Compositions and methods for preventing retinal neurodegeneration

Abstract

In accordance with the present disclosure, there is provided a method for preventing or reversing photoreceptor outer segment destruction, RPE lipid deposition, neurodegeneration, angiogenesis, or any combination thereof, in a patient having macular degenerative disease, comprising administering to the patient a therapeutically effective amount of an ApoM-containing composition.

Description

[Technical field]

[0001] (CROSS REFERENCE TO RELATED APPLICATIONS) This application claims priority to U.S. Provisional Patent Application No. 63 / 226,756, filed July 28, 2021, the entire disclosure of which is incorporated herein by reference.

[0002] (STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT) This invention was made with United States Government support under Grant No. EY019287 awarded by the National Institutes of Health. The United States Government has certain rights in this invention.

[0003] (Technical field) The present disclosure relates generally to compositions and methods for preventing retinal neurodegeneration. [Background technology]

[0004] Age-related macular degeneration (AMD) is the leading cause of blindness in people over the age of 50 in developed countries. Early AMD is characterized by the accumulation of lipid-rich drusen under the retina. AMD can progress to an advanced form characterized by atrophy and neovascularization. Signs of mild or severe neurodegeneration can appear at any stage. Summary of the Invention [Means for solving the problem]

[0005] The present disclosure relates generally to compositions and methods for preventing retinal neurodegeneration.

[0006] In one aspect, the present disclosure provides a method for preventing or reversing macular degenerative disease in a patient having macular degenerative disease, comprising administering to the patient a therapeutically effective amount of an ApoM-containing composition. In some aspects, the macular degenerative disease is selected from the group consisting of age-related macular degeneration (AMD), early-onset macular degeneration, and diabetic retinopathy. In some aspects, the therapeutically effective amount of the ApoM-containing composition prevents or reverses photoreceptor outer segment destruction, RPE lipid deposition, neurodegeneration, angiogenesis, or any combination thereof in the patient.

[0007] Other objects and features will be in part apparent and in part pointed out hereinafter. [Brief description of the drawings]

[0008] The following drawings illustrate various aspects of the present disclosure.

[0009] [Figure 1A] FIG. 1A is a schematic diagram showing the structure of cone and rod photoreceptors. [Figure 1B] FIG. 1B is a schematic diagram showing lipid and cell membrane metabolism. [Diagram 2] FIG. 2 is a schematic showing that lipids, including high density lipoprotein (HDL), are shuttled across the cell membrane by the chaperone ApoM, thereby resulting in further cellular effects such as cell migration, survival, fate, and gene expression that regulate the immune, cardiovascular, and central nervous systems and may lead to organ fibrosis. [Diagram 3] FIG. 3 is a schematic diagram showing the mouse ApoM knockout (KO) experimental design. [Figure 4A] FIG. 4A is a graph of scotopic a-wave amplitude versus intensity in ApoM Tg+ and ApoM KO mice. [Figure 4B] FIG. 4B is a graph of scotopic b-wave amplitude versus intensity in ApoM Tg+ and ApoM KO mice. [Figure 4C]FIG. 4C is a graph of photopic b-wave amplitude versus intensity in ApoM Tg+ and ApoM KO mice. [Figure 5A] FIG. 5A is a transmission electron micrograph of the retinal pigment epithelium of an ApoM KO mouse. [Figure 5B] FIG. 5B is a transmission electron micrograph of the retinal pigment epithelium of an ApoM Tg+ mouse. [Figure 5C] FIG. 5C is a graph quantifying the number of lipid droplets found in the retinal pigment epithelium in the images of FIGS. 5A and 5B. [Figure 6A] FIG. 6A is a transmission electron microscope image of photoreceptors from an ApoM KO mouse. [Figure 6B] FIG. 6B is a transmission electron microscope image of photoreceptors from an ApoM Tg+ mouse. [Figure 7A] FIG. 7A is another transmission electron microscopy image of photoreceptors from an ApoM KO mouse. [Figure 7B] FIG. 7B is another transmission electron microscopy image of photoreceptors from an ApoM Tg+ mouse. [Figure 8] FIG. 8 is a schematic diagram showing the structures of the eye, including the outer plexiform layer, photoreceptors, pigment epithelium, branch membrane, and choroid. [Figure 9] FIG. 9 is a schematic diagram showing the generation of transgenic S1P1R-RPE / -RPE knockout mice and their use in subsequent experiments and analyses. [Figure 10] FIG. 10 is a schematic diagram showing the use of germline ApoM knockout mice in a series of experiments. [Figure 11A] FIG. 11A is a graph of scotopic a-wave amplitude versus intensity in ApoM control and ApoM mutant mice. [Figure 11B] FIG. 11B is a graph of scotopic b-wave amplitude versus intensity in ApoM control and ApoM mutant mice. [Figure 11C] FIG. 11C is a graph of photopic b-wave amplitude versus intensity in ApoM control and ApoM mutant mice. [Figure 12A]FIG. 12A is a transmission electron microscopy image of the retinal pigment epithelium of an ApoM mutant mouse. [Figure 12B] FIG. 12B is a transmission electron micrograph of the retinal pigment epithelium of an ApoM control mouse. [Figure 13] FIG. 13 is a graph quantifying the number of lipid droplets seen in transmission electron microscopy images of control retinal pigment epithelium, ApoM KO, and ApoM mutant mice. [Figure 14] Figure 14(A) shows a fluorescence contrast image of choroidal neovascularization in a wild-type mouse, Figure 14(B) shows a fluorescence contrast image of choroidal neovascularization in an ApoM heterozygous mouse, and Figure 14(C) shows a fluorescence contrast image of choroidal neovascularization in an ApoM KO mouse. [Figure 15] FIG. 15 is a graph quantifying the size of choroidal neovascular lesions seen in fluorescence contrast images of wild-type mice, ApoM KO mice, and ApoM heterozygous mice. [Figure 16] FIG. 16 is a graph quantifying the ApoM / cholesterol concentration ratio in non-AMD control subjects and AMD patients.

[0010] Those skilled in the art will appreciate that the drawings described below are for illustrative purposes only and are not intended to limit the scope of the present teachings in any way. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0011] The present disclosure is based on the discovery that exogenous administration of apolipoprotein M (ApoM)-rich plasma can reduce both lipid deposition and neurodegeneration in animal models of early age-related macular degeneration (AMD). This effect is ApoM-specific and is not achieved with ApoM-deficient plasma. Anatomically, exogenous administration of ApoM was shown to prevent photoreceptor outer segment destruction and RPE lipid deposition by electron microscopy, and reversed neurodegeneration by visual electrophysiological measurements.

[0012] Exogenous ApoM has previously been found to inhibit ocular neovascularization, but no reversal of lipid deposition or neurodegeneration has been demonstrated. As described in the Examples herein, the compositions and methods of the present disclosure provide treatment for early stage AMD, as well as other diseases associated with retinal neurodegeneration for which no established treatment exists.

