Treatment of macular degeneration using complement factor D (CFD) inhibitors

Abstract

The present disclosure relates generally to methods of treating a subject having or at risk of developing macular degeneration by administering a complement factor D (CFD) inhibitor to the subject and to identifying subjects at increased risk of developing macular degeneration.

Description

Treatment of macular degeneration with complement factor D (CFD) inhibitors Technical Field

[0001] This disclosure generally relates to treating subjects who have macular degeneration or are at risk of developing macular degeneration by administering a complement factor D (CFD) inhibitor, and to methods for identifying subjects at increased risk of developing macular degeneration. Background Technology

[0002] Age-related macular degeneration (AMD) is a serious medical condition that causes central vision loss. It is classified into “dry” (“non-exudative”) and “wet” (“neovascular” or “exudative”) forms. AMD is typically an age-related disease (hence its common name, AMD) that begins with early lipoprotein deposits (called drusen), which progressively accumulate and are accompanied by pigmentary changes in the eye (intermediate AMD). AMD is a leading cause of blindness worldwide, and its pathology includes the accumulation of extracellular deposits in the retina and the progressive loss of retinal pigment epithelium (RPE) and adjacent photoreceptor cells. Neovascular AMD has been successfully treated with VEGF antagonists. Intermediate AMD progresses to neovascular or non-exudative AMD. In neovascular AMD, vision loss may be due to abnormal blood vessel growth (choroidal neovascularization). The proliferation of abnormal blood vessels in the retina is stimulated by vascular endothelial growth factor (VEGF). Newly formed blood vessels are fragile and can cause blood and proteins to leak beneath the macula. Bleeding, leakage, and scarring from these vessels can eventually cause irreversible damage to photoreceptor cells, leading to rapid vision loss. In nonexudative macular degeneration (AMD), vision loss is due to the primary loss of photoreceptor cells in the macula, resulting in extensive cell loss known as "geographic atrophy." There is currently no effective treatment for intermediate to advanced AMD (approximately 90% of all cases).

[0003] Complement factor D (CFD) (NCBI gene ID: 1675) is encoded by an 8 kb gene located at 19p13.3 and is a member of the serine peptidase S1 family or the chymotrypsin family. The CFD protein is 253 amino acids long and 27 kDa. It acts as the rate-limiting enzyme in the alternative pathway of complement activation, a branch of the innate immune system that responds to cellular damage and is known to play a role in macular degeneration. Specifically, CFD, upon complexing with C3b, catalyzes the cleavage of factor B, converting C3bB to C3bBb + Ba. C3bBb then activates downstream pathways, including the formation of the membrane attack complex (MAC), thereby damaging cells, and the Ba fragment can also induce inflammation through other mechanisms. Summary of the Invention

[0004] This disclosure provides a method for treating subjects with macular degeneration or at risk of developing macular degeneration, the method comprising administering a CFD inhibitor to the subject, wherein the macular degeneration is intermediate, wet, or dry in form and may be related to aging.

[0005] This disclosure also provides a method for treating a subject with macular degeneration or at risk of developing macular degeneration by administering a macular degeneration treatment agent or therapy, the method comprising: determining or having determined whether the subject has a CFD variant nucleic acid molecule by: obtaining or having obtained a biological sample from the subject; and performing or having performed sequence analysis on the biological sample to determine whether the subject has a genotype containing a CFD variant nucleic acid molecule; administering or continuing to administer a macular degeneration treatment agent or therapy in an amount equal to or less than a standard dose to a subject serving as a CFD reference and / or administering a CFD inhibitor to the subject; administering or continuing to administer a macular degeneration treatment agent or therapy in an amount equal to or less than a standard dose to a subject heterozygous for a CFD variant nucleic acid molecule and / or administering a CFD inhibitor to the subject; or administering or continuing to administer a standard dose of a macular degeneration treatment agent or therapy to a subject homozygous for a CFD variant nucleic acid molecule; wherein the presence of the CFD variant nucleic acid molecule indicates a reduced risk of the subject developing macular degeneration.

[0006] This disclosure also provides a method for identifying subjects at increased risk of developing macular degeneration, the method comprising: determining or having determined the presence or absence of a CFD variant nucleic acid molecule in a biological sample obtained from the subject; wherein: when the subject is a CFD reference, the subject's risk of developing macular degeneration is increased; and when the subject is heterozygous or homozygous for the CFD variant nucleic acid molecule, the subject's risk of developing macular degeneration is reduced.

[0007] This disclosure also provides a macular degeneration treatment agent for treating or preventing macular degeneration in subjects with CFD variant nucleic acid molecules.

[0008] This disclosure also provides CFD inhibitors for the treatment or prevention of macular degeneration in subjects who are CFD-referenced or heterozygous for CFD variant nucleic acid molecules. Attached Figure Description

[0009] This patent or application document contains at least one color drawing. Upon request and payment of the necessary fees, the Patent Office will provide a published copy of this patent or application with one or more color drawings.

[0010] Figure 1 shows a graphical representation of the analysis results for wet AMD and dry AMD, divided by different cohorts, demonstrating the reproducibility of the effect across different patient cohorts.

[0011] Figure 2 illustrates how CFD missense variants alter the risk of individuals with a high genetic risk of AMD, based on genetic risk score stratification.

[0012] Figure 3 shows that the CFD protective signal remains consistent across the burden mask and top variant in the maximum queue. It has a protective effect on both wet and dry AMD.

[0013] Figure 4 shows that the top variant 19:860766:G:A (E69K) exhibits consistent protection for both AMD and AMD subtypes.

[0014] Figure 5 illustrates the interaction analysis between the CFD protective variant and the CFH Y402H risk variant in the UKB.

[0015] Figure 6 shows the detection of CFD mRNA in the neuroretina and choroid using RNAscope.

[0016] Figure 7 shows the discovery of CFD proteins in retinal pigment epithelial cells and choroid tissue by immunohistochemistry (IHC).

[0017] Figure 8 illustrates the CFD protein production process.

[0018] Figure 9 shows the C3 convertase assay performed using purified CFD.

[0019] Figure 10 shows the measurement verification.

[0020] Figure 11 shows the dose response of CFD WT and its variants.

[0021] Figure 12 illustrates the dynamics of the CFD WT and its variants. Detailed Implementation

[0022] Various terms relating to aspects of this disclosure are used throughout the specification and claims. Unless otherwise specified, such terms shall be given their ordinary meaning in the art. Other specifically defined terms shall be interpreted in a manner consistent with the definitions provided herein.

[0023] Unless otherwise expressly stated, it is never intended that any method or aspect stated herein require its steps to be performed in a particular order. Therefore, in the claims or description, where a method claim does not explicitly state that the steps are limited to a particular order, it is never intended in any way to infer the order. This applies to any possible non-expressive basis of interpretation, including logical matters relative to the arrangement of steps or the flow of operations, the general meaning derived from grammatical organization or punctuation, or the numbering or type of aspects described in the description.

[0024] As used herein, unless the context clearly indicates otherwise, the singular forms “a / an” and “the” include plural referents.

[0025] As used herein, the term "about" means that the listed values ​​are approximate and that small variations will not significantly affect the practice of the disclosed embodiments. When using numerical values, unless the context otherwise indicates, the term "about" means that the values ​​may vary by ±10% and remain within the range of the disclosed embodiments.

[0026] As used herein, in certain embodiments, the term "comprising" may be replaced with "consisting of" or "substantially composed of" as needed.

[0027] As used herein, the terms “nucleic acid,” “nucleic acid molecule,” “nucleic acid sequence,” “polynucleotide,” or “oligonucleotide” can include polymeric forms of nucleotides of any length, can include DNA and / or RNA, and can be single-stranded, double-stranded, or multi-stranded. One strand of a nucleic acid also refers to its complementary sequence.

[0028] As used herein, the term "subject" includes any animal, including mammals. Mammals include, but are not limited to, farm animals (such as, for example, horses, cattle, and pigs), companion animals (such as, for example, dogs and cats), laboratory animals (such as, for example, mice, rats, and rabbits), and non-human primates. In some embodiments, the subject is a human. In some embodiments, the human is a patient under the care of a physician.

