] All publications and patent applications mentioned in this specification are herein incorporated by reference in their entirety to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference. background

[0003] Age-related macular degeneration (AMD) is a chronic metabolic inflammatory disease of the eye. AMD is the main cause of blindness in people over 55 years old, and has high prevalence in the U.S. (e.g., 8.7%) and the whole world. In addition, with the aging of the global population, this problem is expected to increase. Although AMD is classified into various types (e.g., early, middle, wet and dry), most AMD cases are considered to be "dry" AMD, for which there is no approved therapy.

[0004] It is believed that there are many related factors that may cause AMD. The imbalance of lipid transport and processing has been attributed to the outbreak and progress of AMD. The imbalance of lipid homeostasis can cause lipid deposits (drusen (drusen)) in the whole extracellular matrix such as the accumulation in Bruch's membrane (Bruch's membrane). Drusen is the first pathological symptom of AMD that destroys and stresses retinal pigment epithelial (RPE) cells, and the loss of retinal pigment epithelial (RPE) cells causes the serious later stage of photoreceptor degeneration and disease, including geographic atrophy (GA), and can further cause neovascular AMD (nvAMD).

[0005] At present, AMD is incurable, and treatment is usually ineffective. Statins or other potential treatments usually consider the destruction of cholesterol synthesis. However, the current state of the art fails to address the potential defects involved in the pathogenesis of AMD. It is necessary to treat both dry and wet AMD. Summary of the Disclosure

[0006] Compositions and methods of use thereof for treating AMD are described herein.Some compositions and methods consider the lipid outflow of cell, reverse cholesterol transport (RCT) mechanism and lipid from intracellular transportation.For example, compositions comprising one or more small peptides (for example, 80 amino acids or less, 75 amino acids or less, 70 amino acids or less, 65 amino acids or less, 60 amino acids or less, 55 amino acids or less, 50 amino acids or less, 45 amino acids or less, 40 amino acids or less peptides etc.) are described herein, and its simulation apolipoprotein regulates lipid transport and promotes the ability of lipid outflow of cell by RCT mechanism, such as ATP binding cassette (ABC) transporter, high-density lipoprotein (HDL) or B-type 1 scavenger receptor (SR-B1). These peptides may be referred to herein as apolipoprotein peptide mimetics, therapeutic apolipoprotein peptide mimetics, test peptides, peptide candidates, or simply therapeutic peptides, and may include sequences engineered for improved or comparable amphipathic helical structures and functions associated with endogenous forms of one or more apolipoproteins (such as ApoE, ApoA, ApoJ). For example, in some variations described herein, a peptide may include a modified or partial polypeptide sequence corresponding to a sequence associated with a lipid binding or lipid accepting structure of an apolipoprotein.

[0007] Typically, these therapeutic peptides can initiate or mediate lipid transport out of cells via one or more transporters involved in the cholesterol efflux mechanism (illustrated herein by promoting cholesterol efflux from human microglial cell lines and human retinal pigment epithelial cell lines). For example, cholesterol efflux regulatory proteins, ATP-binding cassette transporters (e.g., ABCA1 or ABCG1, e.g., "ATP-binding cassette subfamily A member 1") or scavenger proteins can participate in lipid efflux to remove cholesterol via the RCT mechanism. The therapeutic peptides described herein may have ABCA1-dependent lipid efflux activity (as measured by a reduction in lipid export, e.g., by adding siRNA targeting ABCA1). These therapeutic peptides can treat, prevent or improve dysregulated cholesterol homeostasis associated with defects or mutations in one or more endogenous apolipoproteins, apolipoprotein receptors or lipoprotein particle maturation factors.

[0008] Compositions for treating AMD and methods using them are also described herein.Some compositions and methods consider lipid inflow or transport extracellular lipids via lipid inflow mechanism.For example, compositions comprising one or more small peptides (for example, 80 amino acids or less, 75 amino acids or less, 70 amino acids or less, 65 amino acids or less, 60 amino acids or less, 55 amino acids or less, 50 amino acids or less, 45 amino acids or less, 40 amino acids or less peptides, etc.) are described herein, it simulates the ability of apolipoprotein to solubilize lipids, and lipid transport is introduced into cells by cell lipid uptake mechanism (such as low-density lipoprotein receptor (LDLR), scavenger receptor B class 1 type (SR-B1) or glycosaminoglycan (GAG) dependent mechanism).These peptides may be referred to herein as apolipoprotein peptide mimics, therapeutic apolipoprotein peptide mimics or simply referred to as therapeutic peptides, and may include sequences engineered for improved amphipathic helical properties, which are different from those of the native sequence from apolipoprotein (ApoE, ApoA, ApoJ). For example, in some variations described herein, a peptide can include a modified or partial polypeptide sequence corresponding to helix 4 of ApoE (eg, amino acids 140-150, see, eg, SEQ ID NO: 8).

[0009] Therefore, generally, these therapeutic peptides can be combined with LDLR in a lipid-dependent manner and lipids are transported into cells via LDLR. These therapeutic peptides can overcome the lipid transport defects present in ApoE2 carriers (for example, ApoE2 variants have defective LDLR binding activity). Therapeutic peptides as described herein can have lipid-dependent LDLR binding activity in surprising small polypeptides (for example, 80 or less amino acids, 75 or less amino acids, 70 or less amino acids, 65 or less amino acids, 60 or less amino acids, 55 or less amino acids, 50 or less amino acids, 49 or less amino acids, 48 ​​or less amino acids, 47 or less amino acids, 46 or less amino acids, 45 or less amino acids, 44 or less amino acids, 43 or less amino acids, 42 or less amino acids, 41 or less amino acids, 40 or less amino acids, etc.). These therapeutic polypeptides can retain lipid-dependent LDLR binding and lipid transport activity in smaller peptides.

[0010] In addition, these therapeutic peptides can solubilize lipids (e.g., as shown herein by reducing the turbidity of 1,2-dimyristoyl-sn-glycero-3-phosphocholine or DMPC liposome solutions), and can preferentially bind to oxidized rather than non-oxidized lipids (as measured by surface plasmon resonance SPR). The therapeutic peptides described herein can import lipids into cells (as shown by the import of labeled cholesterol into APRE-19 and HepG2 cells), and this lipid import can be dependent on the GAG ​​binding domain on the peptide (GAG-dependent lipid import activity, as measured by the reduction of lipid import into cells by adding exogenous heparin). The therapeutic peptides described herein may have LDLR-dependent lipid import activity (as measured by the reduction of lipid import into cells, for example, by adding siRNA targeting LDLR). Some exemplary therapeutic peptides described herein may have SR-B1-dependent lipid import activity (as measured by the reduction of lipid import into cells, for example, by adding siRNA targeting SR-B1). In general, the therapeutic polypeptides described herein have minimal cytotoxicity (as measured by membrane permeable dye staining of ARPE-19 cells and hemolysis of human RBCs).

[0011] Therefore, the therapeutic peptides described herein can be used as lipid transport peptides that simulate the behavior of apolipoproteins, lipoprotein particles, lipid exporters, and lipid importers. Generally, the therapeutic peptides described herein can be combined with LDLR, and can increase the input of lipids to LDLR+ cells, resulting in improved or prevented lipid transport, and the lipid transport of disorders is a sign of drusen formation and AMD disease progression. The therapeutic peptides described herein can also be combined with ABCA1, and can increase the output of lipids from ABCA1+ cells, resulting in improved or prevented lipid transport disorders. The methods described herein can include replacing the defective lipid transport function by intravitreal or systemic injection of therapeutic peptide mimics with apolipoprotein function. Therefore, these treatment methods can solve the mechanism of LDLR, GAG and / or SR-B1 dependent lipid import cells. Therefore, these treatment methods can also solve the cellular lipid export mechanism of ABCA1 dependence or ABCA1 independence. These methods and compositions (e.g., therapeutic peptides) can regulate the cholesterol homeostasis of patients. The therapeutic peptides can generally be small (e.g., <50 amino acids, <49 amino acids, <48 amino acids, etc.), amphiphilic and can package lipids. Therapeutic peptides are engineered to combine and isolate lipids, and lipids can be deposited into cells via uptake receptors or lipids can be exported from cells via export receptors. For example, these therapeutic peptides can improve the lipid removal of drusen by increasing the interaction with LDLR and other lipid uptake pathways. As another example, these therapeutic peptides can reduce drusen load by increasing the interaction with ABCA1 and other lipid export pathways, to restore natural lipid transport homeostasis and removal mechanism. Because drusen represent the main risk factor of AMD disease progression, the therapeutic peptides with the potentiality of reducing drusen formation may improve patient results. These peptides can be synthesized by synthetic means. In some instances, these compositions (for example, therapeutic peptides) can be prepared together with one or more pharmaceutically acceptable carriers.

[0012] Also described herein are methods of treating a subject suffering from a disorder associated with undesirable activity of a lipid regulatory pathway, comprising the step of administering to the subject any of the compositions disclosed herein.

[0013] In some instances, the present disclosure provides a method of treating a subject with age-related macular degeneration (AMD), comprising the step of administering to the subject any composition disclosed herein. In some instances, the composition is administered intravitreally. In some instances, the subject is a human. In some instances, the human is at least 40 years old. In some instances, the human is at least 50 years old. In some instances, the human is at least 65 years old. In some instances, the composition is administered topically. In some instances, the composition is administered systemically. In some instances, the composition has an amino acid sequence of any one of SEQ ID NOs: 35-39, 87, 101, or 114. For example, a polypeptide for treating age-related macular degeneration (AMD) is described herein having a length of less than 80 amino acids and having the same amino acid sequence as SEQ ID NO: 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62 , 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 1 13, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 1 52, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177 or 178 has a peptide sequence with 65% or more (e.g., 70% or more, 80% or more, 85% or more, 90% or more) homology.In particular, described herein are polypeptides for treating age-related macular degeneration (AMD) having a peptide sequence less than 80 amino acids in length and having 65% or more (e.g., 70% or more, 80% or more, 85% or more, 90% or more, etc.) homology to one of SEQ ID NOs: 87, 101 or 114.

[0014] In some examples, a polypeptide for treating age-related macular degeneration (AMD) can have a peptide sequence that is at least 85% homologous to one of SEQ ID NOs: 84, 86, 87, 101, 112, 114, or 116.

[0015] In some examples, the polypeptide is more than 90% homologous to one of SEQ ID NOs: 84, 86, 87, 101, 112, 114, or 116.

[0016] The polypeptide for treating age-related macular degeneration (AMD) may have a peptide sequence that is at least 85% homologous to SEQ ID NO: 35, 36, 37, 38 or 39.

[0017] Described herein are examples of one or more polypeptides for treating age-related macular degeneration (AMD) having a peptide sequence of one of SEQ ID NOs: 84, 86, 87, 101, 112, 114, or 116.

[0018] Described herein are examples of one or more polypeptides for treating age-related macular degeneration (AMD) having a peptide sequence of one of SEQ ID NOs: 35, 36, 37, 38, or 39.

[0019] Described herein are apolipoprotein mimetic-related peptide sequences for treating age-related macular degeneration (AMD) having ATP-binding cassette transporter binding activity comprising a sequence at least 65% homologous to one or more of SEQ ID NOs: 84, 86, 87, 101, 112, 114 or 116.

[0020] Described herein is one or more polypeptides having cholesterol efflux regulatory protein (CERP) binding activity for treating age-related macular degeneration (AMD), comprising a sequence at least 65% homologous to one or more of SEQ ID NOs: 84, 86, 87, 101, 112, 114 or 116.

[0021] Described herein is one or more polypeptides having transporter binding activity for treating age-related macular degeneration (AMD), comprising a sequence at least 65% homologous to one or more of SEQ ID NOs: 84, 86, 87, 101, 112, 114 or 116.

[0022] Described herein is one or more polypeptides for treating age-related macular degeneration (AMD) having ATP-binding cassette transporter binding activity comprising a sequence at least 65% homologous to one or more of SEQ ID NOs: 84, 86, 87, 101, 112, 114 or 116.

[0023] The polypeptide for treating age-related macular degeneration (AMD) can have a peptide sequence that is at least 85% homologous to one of SEQ ID NOs: 25, 27, or 29. In some examples, the polypeptide is more than 90% homologous to one of SEQ ID NOs: 25, 27, or 29.

[0024] The polypeptide for treating age-related macular degeneration (AMD) may have a peptide sequence of one of SEQ ID NOs: 25, 27 or 29. For example, the polypeptide for treating age-related macular degeneration (AMD) may have 80 or fewer amino acids, wherein the N-terminus of the polypeptide has 65% or more (e.g., 70% or more, 80% or more, 85% or more, 90% or more, etc.) homology with SEQ ID NO: 8.

[0025] In some examples, a polypeptide for treating age-related macular degeneration (AMD) has an N-terminus of a polypeptide having 65% or more (e.g., 70% or more, 80% or more, 85% or more, 90% or more, etc.) homology to SEQ ID NO: 29, wherein the first eleven amino acids of the polypeptide have four or fewer (e.g., three or fewer, two or fewer, or one) substitutions compared to SEQ ID NO: 29.

[0026] For example, a polypeptide for treating age-related macular degeneration (AMD) may have lipid-dependent low-density lipoprotein receptor (LDLR) binding activity, comprising a sequence at least 65% homologous to one or more of SEQ ID NOs: 25, 27, or 29. In some examples, a polypeptide for treating age-related macular degeneration (AMD) has lipid-dependent low-density lipoprotein receptor (LDLR) binding activity, comprising a sequence at least 65% homologous to one or more of SEQ ID NOs: 5, 7, 9, 13, 18, and 29.

[0027] A polypeptide for treating age-related macular degeneration (AMD), having 80 or fewer amino acids, wherein the N-terminus of the polypeptide has 65% or higher homology with the L-conformation shown in SEQ ID NO: 8, or wherein the C-terminus of the polypeptide has 65% or higher homology with the D-conformation of SEQ ID NO: 8.

[0028] The polypeptide for treating age-related macular degeneration (AMD) can have 80 or fewer amino acids, wherein the N-terminus of the polypeptide is homologous to the L-conformation shown in SEQ ID NO: 8, with less than 2 amino acid substitutions or deletions, or wherein the C-terminus of the polypeptide is homologous to the D-conformation of SEQ ID NO: 8, with less than 2 amino acid substitutions or deletions.

[0029] The polypeptide for treating age-related macular degeneration (AMD) may have 80 amino acids or less, while the N-terminus of the polypeptide has 65% or more homology with SEQ ID NO: 8.

[0030] A polypeptide for treating age-related macular degeneration (AMD) can have an N-terminus of a polypeptide having 65% or more homology to SEQ ID NO: 29, wherein the first eleven amino acids of the polypeptide have two or fewer substitutions compared to SEQ ID NO: 29.

[0031] A polypeptide for treating age-related macular degeneration (AMD) can have 80 or fewer amino acids, wherein the N-terminus of the polypeptide has 65% or more (e.g., 70% or more, 80% or more, 85% or more, 90% or more, etc.) homology with SEQ ID NO: 27.

[0032] Any polypeptide described herein may have lipid-dependent low-density lipoprotein receptor (LDLR) binding activity, such that the binding of the polypeptide to LDLR in the presence of lipid is at least two times higher (e.g., at least 2.5 times, at least 3 times, at least 5 times, at least 10 times, etc.) than the binding to LDLR in the absence of lipid.

[0033] Any of the therapeutic peptides described herein can increase the efflux of lipids from cells (eg, microglia or retinal pigment epithelial cells) through the ATP-binding cassette transporter ABCA1 and increase the presence of ABCA1 on the cell membrane following pro-inflammatory stimulation.

[0034] Any of the therapeutic peptides described herein may include N-terminal acetylation and C-terminal amidation.The therapeutic peptides described herein are also intended to include both the L and D forms of the peptides described herein.

[0035] Also described herein is a pharmaceutical composition for preventing or treating age-related macular degeneration (AMD) in a patient, wherein the composition comprises any polypeptide described herein and a pharmaceutically acceptable excipient. The composition can be used for administration by intraocular injection and / or intravascular (IV) injection and / or subcutaneous (SC) injection. The pharmaceutical composition can comprise two or more of the above polypeptides.

[0036] For example, when a patient shows the signs or symptoms of AMD, the method for treating or preventing the patient's age-related macular degeneration (AMD) using the polypeptide or pharmaceutical composition can be used. These methods for treating or preventing the patient's age-related macular degeneration (AMD) using polypeptide as herein described or pharmaceutical composition can be used to treat early AMD. Any of the methods for treating the patient's age-related macular degeneration (AMD) can include delivering the polypeptide or composition to the patient's eyes, for example, by intraocular injection (and / or by intravascular injection and / or by subcutaneous injection, etc.). As mentioned above, the method can include delivering more than one polypeptide as herein described or composition.

[0037] In particular, described herein are peptides for treating or preventing age-related macular degeneration (AMD) in a patient, comprising or derived from a peptide having the sequence shown below: DAWERFRALFKELADYFR (SEQ ID NO: 87) These polypeptides shown herein have the surprising beneficial properties for the treatment of AMD.As shown herein, there is at least 65% homologous sequence (for example, including six or less conservative and / or conservative hydrophobic replacement) and demonstrate one or more verifiable characteristics (such as: 0.65 or larger hydrophobic moment (μH), ATP binding cassette transporter membrane stabilization and agonist activity, transporter binding activity and / or 17.5% or larger cholesterol outflow) polypeptide for treating AMD can be effective for treatment.With T-087 homology and in key assay, show the limiting examples of the active polypeptide of continuous or improvement and include: T-152, T-160, T-161, T-163 and T-172.

[0038] Therefore, described herein is a polypeptide for treating age-related macular degeneration (AMD) having a peptide sequence with 65% or higher homology to SEQ ID NO: 87, wherein the polypeptide comprises a helical coil with a hydrophobic moment μH of 0.65 or greater. As described above, in the framework of the T-087 polypeptide, the hydrophobic moment is associated with the percentage of cholesterol efflux, such that a hydrophobic moment μH of 0.65 or greater generally results in an increased cholesterol efflux of greater than 15% (e.g., 16% or greater, 17% or greater, 17.5% or greater, 18% or greater, etc.).