[0013] In various aspects, compositions and treatment methods are disclosed for preventing or reversing photoreceptor outer segment destruction, RPE lipid deposition, and / or neurodegeneration associated with various diseases, including, but not limited to, age-related macular degeneration (AMD), early-onset macular degeneration, and diabetic retinopathy. In various aspects, the compositions of the present disclosure comprise exogenously administered apolipoprotein M (ApoM). In some aspects, the compositions of the present disclosure comprise exogenously administered apolipoprotein M (ApoM)-rich plasma.

[0014] As described in the Examples herein, exogenous administration of apolipoprotein M (ApoM)-rich plasma can reduce both lipid deposition and neurodegeneration in animal models of early age-related macular degeneration (AMD). This effect is ApoM-specific, and ApoM-deficient plasma does not produce this effect. Anatomically, exogenous administration of ApoM has been shown to prevent photoreceptor outer segment destruction and RPE lipid deposition by electron microscopy, and reverses neurodegeneration by visual electrophysiological measurements.

[0015] As described in the Examples herein, exogenous ApoM can prevent retinal neurodegeneration and vision loss seen in diseases such as macular degeneration (premature and age-related). Early stage diabetes disease is characterized by neurodegeneration, which can also be prevented using the compositions and methods of the present disclosure.

[0016] Without being bound to any particular theory, the unique structure and function of photoreceptors requires the continuous maintenance of their lamellar profile. For example, photoreceptors need to circadianly shed a portion of their lipid-rich outer segments to maintain proper function. This requires tight control of lipid and cell membrane metabolism. If this metabolism is inhibited, photoreceptors are adversely affected. A pair of cholesterol efflux transporters, ABCA1 and ABCG1, has been shown to be instrumental in maintaining proper photoreceptor function, as evidenced by the phenotype of ABCA1 / ABCG1 knockout mice resembling early age-related macular degeneration (AMD). Patients with AMD were observed to have lower ApoM concentrations in plasma compared to healthy controls (Figure 16).

[0017] In various aspects, treatment of ABCA1 / ABCG1 knockout models with exogenous ApoM has shown therapeutic effects on retinal neurodegeneration. Without being bound to a particular theory, ApoM is the central chaperone of S1P, allowing activation of S1PR in addition to shuttling HDL. S1PR is upstream of many pathways that control important cell survival pathways. Five different isoforms of S1PR have been identified to date, and the different isoforms are known to show different expression in different tissues and have different functions. S1P chaperoned by ApoM interacts with five different S1P receptors, numbered 1 to 5.

[0018] Adjacent to the photoreceptor outer segments is a unique epithelial layer called the retinal pigment epithelium (RPE), which is responsible for the exchange of nutrients and waste products from the systemic circulation with photoreceptor debris and the choroid. Because the RPE maintains close contact with the systemic circulation and is crucial for photoreceptor metabolism, S1P receptors are thought to play an essential role in maintaining the function of the RPE and photoreceptors.

[0019] Molecular Engineering

[0020] The following definitions and methods are provided to better define the present disclosure and to guide those of ordinary skill in the art in the practice of the present disclosure. Unless otherwise specified, terms are to be understood according to conventional usage by those of ordinary skill in the relevant art.

[0021] As used herein, the terms "heterologous DNA sequence," "exogenous DNA fragment," or "heterologous nucleic acid" refer to a sequence that is derived from a source different from a particular host cell, or, if derived from the same source as a particular host cell, is modified from its original form. Thus, a heterologous gene in a host cell includes a gene that is endogenous to a particular host cell, but that has been modified, for example, using DNA shuffling or cloning. These terms also include non-naturally occurring multiple copies of a naturally occurring DNA sequence. Thus, these terms refer to a DNA fragment that is foreign or heterologous to the cell, or that is homologous to the cell, but in a position not normally found within the nucleic acid of the host cell. The exogenous DNA fragment is expressed to produce an exogenous polypeptide. A "homologous" DNA sequence is a DNA sequence that is naturally associated with the host cell into which it is introduced.

[0022] Expression vector, expression construct, plasmid, or recombinant DNA construct is generally understood to refer to a nucleic acid produced by human intervention, including recombinant means or direct chemical synthesis, using a set of specific nucleic acid elements that allows for the transcription or translation of a specific nucleic acid in a host cell.An expression vector can be part of a plasmid, a virus, or a nucleic acid fragment.In general, an expression vector can include the nucleic acid to be transcribed, operably linked to a promoter.

[0023] A "promoter" is generally understood as a nucleic acid control sequence that directs the transcription of a nucleic acid. An inducible promoter is generally understood as a promoter that mediates the transcription of an operably linked gene in response to a specific stimulus. A promoter can include necessary nucleic acid sequences near the transcription start site, such as a TATA element in the case of a polymerase II type promoter. A promoter can optionally include distal enhancer or repressor elements, which can be located several thousand base pairs away from the transcription start site.

[0024] As used herein, "transcribeable nucleic acid molecule" refers to a nucleic acid molecule that can be transcribed into an RNA molecule. Methods are known for introducing constructs into cells so that the transcribable nucleic acid molecule is transcribed into a functional mRNA molecule (i.e., translated and expressed as a protein product). Constructs may also be constructed to express antisense RNA molecules to inhibit the translation of a particular RNA molecule of interest. Conventional compositions and methods for making and using the constructs and host cells to practice the present disclosure are well known to those of skill in the art (see Sambrook and Russel (2006) Condensed Protocols from Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, ISBN-10: 0879697717; Ausubel et al. (2002) Short Protocols in Molecular Biology, 5th ed., Current Protocols, ISBN-10: 0471250929; Sambrook and Russel (2001) Molecular Cloning: A Laboratory Manual, 3d ed., Cold Spring Harbor Laboratory Press, ISBN-10: 0879695773; Elhai, J. and Wolk, CP 1988. Methods in Enzymology 167, 747-754).

[0025] "Transcription start site" or "start site" refers to the position surrounding the first nucleotide that is part of the transcribed sequence, also defined as position +1. All other sequences of the gene and its regulatory regions can be numbered relative to this site. Downstream sequences (i.e., further protein coding sequences in the 3' direction) can be positive and upstream sequences (the majority of the regulatory region in the 5' direction) can be negative.

[0026] "Operably linked" or "functionally linked" preferably refers to the association of nucleic acid sequences on a single nucleic acid fragment such that the function of one is affected by the function of the other. For example, a regulatory DNA sequence is said to be "operably linked" or "associated" with a DNA sequence that codes for an RNA or polypeptide when the regulatory DNA sequence is positioned to affect the expression of the coding DNA sequence (i.e., when the coding sequence or functional RNA is under the transcriptional control of the promoter). The coding sequence can be operably linked to the regulatory sequence in a sense or antisense orientation. The two nucleic acid molecules can be part of a single contiguous nucleic acid molecule or can be adjacent. For example, a promoter is operably linked to a gene of interest if the promoter regulates or mediates the transcription of the gene of interest in a cell.