[0029] Analysis of the association between human genetics and AMD revealed that the alternative pathway of the complement system (AP) is a driver of AMD, demonstrating how multiple gene variants influence the risk of AMD. Specifically, according to this disclosure, rare CFD missense variant nucleic acid molecules (19:860766:G:A, E69K) (regardless of whether these variants are homozygous or heterozygous in a particular subject) are the rate-limiting enzyme for AP activation and are associated with a reduced risk of developing macular degeneration, exhibiting protective effects against AMD (all AMD, OR=0.79, P=5.9×10⁻⁶). -7It is believed that CFD variant nucleic acid molecules, which have not previously been shown to cause reduced function or expression levels of CFD proteins, are associated with macular degeneration in humans. Therefore, CFD inhibitors can be used to treat subjects who are CFD-referenced or heterozygous for CFD variant nucleic acid molecules, thereby inhibiting or preventing macular degeneration, reducing or preventing its symptoms, and / or blocking or preventing the development of symptoms. It is also believed that such subjects with macular degeneration can be further treated with one or more macular degeneration treatments or therapies that treat or inhibit macular degeneration. Additionally, this disclosure provides methods for identifying or stratifying the risk of such subjects developing macular degeneration, or diagnosing an increased risk of developing macular degeneration, using the presence or absence of CFD variant nucleic acid molecules in subjects.

[0030] For the purposes of this disclosure, any particular subject, such as a human, may be classified as having one of three CFD genotypes: i) CFD reference; ii) heterozygous for the CFD variant nucleic acid molecule; or iii) homozygous for the CFD variant nucleic acid molecule. A subject is a CFD reference when it has no copy of the CFD variant nucleic acid molecule. A subject is heterozygous for the CFD variant nucleic acid molecule when it has a single copy. A subject is homozygous for the CFD variant nucleic acid molecule when it has two copies.

[0031] In any of the embodiments described herein, the CFD variant nucleic acid molecule may be any nucleic acid molecule (such as a genomic nucleic acid molecule, mRNA molecule, or cDNA molecule derived from an mRNA molecule) encoding a CFD variant polypeptide having partial loss of function, complete loss of function, predicted partial loss of function, or predicted complete loss of function. The subject having a partially lost (or predicted partially lost) CFD polypeptide is hypomorphic to the CFD. In some embodiments, the CFD variant nucleic acid molecule results in reduced or abnormal expression or activity of the CFD mRNA or polypeptide. In some embodiments, the CFD variant nucleic acid molecule is associated with a weakened in vitro response to the CFD ligand compared to a reference CFD. In some embodiments, the CFD variant nucleic acid molecule is a splice site variant, a stop-gain variant, a start-loss variant, a stop-loss variant, a frameshift variant, an in-frame indel variant, or a variant encoding a truncated CFD variant polypeptide. In some embodiments, the CFD variant nucleic acid molecule is a missense variant nucleic acid molecule. In some embodiments, the CFD variant nucleic acid molecule contains a single nucleotide polymorphism (SNP). In some embodiments, the CFD variant nucleic acid molecule contains a variation in the coding region. In some embodiments, the CFD variant nucleic acid molecule does not contain a variation in the non-coding region, but contains a variation in the splice acceptor region (two bases before the start of any exon except the first exon). In some embodiments, the CFD variant nucleic acid molecule causes or is predicted to cause premature truncation of the CFD peptide compared to a reference CFD. In some embodiments, the CFD variant nucleic acid molecule is a variant that, as predicted by an in vitro prediction algorithm (such as Polyphen, SIFT, or a similar algorithm), causes impairment of protein function (and therefore, in this case, is protective against humans). In some embodiments, the CFD variant nucleic acid molecule is a variant that causes or is predicted to cause non-synonymous amino acid substitutions in the CFD nucleic acid molecule and whose allele frequency is less than 1 / 100 alleles in the population from which subjects are selected. In some implementations, the CFD variant nucleic acid molecule is any rare missense variant (allele frequency <0.1%; or 1 / 1,000 alleles), or any splice site variant, stop codon gain variant, start codon deletion variant, stop codon deletion variant, frameshift variant or in-frame insertion deletion variant, or other frameshift CFD variant.

[0032] In any of the embodiments described herein, the CFD variant genomic nucleic acid molecule may contain one or more variants at any location on human chromosome 19 (i.e., positions 859,453-867,884), using the nucleotide sequence of the CFD reference genomic nucleic acid molecule in the GRCh38 / hg38 human genome assembly (see ENSG00000197766, ENST00000327726.11, UniProt P00746; annotated in the Ensembl database (World Wide Web "https: / / useast.ensembl.org / Homo_sapiens / Gene / Summary?g=ENSG00000197766; r=19:859453-867884") as a reference sequence. The sequences of the CFD genomic nucleic acid molecules provided in these transcripts are merely exemplary sequences. Other sequences of CFD genomic nucleic acid molecules are also possible.

[0033] In any of the embodiments described herein, the CFD variant nucleic acid molecule may comprise any one or more of the following genetic variations in the genomic nucleic acid molecule (refer to the chromosome:position listed in the GRCh38 / hg38 human genome assembly): rs35186399 (19:860766:G:A; Glu69Lys), 19:860741:G:A (Trp60Ter), 19:860775:T:G, 19:863105:GC:G (Pro211ArgfsTer101), 19:860933:C:A (Tyr95Ter), 19:861912:G:T (Glu191Ter), 19:863142:CACCTCGGGCTCGCGCGT:C (Thr223LeufsTer115), 19:860714:C:A(Cys51Ter), 19:861785:C:A(Cys148Ter), 19:861926:C:A(Cys195Ter), 19:860692:AG:A(Gln44HisfsTer2), 19:861826:GCCCGGACAGC:G(Pro163CysfsTer28), 19:860752:CG:C(Ala65ArgfsTer91), 19:859745:G:T (NA) or 19:860686:C:A(Ser42Ter), or mRNA molecules generated from said genomic nucleic acid molecules or cDNA molecules generated from said mRNA molecules. In any of the embodiments described herein, variations that result in the Glu69Lys variant due to variations in variant transcripts or protein residue numbering based on propeptides may also result in the Glu76Lys variant.

[0034] Subjects who are genotyped or identified as CFD references have an increased risk of developing macular degeneration. Subjects who are genotyped or identified as CFD references or who are heterozygous for CFD variant nucleic acid molecules can be treated with CFD inhibitors.

[0035] In any of the embodiments described herein, the subject who prevents macular degeneration by administering a CFD inhibitor can be any subject at risk of developing macular degeneration, including but not limited to subjects with a genetic predisposition to develop macular degeneration. In some embodiments, a CFD inhibitor may be administered to a subject already suffering from macular degeneration to prevent the recurrence of macular degeneration in a subject already suffering from macular degeneration. In any of the embodiments described herein, the method can be used to improve macular degeneration.

[0036] In any of the embodiments described herein, the CFD-predicted loss-of-function peptide may be any CFD peptide having partial loss of function, complete loss of function, predicted partial loss of function, or predicted complete loss of function.

[0037] Any one or more (i.e., any combination) of the CFD variant nucleic acid molecules described herein can be used in any of the methods described herein to determine whether a subject's risk of developing macular degeneration is increased or decreased. Combinations of specific variants can form a mask for statistical analysis of a specific correlation between CFD and an increased or decreased risk of developing macular degeneration. In some embodiments, the mask used for statistical analysis of a specific correlation between CFD and an increased or decreased risk of developing macular degeneration may exclude any one or more of these CFD variant nucleic acid molecules described herein.

[0038] In any of the embodiments described herein, the subject may have macular degeneration (such as, for example, age-related macular degeneration; AMD). In any of the embodiments described herein, the subject may be at risk of developing macular degeneration. In any of the embodiments described herein, the macular degeneration may be atrophic (dry macular degeneration). In any of the embodiments described herein, the macular degeneration may be late neovascular (wet macular degeneration). In any of the embodiments described herein, the macular degeneration may be intermediate macular degeneration.

[0039] In any of the embodiments described herein, the method may be used to treat complications or comorbidities of macular degeneration, or to reduce the risk of developing macular degeneration.

[0040] This disclosure provides a method for treating subjects with macular degeneration or at risk of developing macular degeneration, the method comprising administering a CFD inhibitor to the subject.