[0039] For example, also described herein is a polypeptide for treating age-related macular degeneration (AMD) having a peptide sequence having 65% or greater homology to SEQ ID NO: 87, wherein the polypeptide has ATP-binding cassette transporter membrane stabilizing and agonist activity.

[0040] Also described herein is a polypeptide for treating age-related macular degeneration (AMD) having a sequence at least 65% homologous to SEQ ID NO: 87, wherein the polypeptide has transporter binding activity.

[0041] Any of these polypeptides may have a hydrophobic moment μH of 0.65 or greater, resulting in a cholesterol efflux of 17.5% or greater. Any of these polypeptides may have ATP-binding cassette transporter binding activity.

[0042] Typically, for any of these T-087-based polypeptides, any peptide residues that differ from the sequence of SEQ ID NO: 87 in positions 2-7, 9, and 10-17 are conservative substitutions or conservative hydrophobic substitutions having hydrophobicity values ​​within 0.25 of the hydrophobicity value of the peptide residues in the corresponding positions of SEQ ID NO: 87, as calculated using the method of Fauchere and Pliska, and wherein any of the different peptide residues in positions 1, 8, 10, and 18 are any amino acids. For example, the polypeptide sequence can be one of SEQ ID NO: 87, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, or 178. In particular, the polypeptide sequence can be one of SEQ ID NO: 87, 152, 160, 161, 163 or 172.

[0043] In any of these examples, the polypeptide sequence can be 70% or more (e.g., 80% or more, 85% or more, 90% or more, etc.) homologous to SEQ ID NO: 87. As described above, in any of these polypeptide sequences based on T-087, the polypeptide sequence can be as shown in SEQ ID NO: 35 or 36, wherein X can be any amino acid.

[0044] Also described herein are peptides for treating or preventing age-related macular degeneration (AMD) in a patient, comprising or derived from a peptide having the sequence shown below: RSGADALESALKELKRFIREWT (SEQ ID NO: 101) It is also shown herein that these polypeptides have surprising beneficial properties for treating AMD.Sequences with at least 65% homology (e.g., including eight or less conservative and / or conservative hydrophobic substitutions) and showing one or more verifiable properties (such as: 0.6 or greater hydrophobic moment (μH) (which can give greater ABCA1 stability), ATP binding cassette transporter membrane stabilization and agonist activity, transporter binding activity and / or 0.198 or greater overall hydrophobicity (which can give greater cholesterol efflux)) polypeptides.Non-limiting examples of polypeptides that are homologous to T-087 and show continued or improved activity in key assays include: T-122, T-123, T-129, T-136 and T-139.

[0045] For example, described herein is a polypeptide for treating age-related macular degeneration (AMD), which has a peptide sequence with SEQ ID NO: 101 65% or higher homology, wherein the polypeptide comprises a helical coil with a hydrophobic moment μH of 0.6 or greater. For example, described herein is a polypeptide for treating age-related macular degeneration (AMD), which has a peptide sequence with SEQ ID NO: 101 with 65% or higher homology, wherein the polypeptide has ATP binding cassette transporter membrane stabilization and agonist activity. Also described herein is a polypeptide for treating age-related macular degeneration (AMD), which has a sequence with SEQ ID NO: 101 at least 65% homology, wherein the polypeptide has transporter binding activity. In some instances, the polypeptide for treating age-related macular degeneration (AMD) may have an overall hydrophobicity of 0.198 or greater, thereby resulting in enhanced cholesterol efflux. In any of these examples, the polypeptide may have a hydrophobic moment μH of 0.6 or greater, resulting in an ABCA1 stability greater than 40% of the ABCA1 stability of the polypeptide of SEQ ID NO: 101. The polypeptide may have ATP-binding cassette transporter binding activity.

[0046] In any polypeptide that is 65% or more homologous, any peptide residue that differs from the primary sequence (e.g., the sequence of SEQ ID NO: 101) is substituted, for example, in positions 1, 4-8, 10-15, 17-18 or 20-22, as a conservative substitution or a conservative hydrophobic substitution whose hydrophobicity value is within 0.25 of the hydrophobicity value of the peptide residue in the corresponding position of SEQ ID NO: 101, as calculated using the method of Fauchere and Pliska, and wherein any of the different peptide residues in positions 2, 3, 9, 16 and 19 are any amino acids. For example, the polypeptide sequence can be one of SEQ ID NO: 101, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150 or 151. Specifically, the polypeptide sequence can be one of SEQ ID NO: 101, 122, 123, 129, 136 or 139. The polypeptide sequence can be 70% or more homologous to SEQ ID NO: 101 (e.g., 75% or more homologous, 80% or more homologous, 85% or more homologous, 90% or more homologous, etc.).

[0047] Polypeptides based on the T-114 polypeptide framework are also described herein. For example, polypeptides for treating age-related macular degeneration (AMD) are described herein, which have a peptide sequence 65% or more homologous to SEQ ID NO: 114, wherein the polypeptide has a powerful lipid-dependent binding to the low-density lipoprotein receptor. Polypeptides for treating age-related macular degeneration (AMD) may have a sequence at least 65% homologous to SEQ ID NO: 114, wherein the polypeptide has transporter binding activity. In any of these polypeptides, the polypeptide may also include an ATP-binding cassette transporter binding activity.

[0048] As mentioned above, the pharmaceutical composition for preventing or treating age-related macular degeneration (AMD) of a patient is also generally described herein, wherein the composition comprises the polypeptide described in any of the above examples, and a pharmaceutically acceptable excipient. The composition can be used for administration by intraocular injection, intravascular (IV) injection and / or subcutaneous (SC) injection. Any of these pharmaceutical compositions can include two or more different polypeptides as described herein.

[0049] Also described herein is a method for treating or preventing the age-related macular degeneration (AMD) of a patient using any of these polypeptides or pharmaceutical compositions, wherein prevention or treatment is to prevent the AMD, and wherein the patient is diagnosed as having a tendency to develop AMD. For example, the method for treating or preventing the age-related macular degeneration (AMD) of a patient may include using any polypeptide as described herein, wherein prevention or treatment is treatment and the patient shows the signs or symptoms of AMD. The method for treating or preventing the age-related macular degeneration (AMD) of a patient using one or more of the engineered polypeptides or pharmaceutical compositions as described herein may include treating early AMD. The method for treating the age-related macular degeneration (AMD) of a patient may include delivering polypeptide or compositions to the patient's eyes, for example, by intraocular injection, intravascular (IV) injection and / or subcutaneous (SC) injection. Delivery may include delivering more than one polypeptide or compositions as described herein. As described above, in some instances, the patient may be, for example, 40 years old or larger.

[0050] All methods and apparatus described herein, in any combination, are contemplated herein and can be used to achieve the benefits as described herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0051] The patent or application file contains at least one drawing printed in color. Copies of this patent or patent application publication with color drawings will be provided by the Office upon request and payment of the necessary fee.

[0052] A better understanding of the features and advantages of the methods and apparatus described herein will be obtained by referring to the following detailed description, which sets forth illustrative examples, and the accompanying drawings, in which: Figure 1A-Figure 1I is a list of peptides, including control peptides (T-001 to T-004, T-031 to T-034, and T-081 to T-083) and therapeutic peptides (T-005 to T-030 and T-040 to T-178) described herein, with the hydrophobic moment also shown. The hydrophobic moment is defined by Eisenberg ( Eisenberg et al. , 1982. The helical hydrophobic moment: a measure of the amphiphilicity of a helix. Nature).

[0053] Figure 2A-2D The results of a DMPC solubility assay measuring the solubility of a peptide at a concentration of 100 μM are shown. Figure 1A-Figure 1I Solubilization of 0.5 mM 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) by selected peptides shown in FIG.

[0054] Figure 3A-3C Figure 2 is a graph showing a range of peptide concentrations as described herein for controls (T-001, T-002, T-004, and T-031) and therapeutic polypeptides (T-025, T-027, Figure 3A ;T-051, T-052, T-054, T-059, Figure 3B ;T-062, T-063, T-065, T-069, T-080, Figure 3C ) is a graph showing the results of solubilization of 0.5 mM DMPC.

[0055] Figure 4A-4B A range of peptide concentrations as described herein are shown. Figure 1A-Figure 1I Solubilization of selected peptides shown in 1 mM DMPC.

[0056] Figure 5A-5D is a graph showing the results of an ARPE-19 cell lysis assay of selected controls (T-031, T-032, T-033, and T-034) and therapeutic polypeptides (T-006, T-007, T-009, T-010, T-011, T-012, T-013, T-014, T-018, T-019, T-021, T-025, T-027, T-028, T-029, T-024, T-026, and T-030) as described herein at a single concentration (10 μM).

[0057] Figure 6 is a graph showing ARPE-19 cell lysis titration curves for controls (T-001, T-002, T-004, and T-034) and selected therapeutic polypeptides (T-025 and T-027) as described herein over a range of peptide concentrations.

[0058] Figure 7A-7D A single concentration (100 μM) of Figure 1A-Figure 1I The results of the ARPE-19 cell viability assay for selected peptides are shown.

[0059] Figure 8A-8D Shown is a graph summarizing the results of a human red blood cell (hRBC) lysis assay using a single concentration (10 μM) of control peptides (T-001, T-002, T-004, T-031, T-032, T-033, and T-034) and therapeutic polypeptides (T-006, T-007, T-009, T-010, T-011, T-012, T-013, T-014, T-024, T-026, T-030, T-018, T-019, T-021, T-025, T-027, T-028, T-029) as described herein.

[0060] Figure 9A-9B A single concentration (10 μM) of Figure 1A-Figure 1I Results of hRBC lysis assays for selected peptides are shown.

[0061] Fig.10 is a graph illustrating the hRBC lysis titration curves for a range of concentrations of the selected peptides shown (Controls: T-001, T-002, T-004, and T-034 and Therapeutic Peptides: T-025 and T-027).

[0062] Fig.11 is a graph summarizing the results of hRBC cell lysis titration curves using a range of peptide concentrations for control peptides (T-001 and T-034) and therapeutic polypeptides (T-025, T-086, T-087, T-101, T-112, T-114, and T-116) as described herein.

[0063] Fig.12 A single concentration (10 μM) Figure 1A-Figure 1I Figure 2. ARPE-19 cholesterol input of selected peptides shown in Figure 2.

[0064] Fig.13 is a graph summarizing the results of an ARPE-19 GAG-dependent cholesterol import assay using a single concentration (10 μM) of a control peptide (T-002) and therapeutic polypeptides (T-013, T-021, T-025, T-027, T-028, T-029, and T-030) as described herein.

[0065] Figure 14A-14B is a summary of a single concentration (10 μM) Figure 1A-Figure 1I Figure 2. ARPE-19 cholesterol input assay for selected peptides shown in Figure 2.

[0066] Fig.15 is a graph showing ARPE-19 cholesterol input titration curves for control peptides (T-001, T-002, T-004, and T-032) and selected therapeutic peptides (T-025 and T-027) over a range of peptide concentrations.

[0067] Fig.16 is a graph showing ARPE-19 cholesterol input titration curves for control peptides (T-001, T-002) and selected therapeutic polypeptides (T-086, T-087, T-101, T-112, T-114, and T-116) as described herein over a range of peptide concentrations.

[0068] Fig.17 A single concentration (10 μM) is shown. Figure 1A-Figure 1I Figure 2. HepG2 cholesterol input screening data for selected peptides shown in Figure 2.

[0069] Fig.18 A single concentration (10 μM) is shown. Figure 1A-Figure 1I Figure 2. HepG2 cholesterol input screening data for selected peptides shown in Figure 2.

[0070] Fig.19 A single concentration (10 μM) is shown. Figure 1A-Figure 1I Shown in the figure are the screening results of selected peptides in the HepG2 GAG-dependent cholesterol import assay.

[0071] Fig. 20 Examples of HepG2 cholesterol input titration curves for controls (T-001, T-002, T-004, and T-032) and therapeutic peptides (T-025 and T-027) are shown.

[0072] Fig.21 Examples of HepG2 cholesterol input titration curves for controls (T-001, T-002) and therapeutic peptides (T-086, T-087, T-101, T-112, T-114, and T-116) are shown.

[0073] Fig. 22 A graphical summary of the effects of a single concentration (1 μM) of recombinant ApoE2 and ApoE4 and Figure 1A-Figure 1I Results of lipid-dependent LDLR binding (SPR) assays of control peptides (T-001, T-002, T-031, T-032, T-033, and T-034) and selected test peptides are shown in FIG.

[0074] Fig.23 It shows that for Figure 1A-Figure 1I Table of binding kinetic results of the LDLR binding assay (SPR) using the high surfactant preparation as described herein for selected peptides shown in .

[0075] Figure 24A-24B It shows that for Figure 1A-Figure 1I Table of binding kinetics results of the LDLR binding assay (SPR) using lipid-dependent binding of the low surfactant preparation as described herein for selected peptides shown in FIG.

[0076] Figure 25A-Figure 25O The results show that non-oxidized and oxidized lipids are Figure 1A-Figure 1I Figure 3. Plot of binding activity of the listed peptides (SPR).

[0077] Figure 26A-26B Results from LDL oxidation assays are summarized. Fig.26A The results are shown for a single concentration (25 μM). Figure 1A-Figure 1IFigure 2. Plot of the changes in LDL oxidation over time as measured by UV spectrophotometry for selected peptides shown. Fig.26B The results are shown for a single concentration (25 μM). Figure 1A-Figure 1I Shown are bar graphs of selected peptides, LDL oxidation rates, calculated as maximum absorbance divided by delay phase length.

[0078] Figure 27A-27B A single concentration (20 μM) is shown. Figure 1A-Figure 1I Figure 2. HMC3 cholesterol efflux assay results for selected peptides shown in Figure 2.

[0079] Fig.28 The results of the HMC3 cholesterol efflux assay (compared to control) of variants of peptide T-101 as described herein (peptides T-121 to T-151) at a single concentration (20 μM) are summarized.

[0080] Fig.29 The results of the ARPE-19 cholesterol efflux assay (compared to control) of variants of peptide T-087 as described herein (peptides T-152 to T-178) at a single concentration (20 μM) are summarized.

[0081] Figures 30A-30J is a series of peptide concentrations Figure 1A-Figure 1I Figure 2. HMC3 cholesterol efflux titration curves for selected peptides shown in Figure 2.

[0082] Figure 31A-Figure 31B The graph shows the effect of a single concentration (20 μM) of peptide T-087 ( Fig.31A ) and T-101 ( Fig.31B ), Results of cholesterol efflux assay in ARPE-19 cells after knockdown of various ATP-binding cassette family members and SR-B1 using siRNA.

[0083] Fig.32 is a graph of cholesterol efflux from iPS-RPE cells for a single concentration (10 μM) of peptides T-087 and T-101 as described herein.

[0084] Figure 33A-Figure 33C The graph shows the results of the ABCA1 stability test assay using J774 cells. Fig.33A Shown are representative Western blots of ABCA1 membrane levels after treatment with buffer control or with peptides T-101 or T-087 (10 μM) or with the positive control protein ApoA1 (350 nM) after washout of 8-Br-cAMP. Fig.33B is a graph showing quantitative band densitometry of membrane ABCA1 after treatment with buffer control or peptides T-101 or T-087 (10 μM). Fig.33CGraphically shown is the change in percentage of membrane ABCA1 for different concentrations of peptides T-101 or T-087 as described herein.

[0085] Figure 34A-Figure 34B Graphed are the results of an ABCA1 stability assay using ARPE-19 cells treated with mCRP and peptides T-101 and T-087. Fig.34A Shown are Western blot data from cells treated with 10 μg / mL mCRP and 20 μM of peptides T-101 and T-087. Fig.34B is a graph showing the concentration response of peptides T-087 and T-101 in this assay.

[0086] Fig.35 is a graph showing the results of an ABCA1 stability assay using ARPE-19 cells for a single concentration (20 μM) of peptide T-087 and variants of peptide T-087 as described herein.

[0087] Fig.36 is a graph showing the results of an ABCA1 stability assay using ARPE-19 cells for a single concentration (20 μM) of peptide T-101 and variants of peptide T-101 as described herein.

[0088] Fig.37 is a graph showing cholesterol efflux from ARPE-19 cells ( Fig.29 ) and the hydrophobic moment of variants of peptide T-087 (T-152 to T-178). Linear regression was used to determine the slope deviation from zero.

[0089] Fig.38A Cholesterol efflux from HMC3 cells is shown graphically ( Fig.28 The positive correlation between the hydrophobicity of peptides T-121 to T-151 (variants of peptide T-101) and the hydrophobicity is shown in Figure 1. Hydrophobicity is defined by Fauchere and Pliska (Fauchere and Pliska, 1983. Hydrophobic Parameters II of Amino-Acid Side Chains from the Partitioning of N-Acetyl-Amino-Acid Amides. Eur J Med Chem). Linear regression is used to determine the slope deviation from zero.

[0090] Fig.38B Figure 2 shows the stability of ABCA1 in ARPE-19 cells. Fig.36 A positive correlation between the hydrophobic moments of peptides T-121 to T-151 is shown in Figure 3. Linear regression was used to determine the slope deviation from zero.

[0091] Figure 39A-Figure 39E Shown are representative H&E stained sections of adult C57BL / 6 mouse eyes treated with a subset of the peptides described herein (T-087, T-101, T-112, and T-114), demonstrating in vivo tolerance seven days after intravitreal injection of 1 uL of peptide (520 uM).

[0092] Figure 40A-40B Figure 3 shows the effects of peptides T-087 and T-101 on ApoE - / - Evaluation of peptide-mediated reduction of sub-RPE BODIPY+ lipid deposition in mice Peptides (1 uL, 520 uM or 260 uM) were delivered by intravitreal injection, and sub-RPE lipid deposition was measured by BODIPY staining of retinal sections 14 days after injection.