[0027] A "construct" is generally understood to be a recombinant nucleic acid molecule, such as a plasmid, cosmid, virus, autonomously replicating nucleic acid molecule, phage, or linear or circular single- or double-stranded DNA or RNA nucleic acid molecule, derived from any source, capable of genomic integration or autonomous replication, and consisting of one or more operably linked nucleic acid molecules.

[0028] The constructs of the present disclosure can include a promoter operably linked to a transcribable nucleic acid molecule operably linked to a 3' transcription termination nucleic acid molecule. In addition, the constructs can include additional regulatory nucleic acid molecules, such as, but not limited to, from the 3' untranslated region (3'UTR). The constructs can also include, but are not limited to, the 5' untranslated region (5'UTR) of an mRNA nucleic acid molecule, which plays an important role in translation initiation and is a genetic component in the expression construct. These additional upstream and downstream regulatory nucleic acid molecules can be derived from natural or heterologous sources relative to the other elements present on the promoter construct.

[0029] The term "transformation" refers to the introduction of a nucleic acid fragment into the genome of a host cell, resulting in genetically stable inheritance. Host cells containing the transformed nucleic acid fragments are referred to as "transgenic" cells, and organisms containing transgenic cells are referred to as "transgenic organisms."

[0030] "Transformed," "transgenic," and "recombinant" refer to a host cell or organism, such as a bacterium, cyanobacterium, animal, or plant, into which a heterologous nucleic acid molecule has been introduced. The nucleic acid molecule is generally known in the art and can be stably integrated into the genome (see, for example, Sambrook 1989; Innis 1995; Gelfand 1995; Innis & Gelfand 1999). Known methods of PCR include, but are not limited to, methods using paired primers, nested primers, single specific primers, degenerate primers, gene specific primers, vector specific primers, partial mismatch primers, and the like. The term "untransformed" refers to a normal cell that has not undergone a transformation process.

[0031] "Wild type" refers to a virus or organism that occurs in nature and has no known mutations.

[0032] It is within the skill of the art to design, generate, and test mutant nucleotides and their encoded polypeptides that have the required percent identity and retain the required activity of the expressed protein. For example, directed evolution and rapid isolation of mutants can be performed according to methods described in, but not limited to, Link et al. (2007) Nature Reviews 5(9), 680-688; Sanger et al. (1991) Gene 97(1), 119-123; Ghadessy et al. (2001) Proc Natl Acad Sci USA 98(8) 4552-4557. Thus, one skilled in the art can generate a large number of nucleotide and / or polypeptide mutants that have at least 95-99% identity to the reference sequences described herein, for example, and screen for the desired phenotype according to methods common in the art.

[0033] Percent identity (%) of a nucleotide and / or amino acid sequence is understood as the percentage of nucleotides or amino acid residues that are identical to the nucleotides or amino acid residues in a candidate sequence when the two sequences are aligned, compared to a reference sequence. To determine percent identity, the sequences are aligned, and gaps are introduced if necessary to maximize the percent sequence identity. Sequence alignment procedures for determining percent identity are well known to those skilled in the art. In many cases, commonly available computer software such as BLAST, BLAST2, ALIGN2, or Megalign (DNASTAR) software is used to align sequences. Those skilled in the art can determine the appropriate parameters for measuring alignment, including any algorithm required to achieve maximum alignment over the entire length of the sequences being compared. When sequences are aligned, the percent sequence identity of a sequence A to a sequence B (which can also be rephrased as having a percent sequence identity to a sequence B) can be calculated as follows: percent sequence identity=X / Y100, where X is the number of residues scored as perfect matches by the alignment of A and B by a sequence alignment program or algorithm, and Y is the total number of residues in B. If the length of sequence A is not equal to the length of sequence B, then the percent sequence identity of A to B will not be equal to the percent sequence identity of B to A.

[0034] In general, conservative substitutions can be made at any position as long as the required activity is retained. So-called conservative exchanges can be made in which the substituted amino acid has similar properties to the original amino acid, such as replacement of Glu with Asp, replacement of Gln with Asn, replacement of Val with Ile, replacement of Leu with Ile, and replacement of Ser with Thr. For example, amino acids with similar properties include aliphatic amino acids (e.g., glycine, alanine, valine, leucine, isoleucine), hydroxyl or sulfur / selenium-containing amino acids (e.g., serine, cysteine, selenocysteine, threonine, methionine), cyclic amino acids (e.g., proline), aromatic amino acids (e.g., phenylalanine, tyrosine, tryptophan), basic amino acids (e.g., histidine, lysine, arginine), and acidic amino acids or their amides (e.g., aspartic acid, glutamic acid, asparagine, glutamine). Deletion means that an amino acid is replaced by a direct bond. Positions of deletion include the termini of a polypeptide or the junctions between individual protein domains. An insertion is the introduction of an amino acid into a polypeptide chain, where a direct bond is formally replaced by one or more amino acids. The amino acid sequence can be adjusted with the aid of computer simulation programs known in the art, which can generate, for example, polypeptides with improved activity or altered regulation. Based on these artificially generated polypeptide sequences, the corresponding nucleic acid molecules encoding such adjusted polypeptides can be synthesized in vitro using the specific codon usage of the desired host cell.

[0035] "High stringency hybridization conditions" are defined as hybridization at 65°C in 6XSSC buffer (i.e., 0.9 M sodium chloride and 0.09 M sodium citrate). Given these conditions, it can be determined whether a given set of sequences will hybridize by calculating the melting temperature (Tm) of the DNA duplex between the two sequences. If the melting temperature of a particular duplex is lower than 65°C under the salt conditions of 6XSSC, the two sequences will not hybridize. On the other hand, if the melting temperature is 65°C or higher under the same salt conditions, the two sequences will hybridize. In general, the melting temperature of hybridized DNA:DNA sequences can be determined using the following formula: T m =81.5°C+16.6(log 10 [Na + ]) + 0.41 (fraction G / C content) - 0.63 (% formamide) - (600 / l). Furthermore, the T m decreases by 1–1.5°C for every 1% decrease in nucleotide identity (see, e.g., Sambrook and Russel, 2006).

[0036] Host cells can be transformed using a variety of standard techniques known in the art (see, for example, Sambrook and Russel (2006) Condensed Protocols from Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, ISBN-10: 0879697717; Ausubel et al. (2002) Short Protocols in Molecular Biology, 5th ed., Current Protocols, ISBN-10: 0471250929; Sambrook and Russel (2001) Molecular Cloning: A Laboratory Manual, 3d ed., Cold Spring Harbor Laboratory Press, ISBN-10: 0879695773; Elhai, J. and Wolk, CP 1988. Methods in Enzymology 167, 747-754). Such techniques include, but are not limited to, viral infection, calcium phosphate transfection, liposome-mediated transfection, microprojectile-mediated delivery, receptor-mediated uptake, cell fusion, electroporation, etc. The transfected cells can be selected and propagated to provide recombinant host cells that contain the expression vector stably integrated into the host cell genome.