[0041] In some embodiments, the CFD inhibitor comprises an inhibitory nucleic acid molecule. Examples of inhibitory nucleic acid molecules include, but are not limited to, antisense nucleic acid molecules, small interfering RNA (siRNA), and short hairpin RNA (shRNA). Such inhibitory nucleic acid molecules may be designed to target any region of the CFD nucleic acid molecule. In some embodiments, the antisense RNA, siRNA, or shRNA hybridizes to a sequence within a CFD genomic nucleic acid molecule or mRNA molecule and reduces the expression of the CFD peptide in the subject's cells. In some embodiments, the CFD inhibitor comprises an antisense molecule that hybridizes to a CFD genomic nucleic acid molecule or mRNA molecule and reduces the expression of the CFD peptide in the subject's cells. In some embodiments, the CFD inhibitor comprises siRNA that hybridizes to a CFD genomic nucleic acid molecule or mRNA molecule and reduces the expression of the CFD peptide in the subject's cells. In some embodiments, the CFD inhibitor comprises shRNA that hybridizes to a CFD genomic nucleic acid molecule or mRNA molecule and reduces the expression of the CFD peptide in the subject's cells.

[0042] Repressive nucleic acid molecules may include RNA, DNA, or both. Repressive nucleic acid molecules may also be linked or fused with heterologous nucleic acid sequences (such as heterologous nucleic acid sequences in a vector) or heterologous markers. For example, repressive nucleic acid molecules may be contained within a vector containing a repressive nucleic acid molecule and a heterologous nucleic acid sequence, or as an exogenous donor sequence containing a repressive nucleic acid molecule and a heterologous nucleic acid sequence. Repressive nucleic acid molecules may also be linked or fused with heterologous markers. Markers may be directly detectable (such as fluorophores) or indirectly detectable (such as haptens, enzymes, or fluorophore quenchers). Such markers can be detected by spectroscopic, photochemical, biochemical, immunochemical, or chemical means. Such markers include, for example, radioactive markers, pigments, dyes, chromogens, spin markers, and fluorescent markers. Markers may also be, for example, chemiluminescent substances; metal-containing substances; or enzymes, wherein enzyme-dependent secondary signal generation occurs. The term "marker" may also refer to a "tag" or hapten that selectively binds to a conjugated molecule such that the conjugated molecule is used to generate a detectable signal when subsequently added with a substrate. For example, biotin can be used as a tag along with avidin or streptavidin conjugates of horseradish peroxidase (HRP) to bind to the tag, and the presence of HRP can be detected using a calorimetric substrate (such as, for example, tetramethylbenzidine (TMB)) or a fluorescent substrate. Exemplary tags that can be used as tags to facilitate purification include, but are not limited to, myc, HA, FLAG or 3XFLAG, polyhistidine (i.e., 6XHis, 10XHis), glutathione S-transferase (GST), maltose-binding proteins, epitope tags, or the Fc portion of immunoglobulins. Many tags include, for example, particles, fluorophores, haptens, enzymes and their calorimetric, fluorescent, and chemiluminescent substrates, and other tags.

[0043] Inhibitory nucleic acid molecules may include, for example, nucleotides or non-natural or modified nucleotides, such as nucleotide analogs or nucleotide substitutes. Such nucleotides include nucleotides containing modified bases, sugars, or phosphate groups, or nucleotides incorporating non-natural portions into their structure. Examples of non-natural nucleotides include, but are not limited to, dideoxynucleotides, biotinylated, amination-modified, deamination-modified, alkylated, benzylated, and fluorescently labeled nucleotides.

[0044] Inhibitory nucleic acid molecules may also contain one or more nucleotide analogs or substitutions. Nucleotide analogs are nucleotides containing modifications to their base, sugar, or phosphate moieties. Modifications to the base moieties include, but are not limited to, natural and synthetic modifications to A, C, G, and T / U, as well as to various purine or pyrimidine bases (such as, for example, pseudouridine, uracil-5-yl, hypoxanthine-9-yl (I), and 2-aminoadenine-9-yl). Modified bases include, but are not limited to, 5-methylcytosine (5-me-C), 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyluracil and cytosine, 6-azouracil, cytosine and thymine, 5-Uracil (pseudouracil), 4-thiouracil, 8-halogenated, 8-amino, 8-thio, 8-thioalkyl, 8-hydroxy and other 8-substituted adenine and guanine, 5-halogenated (such as, for example, 5-bromine), 5-trifluoromethyl and other 5-substituted uracil and cytosine, 7-methylguanine, 7-methyladenine, 8-azaguanine, 8-azaadenine, 7-deadenine, 7-deadenine, 3-deadenine and 3-deadenine.

[0045] Nucleotide analogs may also include modifications to the sugar moiety. Modifications to the sugar moiety include, but are not limited to, natural and synthetic modifications of ribose and deoxyribose. Sugar modifications include, but are not limited to, the following modifications at the 2' position: OH; F; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-, S-, or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl, alkenyl, and alkynyl groups may be substituted or unsubstituted C- groups. 1-10 Alkyl or C 2-10 alkenyl and C 2-10 Alkyne group. Exemplary 2' sugar modifications also include, but are not limited to, -O[(CH2)]. n O] m CH3, -O(CH2) n OCH3, -O(CH2) n NH2, -O(CH2) n CH3, -O(CH2) n-ONH2 and -O(CH2) n ON[(CH2) n CH3)]2, where n and m are independently 1 to about 10. Other modifications at the 2' position include, but are not limited to, C 1-10 Alkyl groups, substituted lower alkyl groups, alkylaryl groups, aralkyl groups, O-alkylaryl or O-aralkyl groups, SH, SCH3, OCN, Cl, Br, CN, CF3, OCF3, SOCH3, SO2CH3, ONO2, NO2, N3, NH2, heterocyclic alkyl groups, heterocyclic alkylaryl groups, aminoalkylamino groups, polyalkylamino groups, substituted silyl groups, RNA cleaving groups, reporter groups, intercalating agents, groups used to improve the pharmacokinetic properties of oligonucleotides, or groups used to improve the pharmacodynamic properties of oligonucleotides, and other substituents with similar properties. Similar modifications can also be made at other positions on the sugar, specifically at the 3' position of the sugar on the 3' terminal nucleotide or at the 3' position of the sugar and the 5' position of the 5' terminal nucleotide in 2'-5' linked oligonucleotides. Modified sugars can also include those sugars containing modifications (e.g., CH2 and S) at the bridging epoxy. Nucleotide sugar analogs can also have sugar mimics that replace furanopentoses, such as cyclobutyl moiety.

[0046] Nucleotide analogs may also be modified at the phosphate ester moiety. Modified phosphate ester moieties include, but are not limited to, those that can be modified such that the link between two nucleotides contains the following modified phosphate ester moieties: thiophosphate, chiral thiophosphate, dithiophosphate, phosphate triester, aminoalkyl phosphate triester, methyl and other alkylphosphonates (including 3'-alkylene phosphonates and chiral phosphonates), hypophosphonates, aminophosphates (including 3'-aminoaminophosphate and aminoalkylaminophosphate), thiocarbonyl aminophosphate, thiocarbonyl alkylphosphonate, thiocarbonyl alkyl phosphate triester, and boron phosphate. These phosphate or modified phosphate ester links between two nucleotides may be 3'-5' or 2'-5' links, and said links may contain reversed polarity, such as 3'-5' to 5'-3' or 2'-5' to 5'-2'. Various salts, mixed salts, and free acid forms are also included. Nucleotide substitutes also include peptide nucleic acids (PNAs).

[0047] In some embodiments, the antisense nucleic acid molecule is a gapmer, whereby the first one to seven nucleotides at the 5' and 3' ends are each modified with 2'-methoxyethyl (2'-MOE). In some embodiments, the first five nucleotides at the 5' and 3' ends are each modified with 2'-MOE. In some embodiments, the first one to seven nucleotides at the 5' and 3' ends are RNA nucleotides. In some embodiments, the first five nucleotides at the 5' and 3' ends are RNA nucleotides. In some embodiments, each backbone link between nucleotides is a phosphate thioester linker.

[0048] In some embodiments, the siRNA molecule has a terminal modification. In some embodiments, the 5' end of the antisense strand is phosphorylated. In some embodiments, a non-hydrolyzable 5'-phosphate analogue, such as 5'-(E)-vinylphosphonate, is used.