[0093] Figure 41A-41B Indicator peptide T-087 ( Fig.41A ) and T-101 ( Fig.41B ) can be identical or substituted with conservative amino acid substitutions to maintain the desired therapeutic activity of the peptide (including safety, cholesterol efflux ability, and ABCA1 stabilization ability). The residues shown without boxes indicate positions where any amino acid can be substituted while maintaining the desired therapeutic activity of the peptide (including safety, cholesterol efflux ability, and ABCA1 stabilization ability). Details

[0094] Compositions and methods described herein can be used to treat AMD, and particularly AMD patients whose disease is primarily driven by the dysfunction of lipid transport and processing. For example, synthetic engineered mimetic peptides described herein provide a therapeutic opportunity to safely and effectively address pathophysiology (such as dysregulated lipid homeostasis).

[0095] In particular, apolipoproteins are endogenously synthesized in response to intracellular and extracellular lipid transport and processing requirements. Generally, apolipoproteins are a family of amphipathic molecules that can bind lipids and transport lipids into and out of cells and peripheral tissues as part of the lipid homeostasis mechanism. For example, apolipoproteins are components of HDL / LDL molecules. Apolipoprotein E (ApoE) is usually associated with LDL, while apolipoprotein A1 (ApoA1) is usually associated with HDL.

[0096] The cellular uptake of lipids (such as cholesterol) may be due to the metabolic needs of cells or in response to the extracellular cholesterol concentration of rising. Apolipoprotein E (such as ApoE) is usually related to the mechanism relevant to cholesterol uptake. Low-density lipoprotein receptor (LDLR) is a surface receptor with high affinity for ApoE. Retinal pigment epithelium (RPE) cells have been shown to express LDLR, which makes it possible to take up cholesterol from the LDL / ApoE complex rich in cholesterol.

[0097] Alternatively, cholesterol efflux is a mechanism by which cholesterol is expelled from the cell to the extracellular environment by one or more transporters (e.g., ATP-binding cassette transporter or SR-B1). The transporters efflux cholesterol, which then binds to lipid-poor ApoA1 as a component of HDL formation. When ApoA1 accumulates cholesterol from the transporters, HDL becomes saturated and continues with RCT as part of the lipid transport and processing mechanism for cholesterol homeostasis and lipid regulation.

[0098] Genotypic variation, environmental factors and aging may lead to lipid homeostasis disorders associated with impaired apolipoprotein activity. Synthetic engineered mimetic peptides with lipid transport and processing activity are described herein, as shown in detail below. These synthetic peptides can describe a family of peptides (including conservative substitutions of peptides as described herein) that show significant therapeutic efficacy.

[0099] In some instances, the malfunction of lipid transport and processing may be related to the lipid outflow of one or more cells or tissues relevant to AMD pathogenesis is impaired.For example, the RPE cell-specific deletion of ABCA1 gene in mouse model causes abnormal lipid accumulation, retinal inflammation and RPE / photoreceptor degeneration (PMID 30864945).Apolipoprotein, and particularly apolipoprotein A1 (ApoA1), serves as the regulator of lipid processing and lipid molecule transportation, to maintain cholesterol homeostasis.ApoA1 can interact with one or more cholesterol outflow regulatory proteins such as ABCA1, ABCG1 or SR-B1.Cholesterol outflow regulatory proteins transport cholesterol to ApoA1, and ApoA1 forms nascent HDL in conjunction with cholesterol.Along with the accumulation of cholesterol, lipid-rich HDL circulates to the liver for lipid deposition and metabolism.

[0100] In some instances, where the primary malfunction in lipid processing involves the accumulation of lipids, which improperly exocytose lipids via one or more cholesterol-regulating proteins, patients with AMD may benefit from administering one or more therapeutic peptides described herein (e.g., included in SEQ ID NO: 5-30; or 35-178). For example, when the cholesterol import rate is too fast and the corresponding intracellular lipid concentration is too high, administering one or more therapeutic peptides described herein (e.g., included in SEQ ID NO: 5-30; or 35-178) establishes or restores an effective lipid transport and processing mechanism mediated by one or more apolipoproteins. As will be described in more detail herein, some of these peptides may be more effective than other peptides, however, generally they can provide therapeutic use.

[0101] In some examples, including but not limited to, where the primary contributing factor is due to one or more functional abnormalities associated with lipid transport and processing pathways, patients may be treated with one or more therapeutic peptides as described herein or modified forms of these peptides (e.g., included in SEQ ID NOs: 5-30; or 35-178). In particular, these one or more therapeutic peptides are those that reduce lipid accumulation in Bruch's membrane, subretinal spaces, or reticular pseudodrusen to prevent or reduce AMD-related effects. For example, the methods described herein can replace lipid regulation of defects in Bruch's membrane by intravitreal injection of one or more therapeutic peptides described herein.

[0102] The disclosure herein provides compositions and methods for treating, preventing or inhibiting eye diseases. For example, the disclosure herein provides engineered therapeutic peptides (therapeutic polypeptides) that selectively bind lipids, solubilize lipids, activate lipid efflux mechanisms via ABCA1 and related transporters, and improve cholesterol homeostasis to reduce drusen formation. The disclosure herein provides methods for treating, preventing or inhibiting eye diseases by administering an effective amount of these compositions of the disclosure intraocularly (e.g., intravitreal) to treat or prevent eye diseases using the methods provided herein. Eye diseases that can be treated or prevented using these methods include, but are not limited to, glaucoma, macular degeneration (e.g., age-related macular degeneration, AMD), diabetic retinopathy, hereditary retinal degeneration such as retinitis pigmentosa, retinal detachment or injury, and retinopathy (such as hereditary, caused by surgery, trauma, potential causes such as severe anemia, SLE, hypertension, blood dyscrasias, diabetes, systemic infection, or potential carotid artery disease, toxic compounds or agents, or light-induced retinopathy).

[0103] The disclosure herein provides compositions and methods for treating, preventing or inhibiting eye diseases. For example, the disclosure herein provides engineered therapeutic peptides (therapeutic polypeptides) that selectively bind lipids, solubilize lipids, increase LDLR binding activity, increase lipid import activity through LDLR and related receptors, and improve cholesterol homeostasis to reduce drusen formation. The disclosure herein also provides engineered therapeutic peptides that selectively activate ABCA1 and promote cholesterol efflux from cells through ABCA1-specific mechanisms to improve cholesterol homeostasis and reduce drusen formation and progression. The disclosure herein provides methods for treating, preventing or inhibiting eye diseases by intraocularly (e.g., intravitreal) administering an effective amount of these compositions of the disclosure to treat, prevent or inhibit eye diseases, to treat or prevent eye diseases using the methods provided herein. Ocular diseases that can be treated or prevented using these methods include, but are not limited to, glaucoma, macular degeneration (e.g., age-related macular degeneration, AMD), diabetic retinopathy, inherited retinal degenerations such as retinitis pigmentosa, retinal detachment or injury, and retinopathy (such as inherited, retinopathy induced by surgery, trauma, underlying causes such as severe anemia, SLE, hypertension, blood dyscrasias, diabetes, systemic infection or underlying carotid artery disease, toxic compounds or agents, or light).

[0104] Unless otherwise defined herein, scientific and technical terms used in the present application shall have the meanings commonly understood by one of ordinary skill in the art.

[0105] Generally, nomenclature and techniques used in connection with the pharmacology, cell and tissue culture, molecular biology, cell and cancer biology, neurobiology, neurochemistry, virology, immunology, microbiology, genetics, and protein and nucleic acid chemistry described herein are those well known and commonly used in the art. In case of conflict, the present specification, including definitions, will control.

[0106] The practice of the present disclosure 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. Such techniques are well explained in the literature, such as Molecular Cloning: A Laboratory Manual, second edition (Sambrook et al., 1989) Cold Spring Harbor Press; Oligonucleotide Synthesis (MJ Gait, ed., 1984); Methods in Molecular Biology, Humana Press; CellBiology: A Laboratory Notebook (JE Cellis, ed., 1998) Academic Press; Animal Cell Culture (RI Freshney, ed., 1998) 1987); Introduction to Cell andTissue Culture (JP Mather and PE Roberts, 1998) Plenum Press; Cell andTissue Culture: Laboratory Procedures (A. Doyle, JB Griffiths, and DGNewell, eds., 1993-1998) J. Wiley and Sons; Methods in Enzymology (AcademicPress, Inc.); Gene Transfer Vectors for Mammalian Cells (JM Miller and MPCalos, eds., 1987); Current Protocols in Molecular Biology (FM Ausubel et al., eds., 1987); PCR: The Polymerase Chain Reaction, (Mullis et al., eds., 1994); Sambrook and Russell, Molecular Cloning: A Laboratory Manual, 3rd. ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2001); Ausubel et al., Current Protocols in Molecular Biology, John Wiley&Sons, NY (2002); Harlowand Lane Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1998); Coligan et al., Short Protocols in ProteinScience, John Wiley&Sons, NY (2003); Short Protocols in Molecular Biology (Wiley and Sons, 1999). .

[0107] Enzymatic reactions and purification techniques are performed according to manufacturer's specifications, as commonly accomplished in the art or as described herein. Nomenclature used in connection with, and laboratory procedures and techniques of, analytical chemistry, biochemistry, immunology, molecular biology, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are those well known and commonly used in the art. Standard techniques are used for chemical syntheses and chemical analyses.

[0108] In the case of describing aspects or examples of the present disclosure according to other groupings of Markush groups or alternatives, the present disclosure includes not only the entire group listed as a whole, but also includes each member of the group and all possible subgroups of the main group individually, and also includes the main group without one or more group members. The present disclosure also contemplates explicitly excluding one or more of any group members in the disclosed examples. Exemplary methods and materials are described herein, although methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present disclosure. Materials, methods and examples are illustrative only and are not intended to be limiting.

[0109] definition Unless otherwise indicated, the following terms shall be understood to have the following meanings: As used herein, "residue" refers to a position in a protein and its associated amino acid identity. As known in the art, "polynucleotide" or "nucleic acid" as used interchangeably herein refers to a nucleotide chain of any length, and includes DNA and RNA. Nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases and / or their analogs, or can be incorporated into any substrate in the chain by DNA polymerase or RNA polymerase. Polynucleotides can include modified nucleotides, such as methylated nucleotides and their analogs. If present, the modification of the nucleotide structure can be imparted before or after chain assembly. The nucleotide sequence can be interrupted by non-nucleotide components. After polymerization, the polynucleotide can be further modified, such as by being conjugated with a labeling component. Other types of modifications include, for example, "capping", substitution of one or more naturally occurring nucleotides with an analog, internucleotide modifications such as, for example, those with uncharged linkages (e.g., methylphosphonates, phosphotriesters, phosphoramides, carbamates, etc.) and charged linkages (e.g., phosphorothioates, phosphorodithioates, etc.), those comprising side groups such as, for example, proteins (e.g., nucleases, toxins, antibodies, signal peptides, poly-L-lysine, etc.), those with intercalators (e.g., acridine, psoralen, etc.), those comprising chelators (e.g., metals, radioactive metals, boron, oxidized metals, etc.), those containing alkylating agents, those with modified linkages (e.g., alpha anomeric nucleic acids, etc.), and unmodified forms of polynucleotides. In addition, any hydroxyl groups normally present in the sugar may be replaced, for example, by phosphonate groups, phosphate groups, protected by standard protecting groups, or activated to prepare additional linkages with additional nucleotides, or may be conjugated to a solid support. 5' and 3' terminal OH can be phosphorylated or partially replaced by amine or organic capping groups of 1 to 20 carbon atoms. Other hydroxyls can also be derivatized as standard protecting groups. Polynucleotides can also include analog forms of ribose or deoxyribose sugars commonly known in the art, including, for example, 2'-O-methyl-, 2'-O-allyl, 2'-fluoro-ribose or 2'-azido-ribose, carbocyclic sugar analogs, α-anomeric sugars or β-anomeric sugars, epimeric sugars such as arabinose, xylose or lyxose, pyranose, furanose, sedoheptulose, acyclic analogs and abasic nucleoside analogs such as methyl riboside. One or more phosphodiester connections can be replaced by optional linking groups.These optional linking groups include, but are not limited to, examples where the phosphate is replaced by P(O)S ("thioate"), P(S)S ("dithioate"), "(O)NRi ("amidate"), P(O)R, P(O)OR', CO or CH2 ("formacetal"), wherein each R or R' is independently H or a substituted or unsubstituted alkyl (1-20 C), aryl, alkenyl, cycloalkyl, cycloalkenyl or araldyl optionally containing an ether (-O-) linkage. Not all linkages in a polynucleotide need be identical. The foregoing description applies to all polynucleotides referred to herein, including RNA and DNA.

[0110] The terms "polypeptide", "oligopeptide", "peptide" and "protein" are used interchangeably herein to refer to a chain of amino acids of any length. The chain may be linear or branched, it may contain modified amino acids, and / or may be interrupted by non-amino acids. The term also includes amino acid chains modified naturally or by intervention; for example, amino acid polymers modified by disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification (such as conjugation with a labeling component). The definition also includes, for example, polypeptides containing one or more analogs of amino acids (including, for example, non-natural amino acids, etc.) and other modifications known in the art. It should be understood that polypeptides can exist in the form of single chains or associated chains.

[0111] "Homologous" in all its grammatical forms and spelling variations refers to the relationship between two proteins having a common sequence, including protein sequences from superfamilies in the same species of organism and homologous proteins from different species of organism. Such proteins (and their encoding nucleic acids) have sequence homology, as reflected by their sequence similarity, whether in terms of percentage identity, or by the presence of specific residues or motifs and conserved positions. However, in common usage and in this application, the term "homologous", particularly (but not exclusively) when modified by percentage, may refer to sequence similarity, and may or may not involve a common evolutionary origin.

[0112] The term "sequence similarity" refers to the identity or corresponding degree between nucleic acid or amino acid sequences that may or may not share a common evolutionary origin in all its grammatical forms. "Sequence identity percentage (%)" or "same percentage (%) with reference polypeptide (or nucleotide) sequence" relative to a reference polypeptide (or nucleotide) sequence is defined as the percentage of the amino acid residue (or nucleic acid) identical with the amino acid residue (or nucleic acid) in the reference polypeptide (nucleotide) sequence in the candidate sequence after aligning the sequence and introducing a gap to realize the maximum sequence identity percentage when necessary, and does not consider any conservative substitution as a part for sequence identity. The comparison for determining the amino acid sequence identity percentage can be realized in various ways within the technical scope of the art, for example, using publicly available computer software, such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine the appropriate parameters for aligning the sequence, including any algorithm required for realizing maximum alignment on the total length of the sequence being compared.

[0113] The term "conservative substitution" refers to the substitution of an amino acid in a polypeptide with a natural or non-natural amino acid that is similar in function, structure or chemistry. In certain embodiments, the following groups each contain natural amino acids that are conservative substitutions for each other: 1) glycine (G), alanine (A); 2) aspartic acid (D), glutamic acid (E); 3) asparagine (N), glutamine (Q); 4) arginine (R), lysine (K), histidine (H); 5) isoleucine (I), leucine (L), methionine (M), valine (V), alanine (A); 6) phenylalanine (F), tyrosine (Y), tryptophan (W); and 7) serine (S), threonine (T), cysteine ​​(C).

[0114] The peptides described herein may include substitutions that do not destroy the helical structure of the peptide (in a manner that does not destroy function), as described below. In particular, the peptides (e.g., T-087, T-101, etc.) may include one or more substitutions such that the hydrophobicity of the substituted residue is within 0.25 of the hydrophobicity value of the original amino acid (referred to herein as conservative hydrophobic substitutions). For example: (1) Alanine (A) may be substituted with Threonine (T) or Histidine (H); (2) Glutamic acid (E) may be substituted with Asparagine (N) or Aspartic acid (D); (3) Arginine (R) may be substituted with Aspartic acid (D) or Lysine (K); (4) Phenylalanine (F) may be substituted with Cysteine ​​(C), Leucine (L) or Isoleucine (I); (5) Leucine (L) may be substituted with Isoleucine (I), Phenylalanine (F) or Cysteine ​​(C); (6) Lysine (K) may be substituted with Aspartic acid (D) or Arginine (R); (7) ) Aspartic acid (D) may be substituted with asparagine (N), glutamic acid (E), lysine (K), or arginine (R); (8) tyrosine (Y) may be substituted with proline (P); (9) serine (S) may be substituted with glutamine (Q), glycine (G), or histidine (H); (10) isoleucine (I) may be substituted with leucine (L) or phenylalanine (F); (11) threonine (T) may be substituted with alanine (A) or histidine (H); and (12) serine (S) may be substituted with histidine (H), serine (S), or glutamine (Q). Hydrophobicity is determined using the technique of Fauchere and Pliska (e.g., Fauchere and Pliska, Eur J Med Chem, 1983).

[0115] Alternatively or additionally, the peptides described herein may include substitutions such that the size of the substituted residue is approximately the same as the original amino acid and / or the next closest in size (referred to herein as conservative size substitutions). For example: aspartic acid (D) may be substituted with asparagine (N) or lysine (K); alanine (A) may be substituted with glycine (G) or serine (S); tryptophan (W) may be substituted with tyrosine (Y); glutamic acid (E) may be substituted with glutamine (Q) or methionine (M); arginine (R) may be substituted with phenylalanine (F) or tyrosine (Y); phenylalanine (F) may be substituted with histidine (H) or arginine (R); leucine (L) may be substituted with cysteine ​​(L); Lysine (K) can be replaced by aspartic acid (D) or glutamine (Q); tyrosine (Y) can be replaced by arginine (R) or tryptophan (W); serine (S) can be replaced by alanine (A) or proline (P); glycine (G) can be replaced by alanine (A); isoleucine (I) can be replaced by leucine (L) or asparagine (N); threonine (T) can be replaced by valine (V) or cysteine ​​(C).