[0037] [Table 1]

[0038] [Table 2]

[0039] [Table 3]

[0040] Exemplary nucleic acids that may be introduced into a host cell include, for example, DNA sequences or genes from other species, or genes or sequences that are from or present in the same species but are incorporated into the recipient cell by genetic engineering methods. The term "exogenous" is also intended to refer to genes that are not normally present in the cell being transformed, or perhaps simply not present in the form, structure, etc., as found in the transformed DNA fragment or gene, or that are normally present but that one wishes to express, e.g., overexpress, in a manner different from the natural expression pattern. Thus, the term "exogenous" gene or DNA is intended to refer to a gene or DNA fragment that is introduced into a recipient cell. Types of DNA included in exogenous DNA can include DNA already present in the cell, DNA from another member of the same species of organism, DNA from a different organism, or DNA sequences that contain antisense messages of genes, and DNA that is generated from outside, such as DNA sequences that code for synthetic or modified versions of genes.

[0041] Host strains developed according to the approaches described herein can be evaluated by various means known in the art (see, e.g., Studier (2005) Protein Expr Purif. 41(1), 207-234; Gellissen, ed. (2005) Production of Recombinant Proteins: Novel Microbial and Eukaryotic Expression Systems, Wiley-VCH, ISBN-10: 3527310363; Baneyx (2004) Protein Expression Technologies, Taylor & Francis, ISBN-10: 0954523253).

[0042] Methods for downregulating or silencing genes are known in the art. For example, expressed protein activity can be downregulated or ablated using antisense oligonucleotides (ASOs), protein aptamers, nucleotide aptamers, and RNA interference (RNAi) (e.g., small interfering RNA (siRNA), short hairpin RNA (shRNA), microRNA (miRNA)) (see, e.g., Rinaldi and Wood (2017) Nature Reviews Neurology 14, describing ASO therapies; Fanning and Symonds (2006) Handb Exp Pharmacol. 173, 289-303G, describing hammerhead ribozymes and small hairpin RNA; Helene, et al. (1992) Ann. NY Acad. Sci. 660, 27-36; Maher (1992) Bioassays 14(12): 807-15, describing targeting deoxyribonucleotide sequences; Lee et al. (2006) Curr Opin Chem Biol. 10, 1-8, describing aptamers; Reynolds et al. (2004) Nature Biotechnology 22(3), 326 - 330, describing RNAi; Pushparaj and Melendez (2006) Clinical and Experimental Pharmacology and Physiology 33(5-6), 504-510, describing RNAi; Dillon et al. (2005) Annual Review of Physiology 67, 147-173, describing RNAi; see Dykxhoorn and Lieberman (2005) Annual Review of Medicine 56, 401-423, describing RNAi).RNAi molecules are commercially available from various sources (e.g., Ambion, TX; Sigma Aldrich, MO; Invitrogen). Several siRNA molecule design programs using various algorithms are known in the art (see, e.g., Cenix algorithm, Ambion; BLOCK-iT(tm) RNAi Designer, Invitrogen; siRNA Whitehead Institute Design Tools, Bioinofrmatics & Research Computing). Characteristics that influence the definition of optimal siRNA sequences include the G / C content at the ends of the siRNA, the Tm of certain internal domains of the siRNA, the length of the siRNA, the location of the target sequence within the CDS (coding region), and the nucleotide content of the 3' overhang.

[0043] Genome editing

[0044] As described herein, miR-29 signal can be regulated (e.g., enhanced) by genome editing.Genome editing methods are well known (see, for example, Aldi 2018 Nature Communications 9(1911)).Therefore, unless otherwise stated herein, the method of the present disclosure can be carried out according to such methods.

[0045] For example, genome editing can include CRISPR / Cas9, CRISPR-Cpf1, TALEN, or ZNF. Appropriate blocking of ECM-related gene expression by genome editing to enhance miRNA-29 production can provide protection from bladder fibrosis.

[0046] As an example, CRISPR / CRISPR-associated (Cas) systems are a new class of genome editing tools that target desired genomic sites in mammalian cells. The recently described type II CRISPR / Cas system uses the Cas9 nuclease to hybridize to a 20-nucleotide DNA sequence that contains an NGG motif (hence, (N)) that Cas9 recognizes. 20 It targets genomic sites by complexing with a synthetic guide RNA immediately preceding the NGG target DNA sequence). This results in a double-stranded break three nucleotides upstream of the NGG motif. The double-stranded break induces non-homologous end joining or homology-directed repair. Non-homologous end joining is error-prone and induces frameshift mutations that knock out gene alleles. Homology-directed repair can knock-in or correct genomic mutations using exogenously introduced double-stranded or single-stranded DNA repair templates. Thus, genome editing, for example using the CRISPR / Cas system, could be a useful tool for therapeutic applications to reduce ECM formation in target cells by enhancing miR-29 signals.

[0047] For example, methods of the disclosure include a method of altering a target polynucleotide sequence in a cell, comprising contacting the polynucleotide sequence with a Cas protein.

[0048] formulation

[0049] The agents and compositions of the present disclosure can be formulated in any conventional manner using one or more pharma- ceutically acceptable carriers or excipients, for example, as described in Remington's Pharmaceutical Sciences (AR Gennaro, Ed.), 21st edition, ISBN: 0781746736 (2005), which is incorporated herein by reference in its entirety. Such formulations will contain a therapeutically effective amount of a biologically active agent of the present disclosure, which may be in purified form, together with a suitable amount of carrier to provide the form for proper administration to a subject (patient).

[0050] The term "formulation" refers to preparing a drug in a form suitable for administration to a subject, such as a human. Thus, a "formulation" can include a pharma- ceutically acceptable excipient, including a diluent or carrier.

[0051] As used herein, the term "pharmaceutical acceptable" refers to a substance or ingredient that does not cause unacceptable loss of pharmacological activity or unacceptable side effects. Examples of pharmaceutical acceptable ingredients include those listed in the United States Pharmacopeia (USP29) and National Formulary (NF24), the United States Pharmacopeial Convention, Inc, Rockville, Maryland, 200 ("USP / NF"), or monographs in more recent editions, and ingredients listed in the FDA's continuously updated inactive ingredient search online database. However, other useful ingredients not listed in the USP / NF, etc. may also be used.