[0049] In some embodiments, the siRNA molecule has a backbone modification. In some embodiments, modified phosphodiester groups linking consecutive ribonucleotides have been shown to enhance siRNA stability and in vivo bioavailability. The non-ester groups (-OH, =O) of the phosphodiester bond can be replaced with sulfur, boron, or acetate esters to obtain thiophosphate, borate phosphate, and phosphonoacetate bonds. Additionally, replacing the phosphodiester group with a triphosphate ester can promote cellular uptake of siRNA and retain it on serum components by eliminating its negative charge. In some embodiments, the siRNA molecule has a sugar modification. In some embodiments, the sugar is deprotonated (a reaction catalyzed by exonucleases and endonucleases), thereby allowing the 2'-hydroxyl group to act as a nucleophile and attack the adjacent phosphorus in the phosphodiester bond. Such alternatives include 2'-O-methyl, 2'-O-methoxyethyl, and 2'-fluoro modifications.

[0050] In some embodiments, the siRNA molecule has base modifications. In some embodiments, the bases may be substituted with modified bases such as pseudouridine, 5'-methylcytidine, N6-methyladenosine, inosine, and N7-methylguanosine.

[0051] In some implementations, siRNA molecules are conjugated to lipids. Lipids may conjugate to the 5' or 3' end of the siRNA to enhance its bioavailability in vivo by allowing it to associate with serum lipoproteins. Representative lipids include, but are not limited to, cholesterol and vitamin E, as well as fatty acids such as palmitate and tocopherol.

[0052] In some embodiments, the siRNA molecule is conjugated to an antibody or its antigen-binding fragment. In some embodiments, the antibody is an anti-CFD antibody. In some embodiments, the antibody may promote targeting of specific tissue or cell types (i.e., antibodies targeting protein targets in adipose tissue, blood vessels, the retina, or the choroid).

[0053] In some implementations, a representative siRNA has the following formula: Meaning: mN*mN* / i2FN / mN / i2FN / mN / i2FN / mN / i2FN / mN / i2FN / mN / i2FN / mN / i2FN / mN / i2FN / *mN* / 32FN / Antisense: / 52FN / * / i2FN / *mN / i2FN / mN / i2FN / mN / i2FN / mN / i2FN / mN / i2FN / mN / i2FN / mN / i2FN / mN / i2FN / mN / i2FN / mN*N*N Where: “N” is a base; “2F” is 2'-F modification; “m” is 2'-O-methyl modification; “I” is an internal base; and “*” is a phosphate thioester backbone linker.

[0054] In any of the embodiments described herein, the inhibitory nucleic acid molecule may be administered, for example, as an intravenous infusion or subcutaneous injection over one to two hours. In any of the embodiments described herein, the inhibitory nucleic acid molecule may range from about 50 mg to about 900 mg, about 100 mg to about 800 mg, about 150 mg to about 700 mg, or about 175 mg to about 640 mg (2.5 mg / kg to 9.14 mg / kg; 92.5 to 338 mg / m²). 2 – Based on the assumption of a body weight of 70 kg and according to the dose multiplier of 37 mg / kg for humans, the mg / kg dose level is shifted to mg / m 2 (Dose level conversion) dosage level administration.

[0055] This disclosure also provides vectors comprising one or more of the inhibitory nucleic acid molecules. In some embodiments, the vector comprises one or more of the inhibitory nucleic acid molecules and a heterologous nucleic acid. The vector can be a viral or non-viral vector capable of transporting nucleic acid molecules. In some embodiments, the vector is a plasmid or granule (such as a circular double-stranded DNA in which an additional DNA segment can be linked, for example). In some embodiments, the vector is a viral vector in which an additional DNA segment can be linked to a viral genome. Expression vectors include, but are not limited to, plasmids, granules, retroviruses, adenoviruses, adeno-associated viruses (AAVs), plant viruses (such as cauliflower mosaic virus and tobacco mosaic virus), yeast artificial chromosomes (YACs), episomes derived from Epstein-Barr (EBV), and other expression vectors known in the art. In some embodiments, AAV-based gene transfer vectors can be used to express shRNA. In some embodiments, AAVs can be conjugated to antibodies for cell and tissue-specific targeting, such as conjugation to transferrin receptors to enhance crossing of the blood-brain barrier.

[0056] This disclosure also provides compositions comprising any one or more of the inhibitory nucleic acid molecules. In some embodiments, the composition is a pharmaceutical composition. In some embodiments, the composition comprises a carrier and / or an excipient. Examples of carriers include, but are not limited to, poly(lactic acid) (PLA) microspheres, poly(D,L-lactic acid-coglycolic acid) (PLGA) microspheres, liposomes, micelles, reverse micelles, lipid helices, and lipid microtubules. Carriers may include buffered saline solutions, such as PBS, HBSS, etc.

[0057] In some implementations, the CFD inhibitor comprises a nuclease that induces one or more nicks or double-strand breaks at one or more recognition sequences, or a DNA-binding protein that binds to the recognition sequence within a CFD genomic nucleic acid molecule. The recognition sequence may be located within the coding region of the CFD gene or within a regulatory region affecting gene expression. The recognition sequence of the DNA-binding protein or nuclease may be located in an intron, exon, promoter, enhancer, regulatory region, or any non-protein coding region. The recognition sequence may contain or be close to the start codon of the CFD gene. For example, the recognition sequence may be located approximately 10, 20, 30, 40, 50, 100, 200, 300, 400, 500, or 1,000 nucleotides from the start codon. As another example, two or more nucleases may be used, each targeting a nuclease recognition sequence containing or close to the start codon. As another example, two nuclease agents can be used: one targeting a nuclease recognition sequence containing or near the start codon, and the other targeting a nuclease recognition sequence containing or near the stop codon, wherein cleavage by the nuclease agent results in the deletion of the coding region between the two nuclease recognition sequences. Any nuclease agent that induces a nick or double-strand break to the desired recognition sequence can be used in the methods and compositions disclosed herein. Any DNA-binding protein that binds to the desired recognition sequence can be used in the methods and compositions disclosed herein.

[0058] Suitable nucleases and DNA-binding proteins used in this study include, but are not limited to, zinc finger proteins or zinc finger nucleases (ZFN) pairs, transcription activator-like effector (TALE) proteins or transcription activator-like effector nucleases (TALENs), or clustered, regularly distributed short palindromic repeats (CRISPR) / CRISPR-associated (Cas) systems. The recognition sequence length can vary and includes, for example, recognition sequences of approximately 30-36 bp for zinc finger proteins or ZFN pairs, approximately 15-18 bp for each ZFN, approximately 36 bp for TALE proteins or TALENs, and approximately 20 bp for CRISPR / Cas guide RNAs.

[0059] In some embodiments, the CRISPR / Cas system can be used to modify intracellular CFD genomic nucleic acid molecules. The methods and compositions disclosed herein employ the CRISPR-Cas system using a CRISPR complex (containing a guide RNA (gRNA) complexed with a Cas protein) for site-directed cleavage of CFD nucleic acid molecules.

[0060] Cas proteins typically contain at least one RNA recognition or binding domain that can interact with gRNA. Cas proteins may also contain nuclease domains (such as, for example, DNase or RNase domains), DNA-binding domains, helicase domains, protein-protein interaction domains, dimerization domains, and other domains. Suitable Cas proteins include, for example, wild-type Cas9 proteins and wild-type Cpf1 proteins (such as, for example, FnCpf1). Cas proteins may have full cleavage activity to produce double-strand breaks in CFD genome nucleic acid molecules, or they may be cleavage enzymes that produce single-strand breaks in CFD genome nucleic acid molecules. Additional examples of Cas proteins include, but are not limited to, Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas5e (CasD), Cas6, Cas6e, Cas6f, Cas7, Cas8a1, Cas8a2, Cas8b, Cas8c, Cas9 (Csn1 or Csx12), Cas10, Cas10d, CasF, CasG, CasH, Csy1, Csy2, Csy3, Cse1 (CasA), Cse2 (CasB), Cse3 (CasE), and Cse4. (CasC), Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, Csf4, and Cu1966, and their homologs or modified forms. In some embodiments, the Cas system, such as Cas12a, may have multiple gRNAs encoding a single crRNA. Cas proteins may also be operatively linked as fusion proteins to heterologous peptides. For example, Cas proteins may be fused to a cleavage domain, an epigenetic modification domain, a transcriptional activation domain, or a transcriptional repression domain. Cas proteins may be provided in any form. For example, Cas proteins can be provided in the form of proteins, such as Cas proteins complexed with gRNA. Alternatively, Cas proteins can be provided in the form of nucleic acid molecules encoding Cas proteins, such as RNA or DNA. In some embodiments, transduction can be performed using an AAV vector encoding a CRISPR-Cas nuclease.