[0116] As used herein, an "isolated molecule" (wherein the molecule is, for example, a polypeptide, polynucleotide, or fragment thereof) is a molecule that, by its origin or source of derivation: (1) is not associated with one or more naturally associated components with which it accompanies it in its native state, (2) is substantially free of one or more other molecules from the same species; (3) is expressed by a cell from a different species, or (4) does not occur in nature. The therapeutic polypeptides described herein can be isolated.

[0117] As used herein, "purification" and grammatical variations thereof refer to the complete or partial removal of at least one impurity from a mixture containing a polypeptide and one or more impurities, thereby increasing the purity level of the polypeptide in the composition (i.e., by reducing the amount (ppm) of the impurity in the composition). The therapeutic polypeptides described herein can be referred to as purified.

[0118] As used herein, "substantially pure" refers to material that is at least 50% pure (i.e., free of contaminants), more preferably at least 90% pure, more preferably at least 95% pure, still more preferably at least 98% pure, and most preferably at least 99% pure. The therapeutic polypeptides described herein can be substantially pure.

[0119] The terms "patient", "subject" or "individual" are used interchangeably herein and refer to humans or non-human animals. These terms include mammals, such as humans, non-human primates, laboratory animals, livestock (including bovines, porcines, camels, etc.), companion animals (e.g., canines, felines, other domestic animals, etc.), and rodents (e.g., mice and rats). In some instances, the subject is a human who is at least 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, or 95 years old.

[0120] In one example, the subject has or is at risk of developing an ocular disease. Eye diseases include, but are not limited to, retinitis pigmentosa, rod-coned dystrophy, Leber's congenital amaurosis, Usher's syndrome, Bardet-Biedl Syndrome, Best disease, retinoschisis, Stargardt disease (autosomal dominant or autosomal recessive), untreated retinal detachment, pattern dystrophy, cone-rod dystrophy, achromatopsia, ocular albinism, enhanced S cone syndrome, diabetic retinopathy, age-related macular degeneration, retinopathy of prematurity. In some examples, the subject has clinical signs of an ocular disease.

[0121] In some instances, the subject suffers from renal disease or complication or is in the risk of developing renal disease or complication. In some instances, renal disease or complication is relevant to AMD or atypical hemolytic uremic syndrome (aHUS). In some instances, the subject suffers from AMD or aHUS or is in the risk of developing AMD or aHUS.

[0122] Clinical signs of eye disease include, but are not limited to, decreased peripheral vision, decreased central (reading) vision, decreased night vision, color vision loss, decreased visual acuity, decreased photoreceptor function, and pigment changes. In one example, the subject shows degeneration of the outer nuclear layer (ONL). In another example, the subject has been diagnosed with an eye disease. In yet another example, the subject has not yet shown clinical signs of an eye disease.

[0123] As used herein, the terms "prevent", "preventing" and "prevention" refer to preventing the recurrence or onset of a disease or disorder (e.g., an ocular disease) in a subject, or alleviating one or more symptoms thereof, as a result of administering a therapy (e.g., a prophylactic or therapeutic agent). For example, in the context of administering a therapy to a subject, "prevent", "preventing" and "prevention" refer to inhibiting or reducing the development or onset or progression of a disease or disorder (e.g., an ocular disease) in a subject, or preventing the recurrence, onset or development of one or more symptoms of a disease or disorder (e.g., an ocular disease) as a result of administering a therapy (e.g., a prophylactic or therapeutic agent) or administering a combination of therapies (e.g., a combination of prophylactic or therapeutic agents).

[0124] "Treating" a condition or patient means taking steps to obtain beneficial or desired results, including clinical results. With respect to a disease or condition (e.g., an ocular disease), treatment refers to a reduction in the progression, severity, and / or duration or improvement in the condition (e.g., an ocular disease or symptoms associated therewith) or an improvement in one or more symptoms resulting from the administration of one or more therapies (including, but not limited to, the administration of one or more prophylactic or therapeutic agents).

[0125] "Administering" or "administration of" a substance, compound, or agent to a subject can be performed using one of a variety of methods known to those skilled in the art. For example, the compound or agent can be administered intravitreally or subretinally or systemically. In specific examples, the compound or agent is administered intravitreally. In some examples, administration can be local. In other examples, administration can be systemic. Administration can also be performed, for example, once, more than once, and / or over one or more extended time periods. In certain aspects, administration includes direct administration (including self-administration) and indirect administration (including the act of prescribing a drug). For example, as used herein, a physician who instructs a patient to self-administer a drug or who administers a drug by another person and / or provides a drug prescription to a patient administers a drug to a patient.

[0126] Each example described herein may be used alone or in combination with any other example described herein.

[0127] In some instances, therapeutic peptides as described herein are synthetic engineered mimics related to the structure or function of one or more endogenous molecules.For example, the endogenous apolipoproteins participating in lipid transport and processing mechanisms are responsible for the solubilization and transportation of lipid molecules for cellular uptake or outflow.Especially, apolipoproteins directly participate in the uptake of lipids such as cholesterol or the cholesterol homeostasis related to outflow in RPE.

[0128] The therapeutic compositions described herein may include one or more therapeutic peptides having lipid solubilizing and lipid efflux activities; these therapeutic compositions may have minimal cytotoxicity.

[0129] As mentioned, the therapeutic peptides described herein can be short amphipathic polypeptides (e.g., 80 or fewer amino acids, 75 or fewer amino acids, 70 or fewer amino acids, 65 or fewer amino acids, 60 or fewer amino acids, 55 or fewer amino acids, 50 or fewer amino acids, 45 or fewer amino acids, 40 or fewer amino acids, less than 35 amino acids, less than 30 amino acids, etc.), which can bind to one or more cholesterol efflux transporters such as ABCA1 or other transporters and promote lipid efflux out of cells. Therefore, the therapeutic peptide mimetics described herein can have a structure that is functionally homologous to endogenous apolipoprotein A (e.g., ApoA1), which can confer therapeutic benefits to disorders based on dysregulation of lipid efflux mechanisms to prevent lipid accumulation or increased drusen formation.

[0130] The therapeutic compositions described herein may include one or more therapeutic peptides having lipid solubilizing and lipid importing activities; these therapeutic compositions may have minimal cytotoxicity.

[0131] As described above, the therapeutic peptides described herein can be short, amphipathic, which can bind to LDLR in a lipid-dependent manner and transport lipids into cells via the LDLR receptor. These therapeutic peptides can overcome the lipid transport defects present in the ApoE2 carrier, such as ApoE2 variants with defective LDLR binding activity. These therapeutic peptides retain lipid-dependent LDLR binding and lipid transport activity in smaller peptides. Therefore, the therapeutic peptide mimetics described herein can have a structure homologous to endogenous apolipoprotein E (e.g., ApoE) function, which can confer therapeutic benefits to disorders based on lipid import mechanisms to prevent lipid accumulation or increased formation of drusen.

[0132] Figure 1A-Figure 1IPeptides including control peptides (T-001 to T-004 and T-031 to T-034) and therapeutic peptides (T-005 to T-030 and T-35 to T-178 corresponding to SEQ ID NOs: 5-30 and 35-178, respectively) are shown. These peptides or modified forms of these peptides can be used as therapeutic peptides as described herein. Figure 1A-Figure 1I The table shown in also includes the helical hydrophobic moment (μH), which is a measure of the amphipathicity of the peptide helix.

[0133] As described above, any peptide in T-005 to T-030, T-35 to T-080 and T-084 to T-178 and related peptides (e.g., peptides with 65% or higher homology) can be used for treatment as described herein. A peptide at least 65% homologous (in some instances at least 75% homologous, at least 80% homologous, at least 85% homologous, at least 90% homologous) to any peptide in T-005 to T-030, T-35 to T-080 and T-084 to T-178 can refer to a homologous peptide in which different amino acid residues are conservatively substituted. In any of these examples, a homologous peptide can refer to a peptide in which the amino acid different from the peptides of T-005 to T-030, T-035 to T-080 and T-084 to T-178 is a conservative hydrophobic substitution and / or a conservative size substitution and / or a conservative charge substitution. In any of these examples, the conservative substitutions (and / or conservative hydrophobicity substitutions and / or conservative size substitutions and / or conservative charge substitutions) maintain the helical structure as described herein.

[0134] Specifically, in one example, a T-087 (SEQ ID NO: 87) peptide or a peptide at least 65% homologous to a T-087 peptide can be used therapeutically as described herein. For example, Fig.41A The sequence of a family of homologous peptides based on the T-087 peptide is shown. Fig.41A In FIG, the boxed amino acids represent residues that contribute to the desired therapeutic activity of the peptide, including safety, cholesterol efflux ability, and ABCA1 stabilization ability. Fig.7D , Fig.29 and Fig.35 Derivatives of T-087 generated by alanine scanning (single and triple) mutagenesis were tested in the functional assays described to determine these residues. Peptides T-152 to T-178 illustrate some exemplary peptides corresponding to variants of T-087. Fig.41AIn the examples, the unboxed amino acids can correspond to any amino acid. In some examples, the boxed amino acids correspond to conservative substitutions (e.g., use of glycine (G) at the second position, phenylalanine (F) at the third position, aspartic acid (D) at the fourth position, lysine (K) at the fifth position, leucine (L) at the sixth position, arginine (R) at the seventh position, isoleucine (I) at the ninth position, arginine (R) at the eleventh position, aspartic acid (D) at the twelfth position, valine (V) at the thirteenth position, glycine (G) at the fourteenth position, at the fifteenth position, glutamic acid (E), tryptophan (W), and tyrosine (Y) at the sixteenth position), conservative hydrophobic substitutions (e.g., threonine (T) at the second position, aspartic acid (D) at the fourth position, aspartic acid (D) at the fifth position, isoleucine (I) at the sixth position, aspartic acid (D) at the seventh position, phenylalanine (F) at the ninth position, aspartic acid (D) at the eleventh position, asparagine (N) at the twelfth position, and amino acid (N) at the thirteenth position). 14 position, threonine (T), lysine (K), proline (P), and leucine (L) at the 17th position), or conservative size substitutions (e.g., glycine (G) or serine (S) at the second position, tyrosine (Y) at the third position, glutamine (Q) or methionine (M) at the fourth position, phenylalanine (F) or tyrosine (Y) at the fifth position, histidine (H) or arginine (R) at the sixth position, phenylalanine (F) or tyrosine (Y) at the seventh position, and threonine (T) at the fifth position. The amino acids in the amino acids 1 and 2 may be selected from the group consisting of amino acids 1 and 2 (i) , 1 and 3 (f), 1 and 4 (h), 1 and 2 (w), 1 and 3 (h), 1 and 4 (h ...

[0135] In another example, the T-101 (SEQ ID NO: 101) peptide or a peptide at least 65% homologous to the T-101 peptide can be used for treatment as described herein. For example, Fig.41B The sequence of a family of homologous peptides based on the T-101 peptide is shown. Fig.41B In FIG, the boxed amino acids represent residues that contribute to the desired therapeutic activity of the peptide, including safety, cholesterol efflux ability, and ABCA1 stabilization ability. Figure 7C , Fig.28 and Fig.36 Derivatives of T-101 generated by alanine scanning mutagenesis (single and triple) were tested in the functional assays described to determine these residues. Peptides T-121 to T-151 illustrate some exemplary peptides corresponding to variants of T-101. Fig.41B In the examples, the unboxed amino acids can correspond to any amino acids. In some examples, the boxed amino acids correspond to conservative substitutions (e.g., lysine (K) at position 1, glycine (G) at position 4, glutamic acid (E) at position 5, glycine (G) at position 6, valine (V) at position 7, aspartic acid (D) at position 8, glycine (G) at position 10, isoleucine (I) at position 11, arginine (R) at position 12, aspartic acid (D) at position 13, valine (V) at position 14, arginine (R) at position 15, tryptophan (W) at position 17, leucine (L) at position 18, 20 aspartic acid (D), phenylalanine (F) at position 21 and serine (S) at position 22), conservative hydrophobic substitutions (e.g., aspartic acid (D) at position 1, threonine (T) at position 4, lysine (K) or arginine (R) at position 5, histidine (H) at position 6, phenylalanine (F) at position 7, asparagine (N) at position 8, threonine (T) at position 10, isoleucine (I) at position 11, aspartic acid (D) at position 12, asparagine (N) at position 13, cysteine ​​(C) at position 14, aspartic acid (D) at position 15, arginine (R) at position 16, histidine (H) at position 17, phenylalanine (F) at position 18, asparagine (N) at position 19, 7, leucine (L), phenylalanine (F), asparagine (N), and histidine (H) at position 22), or conservative size substitutions (e.g., phenylalanine (F) or tyrosine (Y) at position 1, glycine (G) or serine (S) at position 4, asparagine (N) or lysine (K) at position 5, glycine (G) or serine (S) at position 6, cysteine ​​(C) or isoleucine (I) at position 7, glutamine (Q) or methionine (M) at position 8, glycine (G) or serine (S) at position 10, and histidine (H) at position 11). 14, cysteine ​​(C) or isoleucine (I) at position 1, aspartic acid (D) or glutamine (Q) at position 12, glutamine (Q) or methionine (M) at position 13, cysteine ​​(C) or isoleucine (I) at position 14, aspartic acid (D) or glutamine (Q) at position 15, histidine (H) or arginine (R) at position 17, leucine (L) or asparagine (N) at position 18, glutamine (Q) or methionine (M) at position 20, tyrosine (Y) at position 21, and valine (V) or cysteine ​​(C) at position 22).

[0136] The safety and functional activity of the peptides described herein are illustrated in the figures and data provided herein. For example, Figure 2A-2D The lipid solubilizing activity of each peptide shown is measured by reducing the turbidity of a 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) liposome solution. In this example, the peptide stock solution was diluted to 200 μM (2x solution) in H2O, and 50 μL of the peptide solution was transferred to a 96-well plate in triplicate. A 1 mM solution of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) was prepared in H2O, and 50 μL of the DMPC solution was quickly added to the plate using a multichannel pipette. The peptide and lipid solutions were incubated at room temperature for 15 minutes, with a final concentration of 100 μM peptide and 0.5 mM DMPC. After 15 minutes, the absorbance was read at 405 nm on a plate reader. Background correction was performed using a HO blank and 100% lipid clarification was determined using the absorbance of DMPC incubated in 1% Triton-X-100. Data are presented as the mean + SD of two runs. Almost all peptides tested cleared more than 50% of lipids during the assay.

[0137] The 11 tested peptides (T-025, T-027, T-051, T-052, T-054, T-059, T-062, T-063, T-065, T-069, and T-080) were examined in more detail. Figure 3A-3CShown. In these experiments, DMPC solubility titration curves were generated. The peptide stock solution was diluted to 200 μM (2X solution) in H2O and serially diluted in H2O through 4 concentrations, and then 50 μL of the peptide solution was dispensed in triplicate into a 96-well plate. A 1 mM solution of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) was prepared in H2O, and 50 μL of the DMPC solution was quickly added to the plate using a multichannel pipette. The peptide and lipid solutions were incubated at room temperature for 15 minutes with a final concentration of 3.16 - 100 μM peptide and 0.5 mM DMPC. After 15 minutes, the absorbance was read at 405 nm on a plate reader. Background correction was performed using an H2O blank, and 100% lipid clarification was determined using the absorbance of DMPC incubated in 1% Triton-X-100. Data are presented as the mean + SD of two runs. EC50 values ​​were calculated using nonlinear regression. The EC50 values ​​for the peptides tested in the assay were (T-025, 25.33 μM; T-027, 56.55 μM; T-051, 15.6 μM; T-052, 20.6 μM; T-054, 122.6 μM; T-062, 213.4 μM; T-063, 110.6 μM; T-065, 24.4 μM; T-069, 38.77 μM; T-080, 154.8 μM).

[0138] according to Figure 2A-2D and Figure 3A-3C Based on these results shown in , if the peptide causes more than 50% clarification at 100 μM at 15 minutes (and especially if there is activity at 10 μM), the peptide may be particularly useful therapeutically. Many non-control peptides have significant activity. See, for example, T-006, T-007, T-009, T-010, T-011, T-012, T-013, T-014, T-024, T-026, T-030, T-018, T-019, T-021, T-025, T-027, T-028, T-029, T-086, T-087, T-101, T-112, T-114, T-116. Some of these peptides (T-005, T-008, T-015, T-017, T-020, and T-099) did not have significant solubilizing activity.

[0139] Figure 4A-4B The diagram shows Figure 1B-Figure 1I A subset of peptides in a DMPC solubility-based screening assay, measured by reducing the turbidity of DMPC liposome solutions. Figure 4A-4BDMPC solubility titration curves are shown. The peptide stock solution was diluted to 200 μM (2x solution) in H2O and serially diluted in H2O over 4 concentrations, and 50 μL of the peptide solution was dispensed in triplicate into a 96-well plate. A 2 mM solution of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) was prepared in H2O, and 50 μL of the DMPC solution was quickly added to the plate using a multichannel pipette. The peptide and lipid solutions were incubated at room temperature for 15 minutes to a final concentration of 3.16 - 100 μM peptide and 1 mM DMPC. After 15 minutes, the absorbance was read at 405 nm on a plate reader. Background correction was performed using an H2O blank, and 100% lipid clearing was determined using the absorbance of DMPC incubated in 1% Triton-X-100. Data are presented as the mean + SD of two runs. EC50 values ​​were calculated using nonlinear regression. The EC50 values ​​of the tested peptides in the assay were (T-084, 16.15 μM; T-086, 15.81 μM; T-087, 17.82 μM; T-101, 10.50 μM; T-112, 8.26 μM; T-113, 8.35 μM; T-114, 12.21 μM; T-116, 7.54 μM).