[0052] As used herein, the term "pharmaceutical acceptable excipient" can include any and all solvents, dispersion media, coatings, antibacterial agents, antifungal agents, isotonic agents, or absorption delaying agents. The use of such media and agents for pharma-ceutically active substances is known in the art (see, for example, Remington's Pharmaceutical Sciences (AR Gennaro, Ed.), 21st edition, ISBN: 0781746736 (2005)). Except insofar as any conventional media or agent is incompatible with the active ingredient, its use in the therapeutic compositions is contemplated. Supplementary active ingredients can also be incorporated into the compositions.

[0053] A "stable" formulation or composition refers to a composition that has sufficient stability to permit storage at an appropriate temperature, e.g., from about 0°C to about 60°C, for a commercially reasonable period of time, e.g., at least about 1 day, at least about 1 week, at least about 1 month, at least about 3 months, at least about 6 months, at least about 1 year, or at least about 2 years.

[0054] The formulation should be compatible with the mode of administration. The agents used in the present disclosure can be formulated according to known methods for administration to a subject (patient) using various routes, including, but not limited to, parenteral, pulmonary, oral, topical, intradermal, intratumoral, intranasal, inhalation (e.g., aerosol), implantation, intramuscular, intraperitoneal, intravenous, intrathecal, intracranial, intraventricular, subcutaneous, intranasal, epidural, intraocular, transdermal, buccal, and rectal. Individual agents can also be administered in combination with one or more additional agents, or with other biologically active or biologically inactive agents. Such biologically active or inactive agents may be in fluid or mechanical communication with the agent, or may be bound to the agent by ionic, covalent, van der Waals, hydrophobic, hydrophilic, or other physical forces.

[0055] Controlled release (or sustained release) formulations can be formulated to extend the activity of a drug and reduce the frequency of administration. Controlled release formulations can also be used to affect the time of onset of action or other characteristics, such as the blood concentration of the drug, and thus the occurrence of side effects. Controlled release formulations can be designed to initially release an amount of drug that produces a desired therapeutic effect, and then slowly or continuously release other amounts of the drug, thereby maintaining a level of therapeutic effect over an extended period of time. To maintain a nearly constant concentration of the drug in the body, the drug is released from the formulation in an amount that replaces the amount of drug metabolized or excreted in the body. The controlled release of the drug can be stimulated by various inducers, such as pH change, temperature change, enzymes, water, or other physiological conditions or molecules.

[0056] The agents or compositions of the present disclosure can also be used in combination with other therapies, as described further below. Thus, in addition to the therapies described herein, the subject (patient) can also be provided with other therapies known to be effective in treating the disease, disorder, or condition.

[0057] Treatment method

[0058] The present disclosure also provides methods for treating, preventing, or reversing bladder fibrosis in a patient in need of administration of a therapeutically effective amount of ApoM or ApoM-containing plasma to prevent, reduce, or reverse photoreceptor outer segment destruction, RPE lipid deposition, and / or neurodegeneration in the patient's retina.

[0059] The therapeutic method of the present disclosure is generally administered to a patient in need thereof. The patient in need of the therapeutic method of the present disclosure may be a patient with bladder fibrosis, a patient diagnosed with bladder fibrosis, a patient suspected of having bladder fibrosis, or a patient at risk of developing bladder fibrosis. The determination of the need for treatment is generally assessed by a medical history, physical examination, or diagnostic test consistent with the disease or condition in question. Diagnosis of various conditions treatable by the method of the present disclosure is within the skill of those of ordinary skill in the art. The subject may be an animal subject, including mammals such as horses, cows, dogs, cats, sheep, pigs, mice, rats, monkeys, hamsters, guinea pigs, humans, chickens, etc. For example, the subject may be a human subject.

[0060] In general, a safe and effective amount of ApoM or ApoM-containing plasma is, for example, an amount that provides a desired therapeutic effect in a subject while minimizing undesirable side effects. In various embodiments, an effective amount of the disclosed ApoM or ApoM-containing plasma can substantially inhibit photoreceptor outer segment destruction, RPE lipid deposition, and / or neurodegeneration, substantially slow the progression of photoreceptor outer segment destruction, RPE lipid deposition, and / or neurodegeneration, or substantially limit the onset of photoreceptor outer segment destruction, RPE lipid deposition, and / or neurodegeneration.

[0061] According to the methods of the present disclosure, administration can be parenteral, pulmonary, oral, topical, intradermal, intramuscular, intraperitoneal, intravenous, intratumoral, intrathecal, intracranial, intraventricular, subcutaneous, intranasal, epidural, intraocular, buccal, or rectal.

[0062] When used in the treatments of the present disclosure, therapeutically effective amounts of ApoM or ApoM-containing plasma can be used in pure form, or, where such forms exist, in pharma- ceutically acceptable salt form, with or without pharma- ceutically acceptable excipients. For example, compounds of the present disclosure can be administered in an amount sufficient to prevent, reduce, or reverse bladder fibrosis, at a reasonable benefit / risk ratio applicable to any medical treatment.

[0063] The amount of the composition of the present disclosure that can be combined with a pharma- ceutically acceptable carrier to produce a single dosage form (formulation) varies depending on the subject or host to be treated and the particular method of administration. Those skilled in the art will understand that the unit content of the drug contained in each individual dose of each dosage form does not necessarily constitute a therapeutically effective amount in itself, since the required therapeutically effective amount can be reached by administration of multiple individual doses.

[0064] The toxicity and therapeutic efficacy of the compositions of the present disclosure are 50 (lethal dose for 50% of the population) and ED 50 The LD (the dose that is therapeutically effective in 50% of a population) can be determined by standard pharmaceutical procedures in cell cultures or experimental animals. Toxic and therapeutic effects and dose ratios can be determined using the LD 50 / ED 50 and it is generally understood in the art that the larger therapeutic index is optimal.

[0065] The specific therapeutically effective dosage level for any particular subject will depend on a variety of factors, such as the disease being treated and its severity; the activity of the specific compound used; the specific composition used; the age, weight, relative health, sex and diet of the subject; the timing of administration; the route of administration; the excretion rate of the composition used; the duration of treatment; drugs used in combination with or concomitantly with the particular compound used; and similar factors well known in the medical arts (see, e.g., Koda-Kimble et al. (2004) Applied Therapeutics: The Clinical Use of Drugs, Lippincott Williams & Wilkins, ISBN 0781748453; Winter (2003) Basic Clinical Pharmacokinetics, 4th ed., Lippincott Williams & Wilkins, ISBN 0781741475; Sharqel (2004) Applied Biopharmaceutics & Pharmacokinetics, McGraw-Hill / Appleton & Lange, ISBN 0071375503). For example, it is within the skill of the art to start the dosage of the composition at a level lower than that required to achieve the desired therapeutic effect, and gradually increase the dosage of the composition until the desired effect is achieved. If necessary, the effective amount may be administered in multiple doses. Thus, a single-dose composition may contain such amounts or submultiples thereof to make up a daily dose. However, it will be understood that the total daily usage of the compounds and compositions of the present disclosure will be determined by the attending physician within the scope of sound medical judgment.