[0061] In some implementations, targeted genetic modifications to a CFD genomic nucleic acid molecule can be generated by contacting cells with a Cas protein and one or more gRNAs, wherein the one or more gRNAs hybridize to one or more gRNA recognition sequences within a target genomic locus in the CFD genomic nucleic acid molecule. The gRNA recognition sequence may include or be located near the start codon or stop codon of the CFD genomic nucleic acid molecule. For example, the gRNA recognition sequence may be located approximately 10, 20, 30, 40, 50, 100, 200, 300, 400, 500, or 1,000 nucleotides from the start or stop codon.

[0062] In CFD genomic nucleic acid molecules, the gRNA recognition sequence within the target genomic locus is located near a protospacer adjacent motif (PAM) sequence, which is a 2-6 base pair DNA sequence immediately following the DNA sequence targeted by the Cas9 nuclease. A typical PAM is the sequence 5'-NGG-3', where "N" is any nucleobase followed by two guanine ("G") nucleotides. The gRNA can transport Cas9 to any location in the genome for gene editing, but editing may not occur at any site other than the Cas9-recognized PAM site. Alternatively, 5'-NGA-3' can serve as a highly efficient atypical PAM for human cells. Generally, the PAM is located approximately 2-6 nucleotides downstream of the gRNA-targeted DNA sequence. The PAM can be side-attached to the gRNA recognition sequence. In some embodiments, the gRNA recognition sequence may be side-attached to the 3' end by the PAM. In some embodiments, the gRNA recognition sequence may be side-attached to the 5' end by the PAM. For example, the cleavage site of the Cas protein can be approximately one to ten, two to five, or three base pairs upstream or downstream of the PAM sequence. In some embodiments (such as when using Cas9 from *S. pyogenes* or closely related Cas9), the PAM sequence of the non-complementary strand can be 5'-NGG-3', where N is any DNA nucleotide and is immediately adjacent to the 3' of the gRNA recognition sequence of the non-complementary strand of the target DNA. Thus, the PAM sequence of the complementary strand would be 5'-CCN-3', where N is any DNA nucleotide and is immediately adjacent to the 5' of the gRNA recognition sequence of the complementary strand of the target DNA.

[0063] gRNA is an RNA molecule that binds to the Cas protein and targets the Cas protein to a specific location within a CFD genome nucleic acid molecule. An exemplary gRNA is one that effectively guides the Cas enzyme to bind to or cleave a CFD genome nucleic acid molecule, wherein the gRNA contains a DNA targeting fragment that hybridizes to a gRNA recognition sequence within the CFD genome nucleic acid molecule. An exemplary gRNA contains a DNA targeting fragment that hybridizes to a gRNA recognition sequence present within the CFD genome nucleic acid molecule, said gRNA recognition sequence including or near a start codon or stop codon. For example, the gRNA can be selected such that it hybridizes with a gRNA recognition sequence located at approximately 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 100, 200, 300, 400, 500, or 1,000 nucleotides from the start codon or approximately 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 100, 200, 300, 400, 500, or 1,000 nucleotides from the stop codon. Suitable gRNAs may contain approximately 17 to approximately 25 nucleotides, approximately 17 to approximately 23 nucleotides, approximately 18 to approximately 22 nucleotides, or approximately 19 to approximately 21 nucleotides. In some implementations, gRNA may contain 20 nucleotides.

[0064] The Cas protein and gRNA form a complex, and the Cas protein cleaves the CFD genome nucleic acid molecule. The Cas protein can cleave the nucleic acid molecule at sites within or outside the DNA target region of the gRNA present in the CFD genome nucleic acid molecule. For example, the formation of a CRISPR complex (containing gRNA that hybridizes to the gRNA recognition sequence and complexes with the Cas protein) can result in the cleavage of one or both strands within or near the DNA target region of the gRNA present in the CFD genome nucleic acid molecule (e.g., within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50 or more base pairs).

[0065] Such methods can produce CFD genomic nucleic acid molecules in which, for example, regions are disrupted, start codons are disrupted, stop codons are disrupted, or coding sequences are disrupted or deleted. Optionally, the cell can be further contacted with one or more additional gRNAs that hybridize to additional gRNA recognition sequences within target genomic loci in the CFD genomic nucleic acid molecule. By contacting the cell with one or more additional gRNAs (such as a second gRNA that hybridizes to a second gRNA recognition sequence, cleavage by the Cas protein can produce two or more double-strand breaks or two or more single-strand breaks.

[0066] In any of the treatment or prevention methods described herein, the subject receiving treatment may contain a CFD variant nucleic acid molecule. In some embodiments, the subject receiving treatment is heterozygous for a CFD variant nucleic acid molecule. In some embodiments, the subject receiving treatment is a CFD reference. The CFD variant nucleic acid molecule may be any CFD variant nucleic acid molecule disclosed herein. In some implementations, the CFD variant nucleic acid molecule is a CFD variant genomic nucleic acid molecule containing the following genetic variations: rs35186399 (19:860766:G:A; Glu69Lys), 19:860741:G:A (Trp60Ter), 19:860775:T:G, 19:863105:GC:G (Pro211ArgfsTer101), 19:860933:C:A (Tyr95Ter), 19:861912:G:T (Glu191Ter), 19:863142:CACCTCGGGCTCGCGCGT:C (Thr223LeufsTer115), 19:860714:C:A (Cys51Ter), 19:861785:C:A (Cys148Ter), 19:861926:C:A (Cys195Ter), 19:860692:AG:A(Gln44HisfsTer2), 19:861826:GCCCGGACAGC:G (Pro163CysfsTer28), 19:860752:CG:C(Ala65ArgfsTer91), 19:859745:G:T (NA) or 19:860686:C:A (Ser42Ter), or an mRNA molecule produced from the said genomic nucleic acid molecule or a cDNA molecule produced from the said mRNA molecule.

[0067] In some embodiments, the treatment or prevention method further includes detecting the presence or absence of a CFD variant nucleic acid molecule in a biological sample from the subject. In some embodiments, the CFD variant nucleic acid molecule may be any CFD variant nucleic acid molecule disclosed herein. In some implementations, the CFD variant nucleic acid molecule is a CFD variant genomic nucleic acid molecule containing the following genetic variations: rs35186399 (19:860766:G:A; Glu69Lys), 19:860741:G:A (Trp60Ter), 19:860775:T:G, 19:863105:GC:G (Pro211ArgfsTer101), 19:860933:C:A (Tyr95Ter), 19:861912:G:T (Glu191Ter), 19:863142:CACCTCGGGCTCGCGCGT:C (Thr223LeufsTer115), 19:860714:C:A (Cys51Ter), 19:861785:C:A (Cys148Ter), 19:861926:C:A (Cys195Ter), 19:860692:AG:A (Gln44HisfsTer2), 19:861826:GCCCGGACAGC:G (Pro163CysfsTer28), 19:860752:CG:C (Ala65ArgfsTer91), 19:859745:G:T (NA) or 19:860686:C:A (Ser42Ter), or an mRNA molecule produced from the said genomic nucleic acid molecule or a cDNA molecule produced from the said mRNA molecule.