[0140] Selected test peptides were further evaluated, e.g. Figure 5A-5D . These figures illustrate ARPE-19 cell lysis. ARPE-19 cells were grown in 96-well plates to 75%-85% confluence. 10 mM of peptide was diluted to a 20 μM working solution (2X solution) in serum-free DMEM:F12 medium. Sytox Green was prepared as a 1 μM working solution (2X solution) in serum-free DMEM:F12. Equal volumes of peptide and Sytox Green solution were added to an empty 96-well plate and gently mixed. The final peptide concentration was 10 μM. ARPE-19 cells (approximately 80% confluence in a 96-well plate) were washed with DPBS and then incubated in 100 μL of peptide / SytoxGreen mixture at 37°C for 2h. Cells were fixed in 4% paraformaldehyde solution in DPBS, then incubated in 10 μM Hoechst solution for 10 min, washed, and imaged on a Yokogawa CQ1 high-content imager. Live / dead cells were counted in 4 fields of view at 20x magnification using CellPathfinder software. The percentage of lysis (%) was calculated by dividing the number of Sytox Green+ nuclei by the total number of Hoechst+ nuclei. In this assay, the test peptides did not induce cell lysis except T-021, T-028, and T-034.

[0141] Figure 6 Shows Figure 1B-Figure 1I Figure 2. ARPE-19 cell lysis titration curves of a subset of peptides. ARPE-19 cells were grown in 96-well plates to 75%-85% confluence. 10 mM of peptide was diluted in serum-free DMEM:F12 medium to a solution between 6.32 - 200 μM. Sytox Green was prepared as a 1 μM working solution in serum-free DMEM:F12 medium. Equal volumes of peptide and Sytox Green solutions were added to an empty 96-well plate and mixed gently. The final peptide concentration was 3.16 - 100 μM. ARPE-19 cells (~80% confluence in 96-well plates) were washed with DPBS and then incubated in 100 μL of the peptide / Sytox Green mixture at 37°C for 2 h. Cells were fixed in 4% paraformaldehyde solution in DPBS, then incubated in 10 μM Hoechst solution for 10 min, washed, and imaged on a Yokogawa CQ1 high-content imager. Live / dead cells were counted in 4 fields at 20x magnification using CellPathfinder software. The percentage of lysis (%) was calculated by dividing the number of Sytox Green+ nuclei by the total number of Hoechst+ nuclei. EC50 values ​​were calculated using nonlinear regression. The EC50 values ​​of the tested peptides in the assay were (T-025, 144.1 μM; T-027, 40.14 μM).

[0142] Figure 7A-7D The results of the ARPE-19 cell viability assay for the peptides described herein are shown. ARPE-19 cells were seeded in 96-well plates at 25,000 cells / well and allowed to adhere to the plate surface for at least 24 hours. The peptides were diluted to a final concentration of 100 μM in serum-free DMEM:F12. The cells were washed and then incubated at 37°C in a 100 μM peptide solution for 1.5 hours. The plates were placed at room temperature for 30 minutes and then washed with DPBS. The cells were incubated at room temperature for 10 minutes in a 1:1 mixture of DPBS and Cell Titer Glo 2.0 Reagent, and cell luminescence was then read on a plate reader. Viability values ​​were background corrected, and 0% viability was determined by the luminescence value of cells treated with 1% Triton-X-100. Data are represented by the mean + SD of two independent runs. With the exception of test peptides T-062, T-065, T-069, T-077, T-085, T-109, T-110, T-111, and T-115, treatment with the test peptides did not result in substantial changes in viability during the assay.

[0143] Similar results were observed with human red blood cells (RBCs) in lysis assays, e.g. Figure 8A-8D and Figure 9A-9B As shown in . Figure 8A-8D The results of human red blood cell (hRBC) lysis assays for peptide subsets are shown. Human red blood cells (hRBC; washed, suspended in 25% diluted cells in Alsevers solution) were added to 96-well plates containing 50 μL of 40 μM peptide solution in duplicate 50 μL aliquots. The plates were incubated at 37°C for 2 hours, and then the hRBCs were precipitated at 500xg for 5 minutes. 50 μL of supernatant was collected and transferred to a fresh plate containing 50 μL of PBS per well, and the absorbance was read at 560 nm on a plate reader to determine lysis. 100% lysis reflects wells treated with 0.1% Triton-X-100. Data are represented by the mean + SD of two runs. Figure 9A-9B is a graph summarizing the hRBC lysis assay with more peptides. None of the tested peptides induced greater than 10% hRBC lysis in the assay.

[0144] Fig.10 and Fig.11 Human red blood cell (hRBC) lysis titration curves are shown. Human red blood cells (hRBC; washed, suspended at 25% dilution in Alsevers solution) were added in duplicate 50 μL aliquots to a 96-well plate containing 50 μL of 6.32 - 200 μM peptide solution (final peptide concentration of 3.16 μM - 100 μM). The plates were incubated at 37°C for 2 hours, and then the hRBCs were pelleted at 500 xg for 5 minutes. 50 μL of supernatant was collected and transferred to a fresh plate containing 50 μL PBS per well, and the absorbance was read at 560 nm on a plate reader to determine lysis. 100% lysis reflects wells treated with 0.1% Triton-X-100. Data are presented as the mean + SD of two runs. EC50 values ​​were calculated using nonlinear regression. The EC50 values ​​of the peptides tested in this assay were (T-025, >100 μM; T-027, >100 μM; T-086, >100 μM; T-087, >100 μM; T-101, >100 μM; T-112, >100 μM; T-114, >100 μM; T-116, >100 μM).

[0145] Fig.12 and Figure 14A-14BResults of the ARPE-19 cholesterol input assay are shown. ARPE-19 cells were seeded at 25,000 cells / well in a 96-well plate and grown to confluence. A 2X solution of 500 μM DMPC and 50 μM BODIPY-cholesterol was prepared in serum-free DMEM:F12. Peptides were diluted to 20 μM (2X solution) in serum-free DMEM:F12. Peptide aliquots (60 μl each) were added to the lipid solution and incubated at 37°C for 1 hour. ARPE-19 cells were serum starved in serum-free DMEM:F12 for 1 hour at 37°C. Peptide:lipid complexes (100 μL total volume per well) were transferred to ARPE-19 cells for 2 hours at 37°C. Cells were washed, fixed, and intracellular fluorescence was read on a Promega GloMax at 475 nm excitation, 500 nm - 550 nm emission. Fluorescence values ​​were background corrected for the no treatment condition. Data are presented as mean ± SD of two runs. Fig.12 and Figure 14A-14B The results shown in the graph show that some peptides have cholesterol importing activity (corresponding to fluorescence values ​​of at least 10 RFU above baseline), for example: T-013, T-018, T-021, T-025, T-027, T-028, T-030, T-042, T-047, T-065, T-066, T-067, T-068, T-069, T-070, T-073, T-101, T-109, T-111, T-112, T-114, T-115, T-116, T-117, T-118 and T-119.

[0146] Fig.13Results of an ARPE-19 GAG-dependent cholesterol import assay are shown. ARPE-19 cells were seeded at 25,000 cells / well in a 96-well plate and grown to confluence. A 2X solution of 500 μM DMPC and 50 μM BODIPY-cholesterol was prepared in serum-free DMEM:F12, with or without 100 μg / mL heparin. Peptides were diluted to 20 μM (2X solution) in serum-free DMEM:F12. Peptide aliquots (60 μl each) were added to the lipid solution and incubated at 37°C for 1 hour. ARPE-19 cells were serum starved in serum-free DMEM:F12 for 1 hour at 37°C. Peptide:lipid complexes (100 μL total volume per well) were transferred to ARPE-19 cells for 2 hours at 37°C. Cells were washed, fixed, and intracellular fluorescence was read on a Promega GloMax at 475 nm excitation, 500 nm - 550 nm emission. Fluorescence values ​​were background corrected according to the no treatment condition. Data are presented as ± SD of 2 runs. Based on these data, the test peptides T-013, T-021, T-025, T-027 and T-028 were dependent on GAG binding for cholesterol import activity.

[0147] Fig.15 ARPE-19 cholesterol input titration curves for peptide subsets are shown. ARPE-19 cells were seeded at 25,000 cells / well in 96-well plates and grown to confluence. A 2X solution of 500 μM DMPC and 50 μM BODIPY-cholesterol was prepared in serum-free DMEM:F12. The peptides were diluted to 0.74 μM - 60 μM (2X solution) in serum-free DMEM:F12. Peptide aliquots (60 μl each) were added to the lipid solution to produce a final peptide concentration of 0.37 μM - 30 μM and incubated at 37°C for 1 hour. ARPE-19 cells were serum starved for 1 hour at 37°C in serum-free DMEM:F12. The peptide:lipid complexes (100 μL total volume per well) were transferred to ARPE-19 cells for 2 hours at 37°C. Cells were washed, fixed, and intracellular fluorescence was read on a Promega GloMax at 475 nm excitation and 500 nm - 550 nm emission. Fluorescence values ​​were background corrected for the no treatment condition. Data are presented as mean ± SD of three runs. Test peptides T-025 and T-027 showed a concentration-dependent increase in cholesterol import.

[0148] Fig.16ARPE-19 cholesterol input titration curves for another subset of peptides are shown. ARPE-19 cells were seeded at 25,000 cells / well in 96-well plates and grown to confluence. Peptide:lipid complexes were generated by preparing a serum-free DMEM:F12 solution containing 30 μM peptide, 750 μM DMPC, and 75 μM BODIPY-cholesterol. The peptide:lipid complexes were serially diluted to 5 concentrations at 1:3 and incubated at 37°C for 1 hour. ARPE-19 cells were serum starved in serum-free DMEM:F12 at 37°C for 1 hour. Peptide:lipid complexes (100 μL total volume per well) were transferred to ARPE-19 cells and continued at 37°C for 2 hours. Cells were washed, fixed, and intracellular fluorescence was read on a Promega GloMax at 475 nm excitation and 500 nm - 550 nm emission. Fluorescence values ​​were background corrected according to the no treatment condition. Data are presented as the mean ± SD of two runs. The test peptides T-101, T-112, T-114 and T-116 showed concentration-dependent increases in cholesterol import, whereas T-086 and T-087 failed to increase cholesterol import over a range of concentrations.

[0149] Fig.17 and Fig.18 HepG2 cholesterol input screening data for some of the peptides described herein are summarized. HepG2 cells were seeded at 10,000 cells / well in 96-well plates and grown to confluence. Solutions of 500 μM DMPC and 50 μM BODIPY-cholesterol were prepared in serum-free EMEM. Peptides were diluted to 20 μM (2X solution) in serum-free EMEM. Peptide aliquots (60 μl each) were added to the lipid solution to produce a final 1X concentration and incubated at 37°C for 1 hour. HepG2 cells were serum starved in serum-free EMEM for 1 hour at 37°C. Peptide:lipid complexes (100 μL total volume per well) were transferred to HepG2 cells for 2 hours at 37°C. Cells were washed, fixed, and intracellular fluorescence was read on a Promega GloMax at 475 nm excitation, 500 nm - 550 nm emission. Fluorescence values ​​were background corrected for the no treatment condition. Data are presented as mean ± SD of two runs. Fig.17 and Fig.18 The results showed that many of the tested peptides had cholesterol import activity in HepG2 cells (corresponding to cell fluorescence values ​​20RFU above baseline), including T-007, T-009, T-012, T-013, T-018, T-021, T-024, T-025, T-027, T-028, T-029, T-030, T-044, T-065, T-066, T-068, T-070 and T-080.

[0150] Fig.19 HepG2 GAG-dependent cholesterol input in a subset of peptides is shown. HepG2 cells were seeded at 10,000 cells / well in 96-well plates and grown to confluence. A 2X solution of 500 μM DMPC and 50 μM BODIPY-cholesterol was prepared in serum-free EMEM, with or without 100 μg / mL heparin. The peptides were diluted to 20 μM (2X solution) in serum-free EMEM. Peptide aliquots (60 μl each) were added to the lipid solution and incubated at 37°C for 1 hour. HepG2 cells were serum starved in serum-free EMEM at 37°C for 1 hour. Peptide:lipid complexes (100 μL total volume per well) were transferred to HepG2 cells for 2 hours at 37°C. Cells were washed, fixed, and intracellular fluorescence was read on a Promega GloMax at 475 nm excitation and 500 nm - 550 nm emission. Fluorescence values ​​were background corrected for the no treatment condition. Data are presented as ±SD of 2 runs. Based on these data, peptides T-021, T-025, T-027, T-028, and T-029 were tested for their dependence on GAG binding for cholesterol import activity in HepG2 cells.

[0151] Fig. 20 and Fig.21 HepG2 cholesterol input titration curves for some peptides (T-025, T-027, T-032, T-086, T-087, T-101, T-112, T-114, and T-116) are shown as shown. HepG2 cells were seeded at 10,000 cells / well in 96-well plates and grown to confluence. Peptide:lipid complexes were generated by preparing a serum-free EMEM solution containing 30 μM peptide, 750 μM DMPC, and 75 μM BODIPY-cholesterol. Peptide:lipid complexes were serially diluted 1:3 to 5 concentrations and incubated at 37°C for 1 hour. HepG2 cells were serum starved in serum-free EMEM for 1 hour at 37°C. Peptide:lipid complexes (100 μL total volume per well) were transferred to HepG2 cells for 2 hours at 37°C. Cells were washed, fixed, and intracellular fluorescence was read on a PromegaGloMax at 475 nm excitation and 500 nm - 550 nm emission. Fluorescence values ​​were background corrected according to the no treatment condition. Data are presented as mean + SD of two runs. Test peptides T-025, T-027, T-101, T-112, T-114, and T-116 showed concentration-dependent cholesterol import activity in HepG2 cells.

[0152] Fig. 22The lipid-dependent LDLR binding (SPR) of a subset of peptides is shown. The peptides were diluted to a final concentration of 1 μM in 1XHBS-N buffer containing 1 mM CaCl2 and incubated for 1 hour at room temperature (RT) with or without 25 μM DMPC to form lipid complexes. Each peptide was tested in duplicate. LDLR was diluted to 10 μg / mL in 10 mM acetate buffer (pH 4.5) and fixed (~400RU) on a CM5 chip via amine coupling using 1X HBS-P+ buffer and 1 mM CaCl2. The test peptides (with or without DMPC) were exposed to immobilized LDLR for 120 seconds in HBS-N with 1 mM CaCl2 at a flow rate of 30 μL / sec. Between cycles, the surface was regenerated with 3 30-second injections of 10 mM NaOH and 100 mM EDTA. Data were plotted as maximum RU values, averaging from three separate runs. The test peptides T-007, T-009, T-013, T-018 and T-029 showed lipid-dependent LDLR binding activity, wherein the RU values ​​of the peptides binding to LDLR in the presence of lipids were higher than those of the peptides alone without lipids.

[0153] Fig.23 The table shown in illustrates the LDLR binding data (SPR) using high surfactant preparations. Recombinant LDLR was diluted in 10 mM acetate buffer (pH 4.5) and coupled to a CM5 chip with 1000 RU amine. The peptide was diluted to 5 μM in running buffer (HBS-P+, 1 mM CaCl2) and serially diluted to a total of 5 concentrations at 1:3. The peptide was exposed to immobilized LDLR for 120 seconds at a flow rate of 30 μL / sec. Between cycles, the chip surface was regenerated with 3 injections of 50 mM NaOH / 100 mM EDTA. Multi-cycle kinetics was performed on Biacore Insight Evaluation Software, and binding kinetics were analyzed with a 1:1 binding model. In this assay, several tested peptides bound to LDLR with a KD < 10 uM, including T-040, T-041, T-044, T-046, T-047, T-048, T-049, T-052, T-055, T-059, T-062, T-064, T-065, T-066, T-068, T-069, T-072, T-074, T-109, T-110, T-111, T-112, T-113, T-114, T-115, T-116, T-117, T-118, and T-120.

[0154] Figure 24A-24B The table summarizes the results of the LDLR binding data (SPR) for lipid-dependent binding. Recombinant LDLR was diluted in 10 mM acetate buffer (pH 4.5) and amine-coupled to a CM5 chip at 1000 RU. Peptides were diluted to 5 μM in running buffer (HBS-P, 1 mM CaCl2) and serially diluted 1:3 for a total of 5 concentrations. In a separate preparation, peptides were diluted to 1 μM in running buffer containing 25 μM DMPC and serially diluted 1:3 in running buffer for a total of 5 concentrations. Peptide:DMPC complexes were incubated for 1 hour before use. Peptides or peptide:lipid complexes were exposed to immobilized LDLR at a flow rate of 30 μL / sec for 120 seconds. Between cycles, the chip surface was regenerated with 3 injections of 50 mM NaOH / 100 mM EDTA. Multi-Cycle Kinetics was performed on Biacore Insight Evaluation Software, and binding kinetics were analyzed using a 1:1 binding model. In the presence of DMPC lipid, several peptides tested showed >2X changes in LDLR binding, including T-065, T-066, T-068, T-070, T-109, T-110, T-111, T-112, T-113, T-114, T-115, T-116, and T-117.

[0155] Figure 25A-Figure 25O The peptide binding activity of the peptides shown by SPR to non-oxidized and oxidized lipids is shown. The peptides were reconstructed to 50 μg / mL in 10mM acetate buffer (pH 4.5) and fixed to ~200 RU on a CM5 chip by amine coupling. Non-oxidized lipids (DMPC and POPC) and oxidized lipids (POVPC and KOdiA-PC) were diluted to 100 μM in HBS-EP running buffer and exposed to the chip for binding (n=3) at a flow rate of 30 μL / sec for 120 seconds. The surface was regenerated with 20% EtOH. On each chip, T-001 was used as a positive control and T-031 was used as a negative control. For each peptide, the RU responses of all lipids were normalized for DMPC RU values. If the peptide cannot bind DMPC, it is considered to be inactivated due to fixation. Test peptides T-009, T-013, T-021, T-025, T-029 and T-030 all showed binding to at least one oxidized lipid comparable to or better than binding to DMPC.