[0066] Again, each of the conditions, diseases, disorders, and conditions described herein, as well as others, can benefit from the compositions and methods of the present disclosure. Generally, treating a condition, disease, disorder, or condition includes preventing, reversing, or delaying the appearance of clinical symptoms in a mammal that is afflicted with, or may be afflicted with, the condition, disease, disorder, or condition, but has not yet experienced or exhibited clinical or subclinical symptoms thereof. Treatment can also include inhibiting the condition, disease, disorder, or condition, e.g., preventing or reducing the occurrence of the disease or at least one clinical or subclinical symptom thereof. Furthermore, treatment can include alleviating the disease, e.g., causing regression of the condition, disease, disorder, or condition, or at least one clinical or subclinical symptom thereof. The benefit to the treated subject is statistically significant or at least noticeable to the subject or physician.

[0067] The administration of ApoM or ApoM-containing plasma can be a single administration or over a treatment period. For example, ApoM or ApoM-containing plasma can be administered daily, weekly, biweekly, or monthly. For acute disease treatment, the treatment period is usually at least several days. In certain conditions, the treatment period can range from several days to several weeks. For example, the treatment period can range from one week, two weeks, or three weeks. In more chronic conditions, the treatment period can range from several weeks to several months, or even more than one year.

[0068] Treatment according to the methods of the present disclosure can be administered prior to, concurrently with, or following conventional therapy for the prevention, reduction, or reversal of bladder fibrosis.

[0069] ApoM or ApoM-containing plasma can be administered simultaneously or sequentially with other agents, such as antibiotics, anti-inflammatory agents, or other agents. For example, ApoM or ApoM-containing plasma can be administered simultaneously with other agents, such as antibiotics and anti-inflammatory agents. Simultaneous administration can be achieved by administration of separate compositions, each of which contains one or more ApoM or ApoM-containing plasma, antibiotics, anti-inflammatory agents, or other agents. Alternatively, simultaneous administration can be achieved by administration of a single composition containing two or more ApoM or ApoM-containing plasma, antibiotics, anti-inflammatory agents, or other agents. ApoM or ApoM-containing plasma can be administered sequentially with antibiotics, anti-inflammatory agents, or other agents. For example, ApoM or ApoM-containing plasma can be administered before or after administration of antibiotics, anti-inflammatory agents, or other agents.

[0070] Administration

[0071] The agents and compositions of the present disclosure can be administered in a variety of ways known in the art according to the methods of the present disclosure. The agents and compositions can be used in therapy as either exogenous or endogenous agents. An exogenous agent is an agent that is produced or manufactured outside the body and administered to the body. An endogenous agent is an agent that is produced or manufactured within the body by some type of device (biological or otherwise) for delivery to the body or to other organs within the body.

[0072] As described above, administration can be parenteral, pulmonary, oral, topical, intradermal, intratumoral, intranasal, inhaled (e.g., by aerosol), implanted, intramuscular, intraperitoneal, intravenous, intrathecal, intracranial, intraventricular, subcutaneous, intranasal, epidural, intrathecal, intraocular, transdermal, buccal, and rectal.

[0073] The agents and compositions of the present disclosure can be administered in a variety of ways well known in the art, including, for example, oral ingestion, direct injection (e.g., systemic or stereotactic injection), implantation of cells engineered to secrete a factor of interest, drug-releasing biomaterials, polymeric matrices, gels, permeable membranes, osmotic systems, multilayer coatings, microparticles, implantable matrix devices, mini-osmotic pumps, implantable pumps, injectable gels or hydrogels, liposomes, micelles (e.g., 30 μm or less), nanospheres (e.g., less than 1 μm), microspheres (e.g., 1-100 μm), reservoir devices, combinations of any of the above, or other suitable delivery vehicles that provide the desired release profile at various ratios.

[0074] Lipoprotein carriers or larger lipoprotein particles may also be used.

[0075] In some embodiments, ApoM can be fused to an immunoglobulin (eg, ApoM-Fc).

[0076] Other methods of controlled release delivery of agents or compositions are known to those of skill in the art and are within the scope of the present disclosure.

[0077] The delivery system may include, for example, an infusion pump that can be used to administer the agent or composition in a manner similar to that used to deliver insulin or chemotherapy to a specific organ or tumor. Typically, such a system can be used to administer the agent or composition in combination with a biodegradable and biocompatible polymeric implant that releases the agent at a selected site over a controlled period of time. Examples of polymeric materials include polyanhydrides, polyorthoesters, polyglycolic acid, polylactic acid, polyethylenevinylacetate, copolymers thereof, and combinations thereof. In addition, the controlled release system can be placed near the therapeutic target, thus requiring only a fraction of the systemic dose.

[0078] Drugs can be administered by encapsulation in various carrier delivery systems. Examples of carrier delivery systems include microspheres, hydrogels, polymer implants, smart polymer carriers, liposomes, etc. (see, for example, Uchegbu and Schatzlein, eds. (2006) Polymers in Drug Delivery, CRC, ISBN-10: 0849325331). Carrier-based systems for molecular or biomolecule drug delivery can: provide intracellular delivery; tune biomolecule / drug release rates; increase the proportion of biomolecules that reach the site of action; improve transport of drugs to the site of action; allow co-localized deposition with other drugs or excipients; improve the stability of the drug in vivo; extend the residence time of the drug at the site of action by reducing clearance; reduce non-specific delivery of the drug to non-target tissues; reduce drug irritation; reduce toxicity from initial high doses of the drug; change the immunogenicity of the drug; reduce the number of administrations; improve the taste of the product; improve the shelf life of the product.

[0079] screening

[0080] The present disclosure also provides methods of screening.

[0081] The subject methods are utilized to screen a variety of different candidate molecules (e.g., potentially therapeutic candidate molecules). Candidate substances for screening according to the methods of the present disclosure include, but are not limited to, tissue or cell fractions, nucleic acids, polypeptides, siRNAs, antisense molecules, aptamers, ribozymes, triple helix compounds, antibodies, and small (e.g., less than about 2000 mw, or less than about 1000 mw, or less than about 800 mw) organic or inorganic molecules (e.g., salts, metals).

[0082] Candidate molecules encompass a variety of chemical classes, for example organic molecules such as small organic compounds with a molecular weight of 50 Daltons or more and less than about 2500 Daltons. Candidate molecules may contain functional groups necessary for structural interaction with proteins, particularly hydrogen bonding, and generally contain at least an amine, carbonyl, hydroxyl or carboxyl group, and usually contain at least two functional chemical groups. Candidate molecules may contain cyclic carbon or heterocyclic structures and / or aromatic or polyaromatic structures substituted with one or more of the above functional groups.