[0068] This disclosure also provides a method of treating a subject with a macular degeneration treatment agent or macular degeneration therapy to treat or inhibit macular degeneration, wherein the subject has macular degeneration or is at risk of developing macular degeneration. The method includes determining whether the subject has a CFD variant nucleic acid molecule by obtaining or having obtained a biological sample from the subject, and performing or having performed sequence analysis on the biological sample to determine whether the subject has a genotype containing a CFD variant nucleic acid molecule. In an embodiment where the subject is a CFD reference, the method further includes administering or continuing to administer to the subject an amount of macular degeneration treatment agent or macular degeneration therapy equal to or less than a standard dose and / or administering a CFD inhibitor to the subject. In an embodiment where the subject is heterozygous for a CFD variant nucleic acid molecule, the method further includes administering or continuing to administer to the subject an amount of macular degeneration treatment agent or macular degeneration therapy equal to or less than a standard dose and / or administering a CFD inhibitor to the subject. In an embodiment where the subject is homozygous for a CFD variant nucleic acid molecule, the method further includes administering or continuing to administer to the subject a standard dose of macular degeneration treatment agent or macular degeneration therapy. The presence of a CFD variant nucleic acid molecule indicates a reduced risk of developing macular degeneration in the subject. In some embodiments, the subject is a CFD reference. In some embodiments, the subject is heterozygous for a CFD variant nucleic acid molecule. In some embodiments, the subject is homozygous for a CFD variant nucleic acid molecule. In any of the embodiments described herein, a CFD inhibitor is an example of a macular degeneration therapeutic agent.In some implementations, the CFD variant nucleic acid molecule is a CFD variant genomic nucleic acid molecule containing the following genetic variations: rs35186399 (19:860766:G:A; Glu69Lys), 19:860741:G:A (Trp60Ter), 19:860775:T:G, 19:863105:GC:G (Pro211ArgfsTer101), 19:860933:C:A (Tyr95Ter), 19:861912:G:T (Glu191Ter), 19:863142:CACCTCGGGCTCGCGCGT:C (Thr223LeufsTer115), 19:860714:C:A (Cys51Ter), 19:861785:C:A (Cys148Ter), 19:861926:C:A (Cys195Ter), 19:860692:AG:A (Gln44HisfsTer2), 19:861826:GCCCGGACAGC:G (Pro163CysfsTer28), 19:860752:CG:C (Ala65ArgfsTer91), 19:859745:G:T (NA) or 19:860686:C:A (Ser42Ter), or an mRNA molecule produced from the said genomic nucleic acid molecule or a cDNA molecule produced from the said mRNA molecule.

[0069] For subjects who have been genotyped or identified as CFD references or heterozygous for CFD variant nucleic acid molecules, CFD inhibitors may be administered to such subjects as described in this article.

[0070] The detection of the presence or absence of CFD variant nucleic acid molecules in biological samples from a subject and / or the determination of whether a subject possesses CFD variant nucleic acid molecules can be performed by any of the methods described herein. In some embodiments, these methods can be performed in vitro. In some embodiments, these methods can be performed in situ. In some embodiments, these methods can be performed in vivo. In any of these embodiments, the nucleic acid molecules can be present within cells obtained from the subject.

[0071] In some implementations, when the subject is a CFD reference, the subject is administered an amount of macular degeneration treatment agent or macular degeneration therapy and / or CFD inhibitor equal to or less than the standard dose. In some implementations, when the subject is heterozygous for a CFD variant nucleic acid molecule, the subject is administered an amount of macular degeneration treatment agent or macular degeneration therapy and / or CFD inhibitor equal to or less than the standard dose.

[0072] In some embodiments, the treatment or prevention method includes detecting the presence or absence of reduced expression of CFD variant mRNA or peptide in a biological sample from the subject. In some embodiments, when the expression of the subject's CFD variant mRNA or peptide is not reduced, the subject is administered an amount of macular degeneration treatment agent or macular degeneration therapy and / or CFD inhibitor equal to or less than a standard dose. In some embodiments, when the expression of the subject's CFD variant mRNA or peptide is reduced, the subject is administered a standard dose of macular degeneration treatment agent or macular degeneration therapy.

[0073] This disclosure also provides a method of treating a subject with a macular degeneration treatment agent or macular degeneration therapy that treats or inhibits macular degeneration, wherein the subject has macular degeneration or is at risk of developing macular degeneration. The method includes determining whether the expression of a CFD variant mRNA or peptide in the subject is reduced by obtaining or having obtained a biological sample from the subject, and by performing or having performed a assay on the biological sample to determine whether the expression of the subject's CFD variant mRNA or peptide is reduced. In embodiments where the expression of the subject's CFD variant mRNA or peptide is not reduced, the method further includes administering or continuing to administer to the subject an amount of a macular degeneration treatment agent or macular degeneration therapy equal to or less than a standard dose and / or administering a CFD inhibitor to the subject. In embodiments where the expression of the subject's CFD variant mRNA or peptide is reduced, the method further includes administering or continuing to administer to the subject a standard dose of a macular degeneration treatment agent or macular degeneration therapy. The presence of reduced CFD variant mRNA or peptide expression indicates a reduced risk of the subject developing macular degeneration. In some embodiments, the expression of the subject's CFD variant mRNA or peptide is reduced. In some implementations, the expression of CFD variant mRNA or peptide in the subject is not reduced. In any of the implementations described herein, a CFD inhibitor is an example of a macular degeneration treatment. In some implementations, the CFD variant nucleic acid molecule is a CFD variant genomic nucleic acid molecule containing the following genetic variations: rs35186399 (19:860766:G:A; Glu69Lys), 19:860741:G:A (Trp60Ter), 19:860775:T:G, 19:863105:GC:G (Pro211ArgfsTer101), 19:860933:C:A (Tyr95Ter), 19:861912:G:T (Glu191Ter), 19:863142:CACCTCGGGCTCGCGCGT:C (Thr223LeufsTer115), 19:860714:C:A (Cys51Ter), 19:861785:C:A (Cys148Ter), 19:861926:C:A (Cys195Ter), 19:860692:AG:A (Gln44HisfsTer2), 19:861826:GCCCGGACAGC:G (Pro163CysfsTer28), 19:860752:CG:C (Ala65ArgfsTer91), 19:859745:G:T (NA) or 19:860686:C:A (Ser42Ter), or an mRNA molecule produced from the said genomic nucleic acid molecule or a cDNA molecule produced from the said mRNA molecule.

[0074] Detection of reduced expression of CFD variant mRNA or peptide can be performed using a variety of known methods. In some embodiments, these methods are performed in vitro. In some embodiments, these methods are performed in situ. In some embodiments, these methods are performed in vivo. In any of these embodiments, the mRNA or peptide may be present within cells obtained from the subject.

[0075] In some embodiments, the treatment or prevention method includes detecting the presence or absence of a CFD variant peptide in a biological sample from the subject. In some embodiments, when the subject does not have a CFD variant peptide, the subject is administered an amount of a macular degeneration treatment agent or macular degeneration therapy and / or a CFD inhibitor equal to or less than a standard dose. In some embodiments, when the subject has a CFD variant peptide, the subject is administered a standard dose of a macular degeneration treatment agent or macular degeneration therapy.

[0076] This disclosure also provides a method of treating a subject with a macular degeneration treatment agent or macular degeneration therapy to treat or inhibit macular degeneration, wherein the subject has macular degeneration or is at risk of developing macular degeneration. The method includes determining whether the subject has a CFD variant peptide by obtaining or having obtained a biological sample from the subject, and by performing or having performed a assay on the biological sample to determine whether the subject has a CFD variant peptide. When the subject does not have a CFD variant peptide, administering to the subject an amount of a macular degeneration treatment agent or macular degeneration therapy and / or a CFD inhibitor equal to or less than a standard dose. When the subject has a CFD variant peptide, administering to the subject a standard dose of a macular degeneration treatment agent or macular degeneration therapy. The presence of a CFD variant peptide indicates a reduced risk of the subject developing macular degeneration. In some embodiments, the subject has a CFD variant peptide. In some embodiments, the subject does not have a CFD variant peptide.

[0077] This disclosure also provides a method for preventing a subject from developing macular degeneration by administering a macular degeneration treatment agent or therapy. In some embodiments, the method includes determining whether a subject has a CFD variant peptide by obtaining or having obtained a biological sample from the subject, and performing or having performed a assay on the biological sample to determine whether the subject has a CFD variant peptide. When the subject does not have a CFD variant peptide, administering to the subject an amount of a macular degeneration treatment agent or therapy and / or a CFD inhibitor equal to or less than a standard dose. When the subject has a CFD variant peptide, administering to the subject a standard dose of a macular degeneration treatment agent or therapy. The presence of a CFD variant peptide indicates a reduced risk of the subject developing macular degeneration. In some embodiments, the subject has a CFD variant peptide. In some embodiments, the subject does not have a CFD variant peptide.

[0078] Detecting the presence or absence of CFD variant peptides in biological samples from a subject and / or determining whether a subject possesses a CFD variant peptide can be performed by any of the methods described herein. In some embodiments, these methods can be performed in vitro. In some embodiments, these methods can be performed in situ. In some embodiments, these methods can be performed in vivo. In any of these embodiments, the peptide may be present within cells obtained from the subject.