[0156] Figure 26A-26BResults of the oxidized LDL test are shown. In a 96-well microplate, LDL (stock concentration 10 mg / mL), copper (II) sulfate (stock concentration 1.5 mM) and peptide (stock concentration 1 mM) were combined in sextuplicate wells to final concentrations of 100 μg / mL, 15 μM and 25 μM, respectively. Control wells were filled with LDL (100 μg / mL) and copper (II) sulfate (15 μM) or LDL alone. The microplate was immediately read at OD 234 nm in a plate reader every 5 minutes for 240 minutes. An increase in absorbance at 234 nm reflects an increase in conjugated diene formation and is used as a measure of oxidation. Fig.26A Data are presented as mean ± SD of three technical replicates. Fig.26B In the assay, the LDL oxidation rate was calculated as the maximum absorbance divided by the delay phase length, and the data are presented as the mean + SD of three technical replicates. In this assay, in the presence of copper (II) sulfate, the test peptides T-087 and T-101 reduced the oxidation of LDL by 92% and 91.6%, respectively.

[0157] Figure 27A-27B and Fig.28HMC3 cholesterol efflux is shown. A fully confluent 96-well plate of HMC3 cells was treated for 1 hour in serum-free EMEM containing 10 mM methyl-β-cyclodextrin, 100 μM cholesterol, and 25 μM BODIPY cholesterol. The cells were then washed and incubated overnight in serum-free EMEM containing 1 μM LXR agonist GW3965, 2 μg / mL ACAT inhibitor, and 0.2% BSA (LXR agonist treated) or serum-free EMEM containing 2 μg / mL ACAT inhibitor and 0.2% BSA (control). The cells were washed and incubated with 20 μM peptide diluted in serum-free Fluorobrite DMEM at 37°C for 4 hours. The medium was transferred to a new 96-well plate and fresh serum-free Fluorobrite DMEM was placed on the cells. The medium and cell fluorescence were read on a Promega GloMax at 475 nm excitation, 500 nm - 550 nm emission. Fluorescence values ​​were background corrected according to the no treatment condition and the percent efflux was calculated by the following formula: % efflux = (medium fluorescence value) / (medium + cell fluorescence value). Data are presented as mean + SD of two runs for each peptide. Several tested peptides induced greater cholesterol efflux in the presence of LXR agonists in HMC3 cells, including T-084, T-085, T-086, T-087, T-101, and the T-101-derived peptides T-121, T-122, T-123, T-124, T-126, T-128, T-129, T-134, T-135, T-136, T-137, T-138, T-139, T-140, T-141, T-142, T-143, T-144, T-145, T-146, T-148, T-149, T-150, and T-151.

[0158] Fig.29ARPE-19 cholesterol efflux of the peptides shown is shown. A 96-well plate with fully confluent ARPE-19 cells was treated in serum-free DMEM containing 10 mM methyl-β-cyclodextrin, 100 μM cholesterol and 25 μM BODIPY cholesterol for 1 hour. The cells were then washed and incubated overnight in serum-free DMEM (LXR agonist treated) containing 1 μM GW3965, 2 μg / mL ACAT inhibitor and 0.2% BSA or serum-free DMEM (control) containing 2 μg / mL ACAT inhibitor and 0.2% BSA. The cells were washed and incubated at 37°C for 4 hours with 20 μM peptides diluted in serum-free Fluorobrite DMEM. The culture medium was transferred to a new 96-well plate and fresh serum-free Fluorobrite DMEM was placed on the cells. The culture medium and cell fluorescence were read on Promega GloMax with 475 nm excitation and 500 nm - 550 nm emission. Fluorescence values ​​were background corrected according to the no treatment condition, and the percent efflux was calculated by the following formula: % efflux = (fluorescence value of medium) / (fluorescence value of medium + cells). Data are presented as the mean + SD of two runs for each peptide. Several T-087-derived peptides showed increased cholesterol efflux in ARPE-19 cells after LXR agonist treatment, including T-152, T-153, T-154, T-155, T-156, T-157, T-158, T-159, T-160, T-161, T-162, T-163, T-164, T-166, T-167, T-168, T-170, T-171, T-172, T-174, T-175, T-176, and T-178.

[0159] Figures 30A-30JHMC3 cholesterol efflux titration curves of the peptides shown are shown. A 96-well plate with fully confluent HMC3 cells was treated in serum-free EMEM containing 10 mM methyl-β-cyclodextrin, 100 μM cholesterol and 25 μM BODIPY cholesterol for 1 hour. The cells were then washed and incubated overnight in serum-free EMEM (LXR agonist treated) containing 1 μM GW3965, 2 μg / mL ACAT inhibitor and 0.2% BSA or serum-free EMEM (control) containing 2 μg / mL ACAT inhibitor and 0.2% BSA. The cells were washed and incubated at 37°C for 4 hours with 0-100 μM peptides serially diluted in serum-free Fluorobrite DMEM. The culture medium was transferred to a new 96-well plate and fresh serum-free Fluorobrite DMEM was placed on the cells. Culture medium and cell fluorescence were read on Promega GloMax with 475 nm excitation and 500 nm - 550 nm emission. Fluorescence values ​​were background corrected according to the no treatment condition, and the percent efflux was calculated by the following formula: % efflux = (fluorescence value of medium) / (fluorescence value of medium + cells). Data are presented as the mean ± SD of two runs for each peptide. The peptides tested were (A) T-001, positive control; (B) T-082, positive control; (C) T-083, positive control; (D) T-084; (E) T-086; (F) T-087; (G) T-101; (H) T-112; (I) T-114 and (J) T-116. In the presence of LXR agonists, all tested peptides showed a concentration-dependent increase in cholesterol efflux from HMC3 cells.

[0160] Figure 31A-Figure 31BCholesterol efflux following ATP-binding cassette family knockdown assay is shown. ARPE-19 cells were seeded at 25,000 cells / well in 96-well plates or 100,000 cells / well in 12-well plates and allowed to grow to confluence for 1 week. miR-33a (which targets ABCA1) was diluted to 30 nM in Opti-MEM. siRNA for ABCA1, ABCA4, ABCA7, ABCG1, ABCG4, or SRB1 was diluted in Opti-MEM to a final concentration of 100 nM siRNA. RNAiMax Lipofectamine was added to the siRNA or miRNA preparation for 5 minutes, followed by transfection in serum-free DMEM for 24 hours at 37°C. To validate the siRNA or miRNA used in this experiment, qPCR was performed to verify at least 85% knockdown of the target transcript and to verify that there was no effect on other transcripts tested. After siRNA or miRNA treatment, cells were treated in serum-free DMEM containing 10 mM methyl-β-cyclodextrin, 100 μM cholesterol and 25 μM BODIPY cholesterol for 1 hour. Cells were then washed and incubated overnight in serum-free DMEM containing 1 μM LXR agonist GW3965, 2 μg / mL ACAT inhibitor and 0.2% BSA (LXR agonist treated) or serum-free EMEM containing 2 μg / mL ACAT inhibitor and 0.2% BSA (control). Cells were washed and incubated at 37°C for 4 hours with 20 μM peptides T-087 or T-101 diluted in serum-free Fluorobrite DMEM. The culture medium was transferred to a new 96-well plate and fresh serum-free Fluorobrite DMEM was placed on the cells. Culture medium and cell fluorescence were read on a Promega GloMax at 475 nm excitation and 500 nm - 550 nm emission. Fluorescence values ​​were background corrected according to the no treatment condition, and the efflux percentage was calculated by the following formula: % efflux = (medium fluorescence value) / (medium + cell fluorescence value). Data are expressed as mean + SD from two runs (siABCA7, siABCG4, siSCARB1), three runs (miR-33), four runs (siABCA4, siABCG1), or 16 runs (siABCA1). Differences were tested using two-way ANOVA and Tukey post hoc test. *, significant difference (P < 0.05). Peptides T-087 and T-101 showed reduced cholesterol efflux after knockdown of miR-33 and siABCA1, but this was not the case for the other siRNAs tested, indicating that these peptides mainly direct cholesterol efflux activity through ABCA1.

[0161] Fig.32It is the figure of cholesterol efflux in the iPS-RPE cells of two examples T-087 and T-101 illustrated in figure.The RPE cells derived from iPS are obtained from Fujifilm (iCell retinal pigment epithelial cells), and grown 28 days on 96-well culture dishes using vitronectin as coated substrate.Verify the cobblestone morphology and pigmentation (pigmentation) of cells, then process 1 hour in the serum-free DMEM containing 10 mM methyl-β-cyclodextrin, 100 μM cholesterol and 25 μM BODIPY cholesterol.Then wash cells and incubate overnight in the serum-free DMEM (LXR agonist processing) containing 1 μM LXR agonist GW3965, 2 μg / mL ACAT inhibitor and 0.2% BSA or the serum-free EMEM (control) containing 2 μg / mL ACAT inhibitor and 0.2% BSA. The cells were washed and incubated at 37°C for 4 hours with 20 μM peptides serially diluted in serum-free Fluorobrite DMEM. The culture medium was transferred to a new 96-well plate and fresh serum-free Fluorobrite DMEM was placed on the cells. The culture medium and cell fluorescence were read on Promega GloMax at 475 nm excitation and 500 nm - 550 nm emission. The fluorescence values ​​were background corrected according to the untreated conditions, and the efflux percentage was calculated by the following formula: % efflux = (culture medium fluorescence value) / (culture medium + cell fluorescence value) Data are expressed as the mean + SD of two runs. Peptides T-087 and T-101 were able to increase cholesterol efflux in iPS-RPE cells after LXR agonism.

[0162] Figure 33A-Figure 33C The results of the membrane ABCA1 stability test in J774 cells are shown. J774 murine macrophages were seeded at 50,000 cells / well in 12-well plates and allowed to grow to confluence. A vehicle solution of 0.1% BSA was prepared in serum-free DMEM for control wells. An ABCA1 induction solution of 0.1% BSA with 1 mM 8-Br-cAMP and 50 μg / mL AcLDL was prepared in serum-free DMEM and incubated on the cells overnight at 37°C to increase ABCA1 protein levels on the membrane. A solution of 10 μM peptide was prepared in serum-free DMEM and serially diluted 1:10 to 4 concentrations. ApoA1 was prepared at a concentration of 300 nM. Peptides or ApoA1 were added to the cells for 4 hours at 37°C before isolating membrane proteins and measuring membrane ABCA1 levels by western blotting. Blots were quantified in ImageJ and signals were compared against membrane Na + / K +Pump level normalization. Signals were further normalized to the 18-hour cAMP treatment group to determine the percentage (%) of membrane ABCA1 remaining. In the absence of peptide or ApoA1 treatment, membrane ABCA1 levels decreased after 4 hours of 8-Br-cAMP washout, whereas treatment with either peptide T-087, T-101, or ApoA1 preserved membrane ABCA1 levels. The EC50 values ​​for peptides T-087 and T-101 in this assay were 1.51 μM and 3.7 μM, respectively.

[0163] Figure 34 shows the results of membrane ABCA1 stability test of peptides T-087 and T-101 in ARPE-19 cells. ARPE-19 cells were seeded on 12-well plates at 100,000 cells / well and allowed to grow to confluence. In order to prepare monomeric CRP (mCRP), recombinant CRP was prepared in a PBS solution containing 8 M urea and 10 mM EDTA, and incubated at 37°C for 2 hours, and then desalted with Zeba centrifugal columns (7K MW cutoff). mCRP was prepared into a 2X solution of 20 μg / mL. A 2X solution of 40 μM peptides was prepared in serum-free DMEM, and 4 concentrations were diluted in a row at 1:2. mCRP and peptides were added to cells 1:1, continued at 37°C for 24 hours, and then membrane proteins were separated and membrane ABCA1 levels were measured by Western blotting. Quantitative blots were performed in ImageJ. The signal was further normalized to the LXR agonist treatment group to determine the remaining ABCA1 percentage (%). In this assay, treatment with LXR agonists increased membrane ABCA1 levels, but membrane ABCA1 levels were reduced after treatment with mCRP. In the presence of peptides T-087 or T-101 during mCRP incubation, ABCA1 membrane levels were preserved compared to no peptide treatment. The EC50 values ​​for peptides T-087 and T-101 in this assay were 4.6 μM and 3.5 μM, respectively.

[0164] Fig.35 and Fig.36 A membrane ABCA1 stability test using ARPE-19 cells is shown. ARPE-19 cells were seeded at 100,000 cells / well in a 12-well plate and allowed to grow to confluence. To prepare monomeric CRP (mCRP), recombinant CRP was prepared in a PBS solution containing 8 M urea and 10 mM EDTA and incubated at 37°C for 2 hours, then desalted using a Zeba spin column (7K MW cutoff). mCRP was prepared as a 2X solution at 20 μg / mL. A 2X solution of 40 μM peptide was prepared in serum-free DMEM. CRP and peptide were added 1:1 to cells for 24 hours at 37°C, and then membrane proteins were isolated and membrane ABCA1 levels were measured by western blotting. Blots were quantified in ImageJ. Fig.35In , the signal was further normalized to the T-087 treated group to determine the percentage (%) of ABCA1 levels compared to cells treated with peptide T-087. Fig.36 In the figure, the signal was further normalized to the peptide T-101-treated group to determine the percentage (%) of ABCA1 levels compared to peptide T-101-treated cells. Compared to peptide T-087-treated cells, several derivatives of peptide T-087 resulted in decreased membrane ABCA1 levels after mCRP treatment, including peptides T-155, T-156, T-157, T-158, T-159, T-162, T-166, T-167, T-170, T-173, and T-177, indicating that residues 3, 4, 5, 6, 7, 9, 13, 14, and 15 in peptide T-087 contribute to stabilizing the activity of membrane ABCA1 after mCRP treatment. Several derivatives of peptide T-101 also resulted in a decrease in membrane ABCA1 levels after mCRP treatment compared to cells treated with peptide T-101, including peptides T-121, T-124, T-126, T-127, T-128, T-130, T-132, T-134, T-135, T-137, T-138, T-104, T-141, T-145, T-148, T-149, and T-150, indicating that residues at positions 1, 4, 6, 7, 8, 10, 12, 14, 15, 17, 18, 21 and the group of residues between positions 6-19 in T-101 are critical for stabilizing the full activity of ABCA1 after mCRP treatment.

[0165] Fig.37 The correlation of cholesterol efflux and hydrophobic moment for peptides T-152 to T-178 (SEQ ID NO: 152 - 178) is shown. In this example, linear regression was used to determine the slope deviation from zero. The source data in this figure are from Fig.28 , and the calculated hydrophobic moment defined in Eisenberg (Eisenberg et al., 1982. The helical hydrophobic moment: a measure of the amphiphilicity of a helix. Nature). The results of this analysis showed that the hydrophobic moment of the tested peptides was positively correlated with their cholesterol efflux activity.

[0166] Fig.38A The correlation of cholesterol efflux and hydrophobicity of peptides T-121 to T-151 (SEQ ID 121 - 151) is shown. Fig.38B The correlation between ABCA1 membrane stability and the hydrophobic moment of these peptides is shown. Linear regression was used to determine the slope deviation from zero. Source data for these figures are from Fig.29 and Fig.36. Hydrophobicity was defined by Fauchere and Pliska. (Fauchere and Plisk (1983). Hydrophobic Parameters II of Amino-Acid Side Chains from the Partitioning of N-Acetyl-Amino-Acid Amides. Eur J Med Chem). The hydrophobic moment was defined by Eisenberg (Eisenberg et al., 1982. The helical hydrophobic moment: a measure of theamphiphilicity of a helix. Nature). Fig.38A The analysis results shown in show that the hydrophobicity of the tested peptides is positively correlated with their cholesterol efflux activity. Fig.38B The analysis results shown in indicate that membrane ABCA1 stability is positively correlated with its hydrophobic moment.

[0167] Figure 39A-Figure 39E The example of the tolerance test results of representative peptides in mice is shown. T-087, T-101, T-112 and T-114 were prepared to a concentration of 520 μM in sterile water + 0.001% Tween-80 for intravitreal (IVT) delivery to 12-week-old C57BL / 6J mice (n=3 mice per group). 7 days after IVT injection, eyes were harvested and prepared for hematoxylin and eosin staining. Blinded veterinarians performed histopathological analysis on tissue sections to determine whether the peptides induced inflammation or retinal damage. Representative images of each peptide are shown, indicating that all peptides except T-112 are well tolerated.

[0168] Fig.40A and Fig.40B Results of an in vivo study measuring the effects of peptides T-087 and T-101 in reducing ApoE - / - Role of sub-RPE BODIPY+ lipid deposition in mice. Fig.40A Results from a study in which three-month-old ApoE - / - Mouse (B6.129P2- Apoe tm1Unc / J) were given a high cholesterol diet (Research Diets D12079B) for two months. Two weeks before sacrifice, mice were randomized (n=6 mice per group) and received a 1 μL IVT injection of 520 μM peptide T-087 or peptide T-101 or vehicle control. At sacrifice, eyes were enucleated and stained with BODIPY to label neutral lipids and then imaged at 20X magnification. ImageJ was used to quantify the area of ​​BODIPY+ staining under the RPE, and the area of ​​interest (AOI) was then divided by the length of the area to obtain the average width of lipid deposits. Nine images were averaged for each mouse. Data are presented as single data points representing individual mouse values. Differences were tested using one-way ANOVA and Tukey post hoc test. *, different from vehicle (P<0.05). In another study, the results are summarized in Fig.40B ApoE in 3 months - / - Mouse (B6.129P2- Apoe tm1Unc / J) were given a high cholesterol diet (Research Diets D12079B) for two months. Two weeks before sacrifice, mice were randomized (n=4 mice per group) and received a 1 μL IVT injection of 260 μM peptide T-087 or peptide T-101 or vehicle control. At sacrifice, eyes were removed and stained with BODIPY to label neutral lipids, then imaged and quantified as described above. The average of 12 images was taken for each mouse. Data are presented as single data points representing individual mouse values. Differences were tested using one-way analysis of variance and Tukey post hoc test. *, different from vehicle (P<0.05). In Fig.40A and Fig.40B In both, treatment with test peptide T-087 or test peptide T-101 resulted in a statistically significant reduction in lipid deposition under the RPE compared to vehicle-treated mice.