[0083] The candidate molecule may be a compound in a library database of compounds. Those skilled in the art will be generally familiar with, for example, the numerous databases of compounds commercially available for screening (see, for example, the ZINC database (UCSF) with 2.7 million compounds across 12 different molecular subsets; Irwin and Shoichet (2005) J Chem Inf Model 45, 177-182). Those skilled in the art will also be familiar with various search engines for identifying commercial sources and desirable compounds and compound classes for further testing (see, for example, the ZINC database; eMolecules.com; vendor-provided electronic libraries of commercially available compounds (ChemBridge, Princeton BioMolecular, Ambinter SARL, Enamine, ASDI, Life Chemicals, etc.)).

[0084] Candidate molecules for screening according to the methods of the present disclosure include both lead-like compounds and drug-like compounds. Lead-like compounds are generally understood to have relatively small scaffold-like structures (e.g., molecular weights of about 150 to about 350 kD) with relatively few features (e.g., less than about 3 hydrogen donors and / or less than about 6 hydrogen acceptors; hydrophobic character xlogP of about -2 to about 4) (see, e.g., Angewante (1999) Chemie Int. ed. Engl. 24, 3943-3948). In contrast, drug-like compounds are generally understood to have relatively large scaffolds (e.g., molecular weights of about 150 to about 500 kD) with relatively many features (e.g., less than about 10 hydrogen acceptors and / or less than about 8 rotatable bonds; hydrophobic character xlogP of less than about 5) (see, e.g., Lipinski (2000) J. Pharm. Tox. Methods 44, 235-249). Initial screening can be performed using lead-like compounds.

[0085] When designing lead compounds from spatial orientation data, it is useful to understand that a particular molecular structure is "drug-like." Such characterization can be based on a set of empirically recognized properties and is derived by comparing the broad similarities of known drugs in the pharmacopoeia. While a drug does not need to meet all or even any of these characteristics, being drug-like makes a drug candidate much more likely to achieve clinical success.

[0086] Some of these "drug-like" features are summarized in Lipinski's four rules (commonly known as the "rule of fives" due to the prevalence of the number 5.) These rules, which generally relate to oral absorption and are used to predict compound bioavailability during lead optimization, serve as effective guidelines for constructing lead molecules during rational drug design efforts such as may be accomplished by using the methods of the present disclosure.

[0087] The four "rules of five" state that a candidate drug-like compound should have at least three of the following properties: (i) weight less than 500 Daltons; (ii) log P less than 5; (iii) hydrogen bond donors (OH and NH groups combined) less than 5; and (iv) hydrogen bond acceptors (N and O atoms combined) less than 10. Drug-like molecules also typically have a span (width) of about 8 Å to about 15 Å.

[0088] kit

[0089] Also provided according to the present disclosure is a kit. The kit of the present disclosure may include the agent or composition of the present disclosure, and in certain embodiments, instructions for administration. Such a kit may facilitate the performance of the method of the present disclosure. When provided as a kit, different components of the composition of the present disclosure may be packaged in separate containers and mixed immediately before use. The components include, but are not limited to, compositions comprising ApoM or ApoM-containing plasma as described herein. Such separate packaging of components may, if desired, be presented in a pack or dispenser device that includes one or more unit dosage forms comprising the composition of the present disclosure. The pack may, for example, include metal or plastic foils, such as blister packs. Such separate packaging of components may also, in certain cases, allow for long-term storage without loss of activity of the components.

[0090] The kit may also include reagents in separate containers, such as, for example, sterile water or saline to be added to the separately packaged lyophilized active ingredient. For example, a sealed glass ampoule contains the lyophilized ingredient and another contains sterile water or saline, each packaged under a neutral, non-reactive gas, such as nitrogen. The ampoules may be constructed of glass, organic polymers such as polycarbonate, polystyrene, ceramic, metal, or any other material commonly used to hold reagents. Other examples of suitable containers include bottles made of similar materials as the ampoules, and tubes with foil-lined interiors, such as aluminum or alloys. Other containers include test tubes, vials, flasks, bottles, syringes, and the like. The container may have a sterile access port, such as a bottle with a stopper that can be pierced by a hypodermic needle. Other containers have two compartments separated by a readily removable membrane, which allows the ingredients to mix when removed. The removable membrane may be glass, plastic, rubber, and the like.

[0091] In certain embodiments, the kit may be provided with instructions. The instructions may be printed on paper or other substrate, or may be provided as an electronically readable medium or video. The instructions may not be physically associated with the kit. In that case, the user may be directed to an internet website designated by the kit manufacturer or distributor.

[0092] The compositions and methods of the present disclosure utilizing molecular biology protocols can follow a variety of standard techniques known in the art (see, for example, Sambrook and Russel (2006) Condensed Protocols from Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, ISBN-10: 0879697717; Ausubel et al. (2002) Short Protocols in Molecular Biology, 5th ed., Current Protocols, ISBN-10: 0471250929; Sambrook and Russel (2001) Molecular Cloning: A Laboratory Manual, 3d ed., Cold Spring Harbor Laboratory Press, ISBN-10: 0879695773; Elhai, J. and Wolk, CP 1988. Methods in Enzymology 167, 747-754; Studier (2005) Protein Expr Purif. 41(1), 207-234; Gellissen, ed. (2005) Production of Recombinant Proteins: Novel Microbial and Eukaryotic Expression Systems, Wiley-VCH, ISBN-10: 3527310363; Baneyx (2004) Protein Expression Technologies, Taylor & Francis, ISBN-10: 0954523253).

[0093] The definitions and methods set forth herein are provided to better define the present disclosure and to guide those of ordinary skill in the art in the practice of the present disclosure. Unless otherwise specified, terms should be understood according to conventional usage by those of ordinary skill in the relevant art.

[0094] In some embodiments, numbers expressing quantities of ingredients, properties, such as molecular weights, reaction conditions, and the like, used to describe and claim certain embodiments of the present disclosure should be understood in some instances to be modified by the term "about". In some embodiments, the term "about" is used to indicate that a value includes the standard deviation of the average for the device or method being employed to determine the value. In some embodiments, the numerical parameters set forth in this specification and the appended claims are approximations that can vary depending on the desired properties sought to be obtained by a particular embodiment. In some embodiments, the numerical parameters should be construed by taking into account the number of reported significant digits and applying ordinary rounding techniques. Notwithstanding that the numerical ranges and parameters setting forth the broad scope of some embodiments of the present disclosure are approximations, the numerical values ​​set forth in the specific examples are reported as precisely as practicable. The numerical values ​​set forth in some embodiments of the present disclosure may contain certain errors necessarily resulting from the standard deviation found in their respective testing measurements. The recitation of ranges of values ​​herein is merely intended to serve as a shorthand method of individually designating each individual value falling within the range. Unless otherwise specified herein, each individual value is incorporated into the specification as if it were individually set forth herein. A recitation of discrete values ​​is understood to include ranges between each value.