[0079] In some embodiments, the CFD inhibitor is a small molecule. In some embodiments, the small molecule is a low molecular weight (<900 Daltons) organic compound. In some embodiments, the CFD inhibitor includes, but is not limited to, danicopan (ACH-4471), danicopan analogs (ACH-5228 or ALXN-2050), BCX9930, BCX1470, nafamostat, and vemircopan. In some embodiments, the CFD inhibitor includes danicopan (ACH-4471). In some embodiments, the CFD inhibitor includes danicopan analogs (ACH-5228 or ALXN-2050). In some embodiments, the CFD inhibitor includes BCX9930. In some embodiments, the CFD inhibitor includes BCX1470. In some embodiments, the CFD inhibitor includes nafamostat. In some embodiments, the CFD inhibitor includes vemircopan. In some embodiments, the CFD inhibitor comprises a small molecule disclosed in PCT Publication WO2014 / 002051, U.S. Patent No. 10,092,584, U.S. Patent Application Publication 2019 / 0345135, or PCT Publication WO2017 / 136395.

[0080] In some embodiments, the CFD inhibitor comprises an antibody or an antigen-binding fragment thereof. In some embodiments, the antibody or its antigen-binding fragment specifically binds to a human CFD. In some embodiments, the antibody is a fully human monoclonal antibody (mAb) or its antigen-binding fragment that specifically binds to and neutralizes, inhibits, blocks, eliminates, reduces, or interferes with at least one activity of a CFD (especially a human CFD). In some embodiments, the antibody or its fragment can neutralize, inhibit, block, eliminate, reduce, or interfere with the activity of a CFD by binding to an epitope of the CFD that is directly involved in the targeting activity of the CFD. In some embodiments, the antibody or its fragment can neutralize, inhibit, block, eliminate, reduce, or interfere with the activity of a CFD by binding to an epitope of the CFD that is not directly involved in the targeting activity of the CFD, but the antibody or fragment bound to said epitope spatially or conformally inhibits, blocks, eliminates, reduces, or interferes with the targeting activity of the CFD. In some embodiments, the antibody or a fragment thereof binds to an epitope of the CFD that is not directly involved in the targeting activity of the CFD (i.e., a non-blocking antibody). However, the antibody or fragment bound to the epitope results in enhanced clearance of the CFD from circulation compared to the clearance of the CFD in the absence of the antibody or fragment thereof, thereby indirectly inhibiting, blocking, eliminating, reducing, or interfering with the activity of the CFD. CFD clearance from circulation can be particularly enhanced by combining two or more different non-blocking antibodies that do not compete with each other for specific binding to the CFD. The antibody may be full-length (e.g., IgG1 or IgG4 antibody) or may contain only the antigen-binding portion (e.g., Fab, F(ab')2, or scFv fragments) and may be modified to affect function, e.g., to eliminate residual effector function (Reddy et al., J. Immunol., 2000, 164, 1925-1933). In some embodiments, the antibody includes lambalizumab.

[0081] In some implementations, the antibody or its antigen-binding fragment specifically binds to the equilibrium dissociation constant (K) of the CFD. D The values ​​are approximately 7 nM or less, approximately 6 nM or less, approximately 5 nM or less, approximately 4 nM or less, approximately 3 nM or less, approximately 2 nM or less, or approximately 1 nM or less, as determined by surface plasmon resonance (e.g., BIACORE). TM ) as measured. In some implementations, the antibody exhibits K D Approximately 800 pM or less; approximately 700 pM or less; approximately 600 pM or less; approximately 500 pM or less; approximately 400 pM or less; approximately 300 pM or less; approximately 200 pM or less; approximately 100 pM or less; or approximately 50 pM or less.

[0082] In some implementations, anti-CFD antibodies have modified glycosylation patterns. In some applications, modification to remove undesirable glycosylation sites, or, for example, to remove the fucose moiety to increase antibody-dependent cytotoxicity (ADCC) function, may be useful (see Shield et al., J. Biol. Chem., 2002, 277, 26733). In other applications, removal of N-glycosylation sites may reduce undesirable immune responses against therapeutic antibodies or increase antibody affinity. In still other applications, galactosylation may be modified to modify complement-dependent cytotoxicity (CDC).

[0083] This disclosure also provides compositions comprising an antibody or an antigen-binding fragment thereof in combination with a macular degeneration treatment agent.

[0084] In some embodiments, macular degeneration treatment agents include, but are not limited to, aflibercept, ranibizumab, bevacizumab, pegaptanib, conbercept, brolucizumab, faricimab, nepafenac, verteporfin, ketorolac, lidocaine, pegcetacoplan, and avacincaptad pegolintravitreal solution. In some embodiments, the macular degeneration treatment agent includes aflibercept. In some embodiments, the macular degeneration treatment agent includes ranibizumab. In some embodiments, the macular degeneration treatment agent includes bevacizumab. In some embodiments, the macular degeneration treatment agent includes pegaptanib. In some embodiments, the macular degeneration treatment agent includes conbercept. In some embodiments, the macular degeneration treatment agent includes broluximab. In some embodiments, the macular degeneration treatment agent includes fareximab. In some embodiments, the macular degeneration treatment agent includes nepafen. In some embodiments, the macular degeneration treatment agent includes verteporfen. In some embodiments, the macular degeneration treatment agent includes ketoroxyproline. In some embodiments, the macular degeneration treatment agent includes lidocaine. In some embodiments, the macular degeneration treatment agent includes EMPAVELI® (pecetacoplan). In some embodiments, the macular degeneration treatment agent includes IZERVAY. TM(Avacincapate polyethylene glycol intravitreal solution). Additional macular degeneration therapies include any therapy used to reduce or manage risk factors for macular degeneration. In some embodiments, a macular degeneration treatment agent or therapy may be combined with a CFD inhibitor. In some embodiments, the treatment for macular degeneration is laser photocoagulation / photodynamic therapy (PDT), optionally used in combination with verteporfen. These treatments can be delayed or completely avoided by treatment with a CFD inhibitor as described herein.

[0085] In some implementations, the dosage of the macular degeneration treatment agent for treating, preventing, or inhibiting macular degeneration may be reduced by approximately 10%, approximately 20%, approximately 30%, approximately 40%, approximately 50%, approximately 60%, approximately 70%, approximately 80%, or approximately 90% for subjects heterozygous for CFD variant nucleic acid molecules or as a CFD reference (i.e., less than the standard dose) compared to subjects homozygous for CFD variant nucleic acid molecules (whose acceptable standard dose). In some implementations, the dosage of the macular degeneration treatment agent for treating, preventing, or inhibiting macular degeneration may be reduced by approximately 10%, approximately 20%, approximately 30%, approximately 40%, approximately 50%, approximately 60%, approximately 70%, approximately 80%, or approximately 90% for subjects heterozygous for CFD variant nucleic acid molecules or as a CFD reference compared to subjects as a CFD reference. In addition, compared to subjects who are heterozygous for CFD variant nucleic acid molecules, the dosage of macular degeneration treatment agents can be administered at a lower frequency to subjects who are heterozygous for CFD variant nucleic acid molecules or who serve as a CFD reference.

[0086] Treatment, prevention, or inhibition of macular degeneration with macular degeneration agents and / or CFD inhibitors may be repeated, for example, after one, two, three, five, one week, two, three weeks, one month, five, six, seven, eight weeks, two months, or three months. Repeated administration may be at the same dose or at different doses. Administration may be repeated once, twice, three times, four times, five times, six times, seven times, eight times, nine times, ten times, or more. For example, depending on certain dosage regimens, subjects may receive therapy for longer periods, such as, for example, six months, one year, or longer.

[0087] Administering macular degeneration treatments and / or CFD inhibitors may be done via any suitable route, including but not limited to parenteral, intravenous, oral, subcutaneous, intra-arterial, intracranial, intrathecal, intraperitoneal, topical, intranasal, or intramuscular routes. The pharmaceutical composition intended for administration is expected to be sterile and substantially isotonic, and manufactured under GMP conditions. The pharmaceutical composition may be provided in unit dosage forms (i.e., a single-dose dose). The pharmaceutical composition may be formulated using one or more physiologically and pharmaceutically acceptable carriers, diluents, excipients, or adjuvants. The formulation depends on the chosen route of administration. The term "pharmaceutically acceptable" means that the carrier, diluent, excipient, or adjuvant is compatible with the other components of the formulation and is substantially harmless to the recipient.