[0169] Therapeutic Peptide Examples Therefore, the therapeutic peptides described herein can solubilize lipids, can induce lipids to flow out of cells, and can accept and transport lipids released from cells. These therapeutic peptides can be engineered mimics of one or more apolipoproteins such as ApoA1, which have structural homology with endogenous ApoA1, so that the structure and function of the therapeutic peptides are essentially amphipathic, and effectively enhance the activity of cholesterol efflux regulatory proteins or transporters (e.g., ABCA1, ABCG1, SR-B1 or related proteins) involved in lipid efflux or lipid processing mechanisms. The therapeutic peptides described herein can interact with one or more molecules involved in lipid transport and processing pathways, such as lipoproteins, lipid efflux transporters, and lipoprotein particle modifiers. For example, in the case where the therapeutic peptide provides a mimetic similar to ApoA1 in structure, the therapeutic peptide can associate with and support HDL function because it involves lipid processing mechanisms and cholesterol efflux pathways. Figure 1B-Figure 1I Many therapeutic peptides listed in show these properties. Typically, the therapeutic peptide can be 80 amino acids or less (e.g., 75 or less amino acids, 70 or less amino acids, 65 or less amino acids, 60 or less amino acids, 55 or less amino acids, 50 or less amino acids, 49 or less amino acids, 48 ​​or less amino acids, 47 or less amino acids, 46 or less amino acids, 45 or less amino acids, 44 or less amino acids, 43 or less amino acids, 42 or less amino acids, 41 or less amino acids, 40 or less amino acids, 35 or less amino acids, 30 or less amino acids, 25 or less amino acids, 22 or less amino acids, 18 or less amino acids, etc.).

[0170] Therefore, the therapeutic peptides described herein can solubilize lipids and can transport captured lipids into cells. Therapeutic peptides can utilize GAG ​​dependence, GAG independence and / or lipid import receptors (e.g., LDLR, SR-B1 and related receptors) to promote lipid import into cells. Therapeutic peptides can have the property of binding to oxidized lipids, which can enable the removal of pro-inflammatory and / or toxic oxidized lipid substances. Therapeutic peptides can have the property of binding to LDLR in a lipid-dependent manner to ensure that the peptide does not bind to LDLR in the absence of lipids, and this binding can inhibit LDLR function and disrupt cholesterol homeostasis. Figure 1B-Figure 1IMany peptides listed in and shown in the sequence listing as SEQ ID NOs: 5-30, 35-80, and 84-178 exhibit these properties. Some therapeutic peptides include a terminal peptide region that is homologous (in some cases identical; in some instances identical except for four or fewer mismatches in 11 amino acids) to the peptide sequence shown in SEQ ID NO: 8 (e.g., HLRKLRKRLLR). The second region can be attached to the terminal region. Alternatively, the D-conformation of SEQ ID NO: 8 can be located at the C-terminus of the peptide. The middle variable region shown is similar to the middle variable region of SEQ ID NO: 16 (which is shown as attached to the terminal region, such as peptide SEQ ID NO: 27). The therapeutic peptide may include this sequence plus an extended peptide region of up to an additional 16 amino acids or less. The therapeutic peptide may have the properties of binding and activating ABCA1, and also maintain the presence of ABCA1 on the cell membrane in a pro-inflammatory environment. Figure 1B-Figure 1I Many of the peptides listed in and shown in the sequence listing as SEQ ID NOs: 5-30, 35-80, and 84-178 exhibit these properties. The therapeutic peptides may also have N- and C-terminal modifications, including acetylation and amidation.

[0171] In some instances, similar peptides can change 5 or less of the 18 amino acids in the middle variable region. For example, at most five of these amino acids can change. Substitution or deletion can be performed. In some instances, these substitutions can be conservative substitutions. In some instances, the same terminal region is included to be coupled to a second middle variable region. The middle variable region can be similar to the sequence of SEQ ID NO: 20. The peptide comprising the terminal region and the middle variable region is shown in SEQ ID NO: 29.

[0172] Composition Any therapeutic peptide described herein can be used as part of a pharmaceutical composition. A pharmaceutical composition comprising a therapeutic peptide described herein can include one or more pharmaceutically acceptable carriers. The pharmaceutical composition can be suitable for any mode of administration, such as intravitreal administration.

[0173] In some instances, the composition comprises a polypeptide of SEQ ID NO: 5-30, 35-80 or 84-178. For example, in some instances, the pharmaceutical composition includes a peptide with a sequence of SEQ ID NO: 27. In some instances, the pharmaceutical composition includes a peptide with a sequence of SEQ ID NO: 25. In some instances, the pharmaceutical composition includes a peptide with a sequence of SEQ ID NO: 29 (or a modified form thereof). In some instances, the pharmaceutical composition includes a peptide with a sequence of SEQ ID NO: 87 (or a modified form thereof). In some instances, the pharmaceutical composition includes a peptide with a sequence of SEQ ID NO: 101 (or a modified form thereof). In some instances, the pharmaceutical composition includes a peptide with a sequence of SEQ ID NO: 8 at the N-terminus (or at the C-terminus of the D configuration) and a total length of less than 80 amino acids.

[0174] In some instances, the composition comprises a polypeptide of SEQ ID NO: 35-80 or 84-120. For example, in some instances, the pharmaceutical composition comprises a peptide having a sequence of SEQ ID NO: 101 (or a modified form thereof). In some instances, the pharmaceutical composition comprises a peptide having a sequence of SEQ ID NO: 87 (or a modified form thereof). In some instances, the pharmaceutical composition comprises a peptide having a sequence of SEQ ID NO: 100 (or a modified form thereof). In some instances, the pharmaceutical composition comprises a peptide having a sequence of SEQ ID NO: 8 at the N-terminus (or at the C-terminus in a D configuration) and a total length of less than 80 amino acids.

[0175] In some instances, a pharmaceutical composition comprising a peptide as described herein (including but not limited to SEQ ID NO: 5-30, 35-80 or 84-178) and a pharmaceutically acceptable carrier is suitable for administration to a human subject. Such carriers are well known in the art (see, for example, Remington's Pharmaceutical Sciences, 15th Edition, pp.1035-1038 and 1570-1580). In some instances, the pharmaceutical composition is suitable for intravitreal injection. In some instances, the pharmaceutical composition is suitable for subretinal delivery. Such pharmaceutically acceptable carriers can be sterile liquids, such as water and oil, including those of petroleum, animal, plant or synthetic origin, such as peanut oil, soybean oil, mineral oil, etc. Saline solutions and aqueous glucose, polyethylene glycol (PEG) and glycerol solutions can also be used as liquid carriers, particularly for injectable solutions. The pharmaceutical composition may further include additional ingredients, such as preservatives, buffers, tension agents, antioxidants and stabilizers, nonionic wetting agents or clarifying agents, viscosity increasing agents, etc. The pharmaceutical compositions described herein can be packaged in single unit dose or multiple dose forms.The compositions are generally formulated as sterile and substantially isotonic solutions.

[0176] In one example, the therapeutic peptides described herein are formulated into pharmaceutical compositions intended for subretinal or intravitreal injection. Such preparations involve the use of pharmaceutically and / or physiologically acceptable vehicles or carriers, particularly vehicles or carriers suitable for application to eyes, for example, by subretinal injection, such as buffered saline or other buffers, for example, HEPES, to maintain pH at appropriate physiological levels, and optionally, other medical agents, medicaments, stabilizers, buffers, carriers, adjuvants, diluents, etc. For injection, the carrier is generally a liquid. Exemplary physiologically acceptable carriers include sterile, pyrogen-free water and sterile, pyrogen-free phosphate buffered saline. In one example, the carrier is an isotonic sodium chloride solution. In another example, the carrier is a balanced salt solution. In one example, a pharmaceutically acceptable carrier includes a surfactant, for example, Tween-80, Tween-20 or perfluorooctane (Perfluoron liquid). If the peptide solution is to be stored for a long time, it can be frozen in the presence of glycerol or Tween-20.

[0177] In some examples of the methods described herein, the above-mentioned pharmaceutical composition is administered to the subject by subretinal injection. In other examples, the pharmaceutical composition is administered by intravitreal injection. Other forms of administration that can be used for the methods described herein include, but are not limited to, direct delivery to the desired organ (e.g., eyes), oral, inhaled, intranasal, intratracheal, intravenous, intramuscular, subcutaneous, intradermal and other parenteral administration routes. If desired, administration routes can be combined. In some examples, the pharmaceutical composition of the present disclosure is administered after administering an initial loading dose of complement system proteins.

[0178] In some instances, the route of administration is selected so that it reduces the risk of retinal detachment in patients (e.g., intravitreal rather than subretinal administration). In some instances, if the vector / composition is to be administered to an elderly person (e.g., at least 60 years old), intravitreal administration is selected. In a specific instance, any vector / pharmaceutical composition disclosed herein is administered intravitreally to a subject. Procedures for intravitreal injections are known in the art (see, e.g., Peyman, GA et al., (2009) Retina 29(7): 875-912 and Fagan, XJ and Al- Qureshi, S. (2013) Clin. Experiment. Ophthalmol. 41(5): 500-7). In short, subjects for intravitreal injections can be prepared for the procedure by pupil dilation, eye sterilization, and administration of anesthetics. Any suitable mydriatic known in the art can be used for pupil dilation. Sufficient pupil dilation can be confirmed before treatment. Sterilization can be achieved by applying an eye sterilization treatment, e.g., an iodide-containing solution, such as povidone iodine (BETADINE®). Similar solutions can also be used to clean the eyelids, eyelashes, and any other nearby tissue (e.g., skin). Any suitable anesthetic of any suitable concentration can be used, such as lidocaine or proparacaine. The anesthetic can be applied by any method known in the art, including but not limited to topical drops, gels or jellies, and subconjunctival application of anesthetics. Before injection, a sterilized eyelid retractor can be used to clear the eyelashes in the area. The injection site can be marked with a syringe. The injection site can be selected based on the patient's lens. For example, in pseudophakic or aphakic patients, the injection site can be 3 nm-3.5 mm from the limbus, and in phakic patients, the injection site can be 3.5 nm-4 mm from the limbus. The patient can look in the opposite direction to the injection site. During injection, the needle can be inserted perpendicular to the sclera and pointed to the center of the eye. The needle can be inserted so that the tip ends in the vitreous body, rather than the subretinal space. Any suitable volume for injection known in the art can be used. After injection, the eye can be treated with a disinfectant such as an antibiotic. The eye can also be rinsed to remove excess disinfectant.

[0179] In addition, in some instances, it is desirable to carry out non-invasive retinal imaging and functional studies to identify the region of specific eye cells to be targeted for treatment. In these instances, clinical diagnostic tests are used to determine the precise location of one or more subretinal injections. These tests may include ophthalmoscopy, RPE function, electroretinography (ERG) (particularly b wave measurement, c wave measurement), visual field examination, topological projection (topographical mapping) of retinal layers and by means of confocal laser scanning ophthalmoscope (cSLO) and optical coherence tomography (OCT) to measure the thickness of its layer, via the topological projection of the cone density of adaptive optics (AO), functional ophthalmology examination, etc. In some instances, one or more injections are carried out in the same eye to target different regions of the bipolar cells retained.

[0180] The composition can be delivered in a volume of about 0.1 μl to about 1 mL, including all quantities within this range, depending on the size of the area to be treated, the route of administration, and the desired effect of the method. In one example, the volume is about 50 μl. In another example, the volume is about 70 μl. In one example, the volume is about 100 μL. In another example, the volume is about 125 μl. In another example, the volume is about 150 μL. In another example, the volume is about 175 μL. In yet another example, the volume is about 200 μL. In another example, the volume is about 250 μL. In another example, the volume is about 300 μL. In another example, the volume is about 450 μL. In another example, the volume is about 500 μL. In another example, the volume is about 600 μL. In another example, the volume is about 750 μL. In another example, the volume is about 850 μL. In another example, the volume is about 1000 μL.

[0181] For example, the dose can be between about 100 ng / eye and about 10 mg / eye (e.g., about 100 ng / eye, about 150 ng / eye, about 200 ng / eye, about 250 ng / eye, about 300 ng / eye, about 400 ng / eye, about 500 ng / eye, about 600 ng / eye, about 700 ng / eye, about 800 ng / eye, about 900 ng / eye, about 1 μg / eye, about 2 μg / eye, about 3 μg / eye, about 5 μg / eye, about 10 μg / eye, about 15 μg / eye, about 20 μg / eye, about 25 μg / eye, about 30 μg / eye, about 35 μg / eye, about 40 μg / eye, about 50 μg / eye, about 60 μg / eye, about 70 μg / eye, about 80 μg / eye, about 90 about 100 μg / eye, about 120 μg / eye, about 150 μg / eye, about 175 μg / eye, about 200 μg / eye, about 250 μg / eye, about 300 μg / eye, about 350 μg / eye, about 400 μg / eye, about 500 μg / eye, about 750 μg / eye, about 1 mg / eye, about 1.5 mg / eye, about 2 mg / eye, about 2.5 mg / eye, about 3 mg / eye, about 3.5 mg / eye, about 4 mg / eye, about 4.5 mg / eye, about 5 mg / eye, or any range within these ranges).

[0182] Other dosages and administration volumes within these ranges may be selected by the attending physician, taking into account the physical condition of the subject (preferably a human) being treated, the age of the subject, the particular ocular condition, and the extent to which the condition (if progressive) has developed. For extraocular delivery, e.g., oral delivery and / or intravenous delivery, the dosage may be increased proportionally according to the retina.

[0183] Treatment / prevention methods Various methods for preventing, treating, preventing progress or improving ocular disorders and retinal changes associated therewith are described herein. Any of these methods may include identifying patients who may benefit from one or more of these therapies and / or identifying which one or more of the therapies described herein may be most beneficial to a particular patient. Determination as described herein may be based on the scoring or characterization of one or more biomarkers as described herein to produce patients, which may be used to identify patients who may benefit from one or more of these therapies and / or which therapies should be applied. Any of these methods may include determining the dosage to be delivered, the route of delivery, and / or the timetable for delivering one or more dosages. The scoring and / or characterization based on one or more biomarkers described herein may be used to determine the dosage to be delivered, the route of delivery, and / or the timetable for delivering one or more dosages.

[0184] Typically, the method includes administering an effective amount of any composition described herein to a mammalian subject in need. For example, the treatment of age-related macular degeneration can include local delivery of therapeutic compositions described herein to the patient's retina. Cells used as therapeutic targets in these diseases can include cells of the RPE below the photoreceptor cells or the choroidal capillaris layer or Bruch's membrane in the retina. In some aspects, the disclosure provides a method for treating a subject suffering from age-related macular degeneration (AMD), including administering to the subject a step of any composition described herein.

[0185] In a particular example, a method for preventing, preventing, or improving the progress of vision loss associated with an eye disorder in a subject is provided. Vision loss associated with an eye disorder refers to any reduction in peripheral vision, central (reading) vision, night vision, day vision, color vision loss, contrast sensitivity loss, or reduced vision. The methods and compositions described herein may be directed to increasing photoreceptor function. As used herein, "increasing photoreceptor function" means improving the function of photoreceptors or increasing the number or percentage of functional photoreceptors compared to the same eye (suffering from the same eye disease), the same eye at an earlier time point, the untreated portion of the same eye, or the contralateral eye of the same patient. Photoreceptor function can be assessed using functional studies routine in the art (e.g., ERG or visual field examination).

[0186] For each of the methods, treatment can be used to prevent the occurrence of retinal damage or save eyes with mild or advanced disease. As used herein, the term "rescue" means preventing the disease from progressing to complete blindness, preventing damage from spreading to undamaged eye cells, improving damage in damaged eye cells, or providing enhanced vision. In one example, the composition is applied before the disease becomes symptomatic or before photoreceptor loss. "Symptomatic" means the onset of any of the above-mentioned various retinal changes or loss of vision. In another example, the composition is applied after the disease becomes symptomatic. In another example, the composition is applied after the onset of photoreceptor loss. In another example, the composition is applied after the outer nuclear layer (ONL) degeneration begins. In some examples, it is desired to apply the composition when the bipolar cell circuit to the ganglion cells and the optic nerve remains intact. In another example, the composition is applied after the onset of photoreceptor loss. In another example, when less than 90% of the photoreceptors are functional or remaining compared to unaffected eyes, the composition is applied. In another example, the composition is applied when less than 80% of the photoreceptors are functional or remaining. In another example, the composition is applied when less than 70% of the photoreceptors are functioning or remaining. In another example, the composition is applied when less than 60% of the photoreceptors are functioning or remaining. In another example, the composition is applied when less than 50% of the photoreceptors are functioning or remaining. In another example, the composition is applied when less than 40% of the photoreceptors are functioning or remaining. In another example, the composition is applied when less than 30% of the photoreceptors are functioning or remaining. In another example, the composition is applied when less than 20% of the photoreceptors are functioning or remaining. In another example, the composition is applied when less than 10% of the photoreceptors are functioning or remaining. In one example, the composition is applied only to one or more areas of the eye. In another example, the composition is applied to the entire eye. In another example, the method includes performing functional and imaging studies to determine the efficacy of the treatment. These studies include ERG and in vivo retinal imaging, as described in the following examples. In addition, visual field studies, visual field examinations and microperimetry, pupil measurements, activity tests, visual acuity, contrast sensitivity, and color vision tests can be performed.

[0187] In yet another example, any of the methods described herein can be performed in combination with another or secondary therapy. The therapy can be any currently known or yet unknown therapy that helps prevent, arrest or ameliorate any of the described retinal changes and / or vision loss.

[0188] In some instances, the methods of treatment described herein may include patient identification based on one or more diagnostic techniques. In particular, the identification of one or more genetic aberrations associated with the dysfunction of one or more components of lipid transport and processing mechanisms may support the selection of one or more therapeutic peptides described herein. In a particular instance, the abnormality of the genotype of one or more components of lipid processing mechanisms may result in the observed imbalance of lipid processing associated with AMD pathogenesis. Based on the abnormality identified, one or more therapeutic peptides may be administered to support lipid processing mechanisms or cholesterol homeostasis.