[0095] In some embodiments, the terms "a," "an," "the," and similar terms used in the context of describing particular embodiments (especially in the particular context of the claims below) can be construed to encompass both the singular and the plural, unless otherwise indicated. In some embodiments, the term "or" as used herein, including the claims, is used to mean "and / or," unless expressly indicated to refer to alternatives only or the alternatives are mutually exclusive.

[0096] The terms "comprise," "have," and "include" are open-ended linking verbs. Any one or more of the variations or tenses of these verbs, such as "comprises," "comprising," "has," "having," "includes," and "including," are also open-ended. For example, any method that "comprises," "has," or "includes" one or more steps is not limited to having only those one or more steps, but may also include other unrecited steps. Similarly, any composition or device that "comprises," "has," or "includes" one or more features is not limited to having only those one or more features, but may also include other unrecited features.

[0097] All methods described herein can be performed in any suitable order unless otherwise indicated herein or clearly contradicted by context. Any and all examples provided with respect to specific embodiments herein, or the use of exemplary language (e.g., "etc.") are intended merely to better illuminate the disclosure and do not impose limitations on the scope of the invention unless specifically recited in the claims. No language in this specification should be construed as indicating that any non-claimed element is essential to the practice of the disclosure.

[0098] Grouping of alternative elements or embodiments of the disclosure disclosed herein should not be construed as limiting. Each group member may be referred to and claimed individually or in any combination with other members of the group or other elements described herein. One or more members of a group may be included in or deleted from a group for reasons of convenience or patentability. When such inclusion or deletion is made, the specification is deemed to include the modified group and therefore fulfills all Markush group descriptions used in the appended claims.

[0099] All publications, patents, patent applications, and other documents cited in this application are incorporated herein by reference in their entirety for all purposes to the same extent as if each was specifically and individually indicated to be incorporated by reference in its entirety for all purposes. The citation of a document herein should not be construed as an admission that it is prior art to the present disclosure.

[0100] Although the present disclosure has been described in detail, it will be apparent that modifications, variations, and equivalents are possible without departing from the scope of the present disclosure as defined in the appended claims. Moreover, it should be understood that all examples in the present disclosure are provided as non-limiting examples.

[0101] Working Example

[0102] The above non-limiting examples are provided to further illustrate the present disclosure. Those skilled in the art will appreciate that the techniques disclosed in these examples represent approaches that the present inventors have found to work well in implementing the present disclosure, and therefore can be considered to constitute examples of embodiments of the present disclosure. Those skilled in the art will also appreciate that in light of the present disclosure, various changes can be made to the specific embodiments of the present disclosure and still obtain the same or similar results without departing from the spirit and scope of the present disclosure.

[0103] Example 1. ApoM administration to ABCA1 / ABCG1 photoreceptor and macrophage knockouts

[0104] To clarify the mechanism by which ApoM administration improves ABCA1 / ABCG1 photoreceptor and macrophage knockout, the following experiment was performed.

[0105] ABCA1 / ABCG1 photoreceptor knockout mice were fed a high-fat diet. As shown in Figure 3, the first group of mice was administered ApoM-containing plasma (ApoMTg) and the second group was administered ApoM-deficient plasma (ApoMKO).

[0106] Electroretinograms (ERGs) were performed using the UTAS BigShot System (LKC Technologies Inc.). Mice were dark-adapted overnight. Mice were anesthetized with an intraperitoneal injection of 86.9 mg / kg ketamine and 13.4 mg / kg xylazine under red light illumination. Pupils were dilated with 1% atropine sulfate eye drops (Bausch & Lomb). Body temperature was maintained at 37°C with a heating pad. Contact lens electrodes were placed bilaterally with appropriate reference and ground electrodes. Stimuli were delivered in darkness or dim light (30.0 candela [cd] / m) after a 10-min adaptation period. 2 ) with a full-field white light flash (10 μs) in the presence of background illumination. Raw data were processed using MATLAB software (MathWorks). The a-wave amplitude was measured from the average pre-trial baseline to the most negative point of the average trace, and the b-wave amplitude was measured from that point to the most positive point. Electroretinograms (ERGs) revealed higher electroretinogram amplitudes in ApoM Tg mice compared to ABCA1 / ABCG1-rod / -rod knockout ApoM KO mice (Figure 4A,B,C).

[0107] TEM images of the retinal pigment epithelium of ApoM Tg-treated mice were obtained (Figure 5B) and compared with ApoM KO-treated mice (Figure 5A). Significantly fewer lipid droplets were observed in the retinal pigment epithelium of ApoM Tg-treated mice compared with ApoM KO-treated mice (Figure 5C). In addition, destruction of photoreceptor outer segments was observed only in ApoM KO-treated mice (Figures 6A and 7A), but not in ApoM Tg-treated mice (Figures 6B and 7B).

[0108] This effect was ApoM-specific, as it was not rescued by plasma enriched with a mutant ApoM that does not bind S1P. These results demonstrated the specificity and potential mechanism of ApoM.

[0109] As shown in Figure 9, mice with sphingosine-1-phosphate receptor 1 knockout (S1P1R -RPE / -RPE ) and conducted similar experiments.

[0110] Example 2: Effects of APOM knockout on photoreceptors

[0111] To characterize the systemic effects of ApoM knockout on photoreceptors, the following experiments were performed.

[0112] Electroretinograms (ERGs) were performed on ApoM knockout mice (ApoM mutant) and ApoM control mice as described in Example 1. Higher electroretinogram amplitudes were observed in ApoM control mice compared to ApoM mutant mice (FIGS. 11A, 11B, and 11C).

[0113] TEM images of the retinal pigment epithelium of ApoM mutant mice were obtained (Figure 12A) and compared to ApoM control mice (Figure 12B). Significantly fewer lipid droplets were observed in the retinal pigment epithelium of ApoM control-treated mice compared to ApoM KO or ApoM mutant mice (Figure 13). In addition, retinal images showed more extensive choroidal neovascularization (CNV) in ApoM mutant mice (Figure 14(B)) compared to ApoM KO mice (Figure 14(C)) and wild-type mice (Figure 14(A)). Higher CNV lesion size was observed in ApoM mutant mice compared to ApoM KO and wild-type mice (Figure 15).

Claims

Claim 1 A pharmaceutical composition for preventing or reversing age-related macular degeneration in a subject having an age-related macular degeneration disease, wherein the pharmaceutical composition contains ApoM. Claim 2 The pharmaceutical composition according to claim 1, wherein the age-related macular degeneration disease is selected from the group consisting of age-related macular degeneration (AMD), juvenile macular degeneration, and diabetic retinopathy. Claim 3 The pharmaceutical composition according to claim 1, wherein the pharmaceutical composition prevents or reverses photoreceptor outer segment destruction, RPE lipid deposition, neurodegeneration, angiogenesis, or any combination thereof in the subject.

Images