[0189] Any method described herein (including user interfaces) may be implemented as software, hardware, or firmware, and may be described as a non-transitory computer-readable storage medium storing a set of instructions executable by a processor (e.g., a computer, tablet, smartphone, etc.), which, when executed by the processor, causes the processor to control the execution of any steps, including but not limited to: display, communication with a user, analysis, modification of parameters (including timing, frequency, intensity, etc.), determination, alarm, and the like.

[0190] When a feature or element is referred to as "on another feature or element" herein, it can be directly on the other feature or element, or there may also be intermediate features and / or elements. On the contrary, when a feature or element is referred to as "directly on another feature or element", there are no intermediate features or elements. It will also be understood that when a feature or element is referred to as "connected", "attached" or "coupled" to another feature or element, it can be directly connected, attached or coupled to other features or elements, or there may be intermediate features or elements. On the contrary, when a feature or element is referred to as "directly connected", "directly attached" or "directly coupled" to another feature or element, there are no intermediate features or elements. Although described or shown with respect to an example, the features and elements described or shown in this way can be applied to other examples. It will also be understood by those skilled in the art that the structure or feature set with reference to "adjacent" another feature can have a part overlapping with an adjacent feature or below an adjacent feature.

[0191] The terms used herein are only for the purpose of describing specific examples and are not intended to limit the present invention. For example, unless the context clearly indicates otherwise, as used herein, the singular forms "a", "an", and "the" are intended to include the plural forms as well. It will also be understood that the terms "comprises" and / or "comprising" when used in this specification specify the presence of stated features, steps, operations, elements, and / or components, but do not exclude the presence or addition of one or more other features, steps, operations, elements, components, and / or groups thereof. As used herein, the term "and / or" includes any and all combinations of one or more of the associated listed items and may be abbreviated as " / ".

[0192] Spatially related terms, such as "under", "below", "lower", "over", "upper", etc., may be used herein to facilitate description of the relationship of one element or feature to another element or feature as illustrated in the accompanying drawings. It will be understood that spatially related terms are intended to include different orientations of the device in use or operation in addition to the orientation depicted in the accompanying drawings. For example, if the device in the accompanying drawings is inverted, the elements described as "under" or "beneath" other elements or features will then be oriented "over" other elements or features. Thus, the exemplary term "under" may include both orientations of "over" and "under". The device may be oriented in other ways (rotated 90 degrees or in other orientations), and the spatially related descriptors used herein are interpreted accordingly. Similarly, unless otherwise specifically indicated, the terms "upwardly", "downwardly", "vertical", "horizontal", etc. are used herein for explanation purposes.

[0193] Although the terms "first" and "second" may be used herein to describe multiple features / elements (including steps), these features / elements should not be limited by these terms unless the context otherwise indicates. These terms may be used to distinguish one feature / element from another feature / element. Therefore, without departing from the teachings of the present invention, the first feature / element discussed below may be referred to as the second feature / element, and similarly, the second feature / element discussed below may be referred to as the first feature / element.

[0194] Throughout this specification and the appended claims, unless the context requires otherwise, the word "comprise" and variations such as "comprises" and "comprising" mean that various components can be used together in methods and articles (e.g., compositions and devices including apparatuses and methods). For example, the term "comprising" will be understood to imply the inclusion of any stated elements or steps but not the exclusion of any other elements or steps.

[0195] In general, any apparatus and method described herein should be understood to be inclusive, but all or a subset of components and / or steps may alternatively be exclusive and may be expressed as "consisting of" or alternatively "consisting essentially of" a variety of components, steps, sub-components or sub-steps.

[0196] As used herein in the specification and claims, including those used in the examples, and unless otherwise expressly stated, all numbers may be understood to be prefixed with the word "about" or "approximately", even if the term does not appear explicitly. The wording "about" or "approximately" may be used when describing amplitude and / or position to indicate that the value and / or position described are within the reasonable expected range of value and / or position. For example, a numerical value may have a value of + / -0.1% of the stated value (or range of values), + / -1% of the stated value (or range of values), + / -2% of the stated value (or range of values), + / -5% of the stated value (or range of values), + / -10% of the stated value (or range of values), etc. Any numerical value provided herein is also understood to include about or approximately that value, unless otherwise indicated by the context. For example, if the value "10" is disclosed, then "about 10" is also disclosed. Any numerical range listed herein is intended to include all subranges contained therein. It should also be understood that when a value is disclosed, "less than or equal to" the value, "greater than or equal to the value," and possible ranges between values ​​are also disclosed, as appropriately understood by the skilled artisan. For example, if a value "X" is disclosed, then "less than or equal to X" and "greater than or equal to X" (e.g., where X is a numerical value) are also disclosed. It should also be understood that throughout the application, data is provided in a variety of different formats, and that the data represents endpoints and starting points and ranges for any combination of data points. For example, if a specific data point "10" and a specific data point "15" are disclosed, it should be understood that greater than, greater than or equal to, less than, less than or equal to, and equal to 10 and 15, and between 10 and 15 are considered to be disclosed. It should also be understood that every unit between two specific units is also disclosed. For example, if 10 and 15 are disclosed, then 11, 12, 13, and 14 are also disclosed.

[0197] Although various illustrative examples are described above, any of many changes may be made to the various examples without departing from the scope of the invention as described in the claims. For example, in alternative examples, the order in which the various described method steps are performed may generally be changed, and in other alternative examples, one or more method steps may be skipped together. Optional features of the various device and system examples may be included in some examples and not included in other examples. Therefore, the previous description is provided primarily for exemplary purposes and should not be construed as limiting the scope of the invention as set forth in the claims.

[0198] The examples and descriptions included herein show specific examples in which the subject matter can be practiced by way of illustration and not limitation. As mentioned, other examples can be utilized and derived therefrom so that structural and logical replacements and changes can be made without departing from the scope of the present disclosure. For convenience only, such examples of the subject matter of the present invention may be referred to herein individually or collectively by the term "invention", and it is not intended that the scope of the present application is actively limited to any single invention or inventive concept, if in fact more than one is disclosed. Therefore, although specific examples have been illustrated and described herein, any arrangement planned to achieve the same purpose can replace the specific examples shown. The present disclosure is intended to cover any and all modifications or variations of a variety of examples. After reading the above description, the combination of the above examples and other examples not specifically described herein will be apparent to those skilled in the art.

Claims

1. A polypeptide for treating age-related macular degeneration (AMD), having a peptide sequence that is 65% or more homologous to SEQ ID NO: 87, wherein the polypeptide comprises a helical coil having a hydrophobic moment μH of 0.65 or greater.

2. A polypeptide for treating age-related macular degeneration (AMD), having a peptide sequence with 65% or higher homology to SEQ ID NO: 87, wherein the polypeptide has ATP-binding cassette transporter membrane stabilizing and agonist activity.

3. A polypeptide for treating age-related macular degeneration (AMD), having a sequence at least 65% homologous to SEQ ID NO: 87, wherein the polypeptide has transporter binding activity.

4. A polypeptide for treating age-related macular degeneration (AMD), having a peptide sequence of SEQ ID NO:

87.

5. The polypeptide according to any one of claims 1 to 4, wherein the polypeptide has a hydrophobic moment μH of 0.65 or greater, resulting in a cholesterol efflux of 17.5% or greater.

6. The polypeptide according to any one of claims 1 to 4, wherein the polypeptide has ATP-binding cassette transporter binding activity.

7. A polypeptide according to any one of claims 1 to 3, wherein any peptide residue that differs from the sequence of SEQ ID NO: 87 in positions 2-7, 9 and 10-17 is a conservative substitution or a conservative hydrophobic substitution having a hydrophobicity value within 0.25 of the hydrophobicity value of the peptide residue in the corresponding position of SEQ ID NO: 87, as calculated using the method of Fauchere and Pliska, and wherein any different peptide residue in positions 1, 8, 10 and 18 is any amino acid.

8. The polypeptide of any one of claims 1-3, wherein the polypeptide sequence is one of SEQ ID NO: 87, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177 or 178.

9. The polypeptide of any one of claims 1-3, wherein the polypeptide sequence is one of SEQ ID NO: 87, 152, 160, 161, 163 or 172.

10. The polypeptide of any one of claims 1-3, wherein the polypeptide sequence is 70% or more homologous to SEQ ID NO:

87.

11. The polypeptide of any one of claims 1-3, wherein the polypeptide sequence is 80% or more homologous to SEQ ID NO:

87.

12. The polypeptide of any one of claims 1-3, wherein the polypeptide sequence is 85% or more homologous to SEQ ID NO:

87.

13. The polypeptide according to any one of claims 1-3, wherein the polypeptide sequence is SEQ ID NO: 35 or 36, wherein X can be any amino acid.

14. A pharmaceutical composition for preventing or treating age-related macular degeneration (AMD) in a patient, wherein the composition comprises the polypeptide as defined in any one of claims 1 to 14 and a pharmaceutically acceptable excipient.

15. The pharmaceutical composition according to claim 14, wherein the composition is for administration by intraocular injection.

16. The pharmaceutical composition of claim 14, wherein the composition is for administration by intravascular (IV) injection.

17. The pharmaceutical composition according to claim 14, wherein the composition is for administration by subcutaneous (SC) injection.

18. The pharmaceutical composition according to claim 14, further comprising two or more of the polypeptides according to any one of claims 1-13.

19. 1 kind of method using the polypeptide according to any one of claim 14-18 or pharmaceutical composition treatment or prevention patient's age-related macular degeneration (AMD), wherein said prevention or treatment is to prevent said AMD, and wherein said patient is diagnosed as having the tendency of development AMD.

20. A method of treating or preventing age-related macular degeneration (AMD) in a patient using a polypeptide or pharmaceutical composition according to any one of claims 14-18, wherein the prevention or treatment is treatment and the patient exhibits signs or symptoms of AMD.

21. A method of treating or preventing age-related macular degeneration (AMD) in a patient using an engineered polypeptide or pharmaceutical composition according to any one of claims 14-18, wherein the prevention or treatment is treatment of early AMD.

22. A method of treating age-related macular degeneration (AMD) in a patient, the method comprising delivering a polypeptide or composition according to any one of claims 14-18 into the eye of the patient.

23. The method of any one of claims 19-22, wherein delivering comprises intraocular injection.

24. The method of any one of claims 19-22, wherein delivering comprises intravascular (IV) injection.

25. The method of any one of claims 19-22, wherein delivering comprises subcutaneous (SC) injection.

26. The method of any one of claims 19-22, wherein delivering comprises delivering more than one polypeptide or composition of any one of claims 1-13.

27. The method of any one of claims 19-22, wherein the patient is 40 years of age or older.

28. A polypeptide for treating age-related macular degeneration (AMD), having a peptide sequence that is 65% or more homologous to SEQ ID NO: 101, wherein the polypeptide comprises a helical coil with a hydrophobic moment μH of 0.6 or greater.

29. A polypeptide for treating age-related macular degeneration (AMD), having a peptide sequence having 65% or higher homology to SEQ ID NO: 101, wherein the polypeptide has ATP-binding cassette transporter membrane stabilizing and agonist activity.

30. A polypeptide for treating age-related macular degeneration (AMD), having a sequence at least 65% homologous to SEQ ID NO: 101, wherein the polypeptide has transporter binding activity.

31. A polypeptide for treating age-related macular degeneration (AMD), comprising the peptide sequence of SEQ ID NO:

101.

32. The polypeptide of any one of claims 28-31, wherein the polypeptide has an overall hydrophobicity of 0.198 or greater, resulting in enhanced cholesterol efflux.

33. The polypeptide of any one of claims 28-30, wherein the polypeptide has a hydrophobic moment μH of 0.6 or greater, resulting in an ABCA1 stability greater than forty percent of the ABCA1 stability of the polypeptide of SEQ ID NO:

101.

34. The polypeptide according to any one of claims 28-31, wherein the polypeptide has ATP-binding cassette transporter binding activity.

35. A polypeptide according to any one of claims 28 to 30, wherein any peptide residue that differs from the sequence of SEQ ID NO: 101 at positions 1, 4-8, 10-15, 17-18 or 20-22 is a conservative substitution or a conservative hydrophobic substitution having a hydrophobicity value within 0.25 of the hydrophobicity value of the peptide residue in the corresponding position of SEQ ID NO: 101, calculated using the method of Fauchere and Pliska, and wherein any of the different peptide residues in positions 2, 3, 9, 16 and 19 are any amino acids.

36. A polypeptide according to any one of claims 28-30, wherein the polypeptide sequence is one of SEQ ID NO: 101, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150 or 151.

37. A polypeptide according to any one of claims 28-30, wherein the polypeptide sequence is one of SEQ ID NO: 101, 122, 123, 129, 136 or 139.

38. A polypeptide according to any one of claims 28-30, wherein the polypeptide sequence is 70% or more homologous to SEQ ID NO:

101.

39. A polypeptide according to any one of claims 28-30, wherein the polypeptide sequence is 80% or more homologous to SEQ ID NO:

101.

40. The polypeptide of any one of claims 28-30, wherein the polypeptide sequence is 85% or more homologous to SEQ ID NO:

101.

41. The polypeptide of any one of claims 28-30, wherein the polypeptide sequence is SEQ ID NO: 37 or 39, wherein X can be any amino acid.

42. A pharmaceutical composition for preventing or treating age-related macular degeneration (AMD) in a patient, wherein the composition comprises the polypeptide as defined in any one of claims 28-41 and a pharmaceutically acceptable excipient.

43. The pharmaceutical composition of claim 42, wherein the composition is for administration by intraocular injection.

44. The pharmaceutical composition of claim 42, wherein the composition is for administration by intravascular (IV) injection.

45. The pharmaceutical composition of claim 42, wherein the composition is for administration by subcutaneous (SC) injection.

46. ​​The pharmaceutical composition of claim 42, further comprising two or more of the polypeptides of any one of claims 28-41.

47. 1 kind of method using the polypeptide according to any one in claim 42-46 or pharmaceutical composition treatment or prevention patient's age-related macular degeneration (AMD), wherein said prevention or treatment are to prevent said AMD, and wherein said patient is diagnosed as having the tendency of development AMD.

48. A method of treating or preventing age-related macular degeneration (AMD) in a patient using a polypeptide or pharmaceutical composition according to any one of claims 42-46, wherein the prevention or treatment is treatment and the patient exhibits signs or symptoms of AMD.

49. A method of treating or preventing age-related macular degeneration (AMD) in a patient using an engineered polypeptide or pharmaceutical composition according to any one of claims 42-46, wherein the prevention or treatment is treatment of early AMD.

50. A method of treating age-related macular degeneration (AMD) in a patient, the method comprising delivering a polypeptide or composition according to any one of claims 42-46 into an eye of the patient.

51. The method of any one of claims 47-50, wherein delivering comprises intraocular injection.

52. The method of any one of claims 47-50, wherein delivering comprises intravascular (IV) injection.

53. The method of any one of claims 47-50, wherein delivering comprises subcutaneous (SC) injection.

54. The method of any one of claims 47-50, wherein delivering comprises delivering more than one polypeptide or composition according to any one of claims 28-41.

55. The method of any one of claims 28-41, wherein the patient is 40 years of age or older.

56. A polypeptide for treating age-related macular degeneration (AMD), having a peptide sequence that is 65% or more homologous to SEQ ID NO: 114, wherein the polypeptide has enhanced LDLR binding activity in the presence of lipid and cholesterol import activity in cells.

57. A polypeptide for treating age-related macular degeneration (AMD) having a sequence at least 65% homologous to SEQ ID NO: 114, wherein the polypeptide has transporter binding activity.

58. A polypeptide for treating age-related macular degeneration (AMD), comprising the peptide sequence of SEQ ID NO:

114.

59. The polypeptide according to any one of claims 56-58, wherein the polypeptide has ATP-binding cassette transporter binding activity.

60. A polypeptide according to any one of claims 56-57, wherein the polypeptide sequence is 70% or more homologous to SEQ ID NO:

114.

61. A polypeptide according to any one of claims 56-57, wherein the polypeptide sequence is 80% or more homologous to SEQ ID NO:

114.

62. A polypeptide according to any one of claims 56-57, wherein the polypeptide sequence is 85% or more homologous to SEQ ID NO:

114.

63. A pharmaceutical composition for preventing or treating age-related macular degeneration (AMD) in a patient, wherein the composition comprises the polypeptide as defined in any one of claims 56-62 and a pharmaceutically acceptable excipient.

64. The pharmaceutical composition of claim 63, wherein the composition is for administration by intraocular injection.

65. The pharmaceutical composition of claim 63, wherein the composition is for administration by intravascular (IV) injection.

66. The pharmaceutical composition of claim 63, wherein the composition is for administration by subcutaneous (SC) injection.

67. The pharmaceutical composition of claim 63, further comprising two or more of the polypeptides of any one of claims 56-62.

68. 1 kind of method using the polypeptide according to any one of claim 63-67 or pharmaceutical composition treatment or prevention patient's age-related macular degeneration (AMD), wherein said prevention or treatment is to prevent said AMD, and wherein said patient is diagnosed as having the tendency of development AMD.

69. A method of treating or preventing age-related macular degeneration (AMD) in a patient using a polypeptide or pharmaceutical composition according to any one of claims 63-67, wherein the prevention or treatment is treatment and the patient exhibits signs or symptoms of AMD.

70. A method of treating or preventing age-related macular degeneration (AMD) in a patient using an engineered polypeptide or pharmaceutical composition according to any one of claims 63-67, wherein the prevention or treatment is treatment of early AMD.

71. A method of treating age-related macular degeneration (AMD) in a patient, the method comprising delivering a polypeptide or composition according to any one of claims 63-67 into the eye of the patient.

72. The method of any one of claims 68-71, wherein delivering comprises intraocular injection.

73. The method of any one of claims 68-71, wherein delivery comprises intravascular (IV) injection.

74. The method of any one of claims 68-71, wherein delivering comprises subcutaneous (SC) injection.

75. The method of any one of claims 68-71, wherein delivering comprises delivering more than one of the polypeptides.

76. The method of any one of claims 68-71, wherein the patient is 40 years of age or older.

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