[0088] As used herein, the terms “treat,” “treating,” and “treatment,” as well as “prevent,” “preventing,” and “prevention,” refer to eliciting a desired biological response, such as a therapeutic effect and a preventive effect, respectively. In some embodiments, after administration of the agent or a composition containing the agent, the therapeutic effect includes one or more of the following: reduction / alleviation of macular degeneration, reduction / alleviation of the severity of macular degeneration (such as, for example, alleviating or inhibiting the development of macular degeneration), reduction / alleviation of symptom and disease-related effects, delay of the onset of symptom and disease-related effects, alleviation of the severity of symptoms of disease-related effects, reduction of the number of symptom and disease-related effects, reduction of the latency of symptom and disease-related effects, improvement of symptom and disease-related effects, reduction of secondary symptoms, reduction of secondary infections, prevention of macular degeneration recurrence, reduction of the number or frequency of recurrences, increase of the latency between symptom onsets, increase of the duration of continuous progression, accelerated recovery or increased efficacy of alternative treatments or reduction of resistance to alternative treatments and / or increased survival time of affected host animals. Preventive effects may include completely or partially avoiding / inhibiting or delaying the development / progression of macular degeneration (e.g., completely or partially avoiding / inhibiting or delaying it) after the administration of a treatment regimen, and increasing the survival time of affected host animals. Treatment for macular degeneration covers treating subjects diagnosed with any form of macular degeneration at any clinical stage or presentation, delaying the onset or evolution or exacerbation or worsening of symptoms or signs of macular degeneration, and / or preventing and / or reducing the severity of macular degeneration.

[0089] In some embodiments, a CFD inhibitor and a macular degeneration treatment agent are contained within a pharmaceutical composition. In some embodiments, the CFD inhibitor is contained within a first pharmaceutical composition and the macular degeneration treatment agent is contained within a second pharmaceutical composition. In some embodiments, the first pharmaceutical composition and the second pharmaceutical composition are administered simultaneously. In some embodiments, the first pharmaceutical composition is administered before the second pharmaceutical composition. In some embodiments, the first pharmaceutical composition is administered after the second pharmaceutical composition.

[0090] This disclosure also provides methods for identifying subjects at increased risk of developing macular degeneration. In some embodiments, the method includes determining, or having determined, the presence or absence of CFD variant nucleic acid molecules (such as genomic nucleic acid molecules, mRNA molecules, and / or cDNA molecules) in a biological sample obtained from the subject. When a subject lacks a CFD variant nucleic acid molecule (i.e., the subject's genotype is classified as CFD reference), the subject's risk of developing macular degeneration is increased. When a subject has a CFD variant nucleic acid molecule (i.e., the subject is heterozygous or homozygous for a CFD variant nucleic acid molecule), the subject's risk of developing macular degeneration is decreased. In some implementations, the CFD variant nucleic acid molecule is a CFD variant genomic nucleic acid molecule containing the following genetic variations: rs35186399 (19:860766:G:A; Glu69Lys), 19:860741:G:A (Trp60Ter), 19:860775:T:G, 19:863105:GC:G (Pro211ArgfsTer101), 19:860933:C:A (Tyr95Ter), 19:861912:G:T (Glu191Ter), 19:863142:CACCTCGGGCTCGCGCGT:C (Thr223LeufsTer115), 19:860714:C:A (Cys51Ter), 19:861785:C:A (Cys148Ter), 19:861926:C:A (Cys195Ter), 19:860692:AG:A (Gln44HisfsTer2), 19:861826:GCCCGGACAGC:G (Pro163CysfsTer28), 19:860752:CG:C (Ala65ArgfsTer91), 19:859745:G:T (NA) or 19:860686:C:A (Ser42Ter), or an mRNA molecule produced from the said genomic nucleic acid molecule or a cDNA molecule produced from the said mRNA molecule.

[0091] A single copy of a CFD-variant nucleic acid molecule provides better protection against macular degeneration than a copy without the CFD-variant. Without intending to be limited to any particular theory or mechanism of action, it is believed that a single copy of a CFD-variant nucleic acid molecule (i.e., heterozygous for the CFD-variant) protects a subject from developing macular degeneration, and it is also believed that two copies of a CFD-variant nucleic acid molecule (i.e., homozygous for the CFD-variant) provide better protection against macular degeneration than a single copy. Therefore, in some implementations, a single copy of a CFD-variant nucleic acid molecule may not provide complete protection, but may provide partial or incomplete protection against macular degeneration. While not wishing to be bound by any particular theory, additional factors or molecules involved in the development of macular degeneration may exist in subjects with a single copy of the CFD-variant nucleic acid molecule, thus resulting in less complete protection against macular degeneration.

[0092] Determining whether a subject possesses CFD-variant nucleic acid molecules in a sample from the subject and / or determining whether a subject possesses CFD-variant nucleic acid molecules can be performed by any of the methods described herein. In some embodiments, these methods can be performed in vitro. In some embodiments, these methods can be performed in situ. In some embodiments, these methods can be performed in vivo. In any of these embodiments, the nucleic acid molecules may be present within cells obtained from the subject.

[0093] In some embodiments, when a subject is identified as having an increased risk of developing macular degeneration, a macular degeneration treatment agent or therapy and / or a CFD inhibitor is administered to the subject, as described herein. For example, when a subject is a CFD reference and therefore has an increased risk of developing macular degeneration, an amount of a macular degeneration treatment agent or therapy and / or a CFD inhibitor is administered to the subject, which is the same as or less than the standard dose. In some embodiments, when a subject is heterozygous for a CFD variant nucleic acid molecule, an amount of a macular degeneration treatment agent or therapy and / or a CFD inhibitor is administered to the subject, which is the same as or less than the standard dose. In some embodiments, when a subject is homozygous for a CFD variant nucleic acid molecule, a standard dose of a macular degeneration treatment agent or therapy is administered to the subject. In some embodiments, the subject is a CFD reference. In some embodiments, the subject is heterozygous for a CFD variant nucleic acid molecule. In some embodiments, the subject is homozygous for a CFD variant nucleic acid molecule.

[0094] This disclosure also provides a total burden or risk score for determining whether a subject has a total burden or risk score for two or more CFD variant nucleic acid molecules and / or two or more CFD variant peptides associated with a reduced risk of developing macular degeneration. Total burden is the sum of two or more genetic variants that can be analyzed in association with macular degeneration. In some embodiments, the subject is homozygous for one or more CFD variant nucleic acid molecules associated with a reduced risk of developing macular degeneration. In some embodiments, the subject is heterozygous for one or more CFD variant nucleic acid molecules associated with a reduced risk of developing macular degeneration. When a subject has a low total burden, the risk of developing macular degeneration is increased, and the subject is given or continues to be given an amount of macular degeneration treatment agent or macular degeneration therapy and / or CFD inhibitor equal to or less than a standard dose. When a subject has a high total burden, the risk of developing macular degeneration is reduced, and the subject is given or continues to be given a standard dose of macular degeneration treatment agent or macular degeneration therapy. The higher the total burden, the lower the risk of developing macular degeneration.

[0095] In some implementations, the total load of any two or more CFD variant nucleic acid molecules in the subject represents a weighted sum of multiple CFD variant nucleic acid molecules. In some implementations, the total load is calculated using at least about 2, at least about 3, at least about 4, at least about 5, at least about 10, at least about 20, at least about 30, at least about 40, at least about 50, at least about 60, at least about 70, at least about 80, at least about 100, at least about 120, at least about 150, at least about 200, at least about 250, at least about 300, at least about 400, at least about 500, at least about 1,000, at least about 10,000, at least about 100,000, or at least about or greater than 1,000,000 genetic variants present in or around the CFD gene (up to 10 Mb), wherein the genetic load is the number of alleles multiplied by an association estimate with macular degeneration or a result associated with each allele (e.g., a weighted polygenic load score). In some implementations, the risk of developing macular degeneration is reduced when the subject's total workload is above the expected threshold score. In other implementations, the risk of developing macular degeneration is increased when the subject's total workload is below the expected threshold score.

[0096] In some implementations, the total burden may be divided into quintiles, such as the highest quintile, second quintile, middle quintile, fourth quintile, and lowest quintile, wherein the highest quintile of the total burden corresponds to the lowest risk group, and the lowest quintile of the total burden corresponds to the highest risk group. In some implementations, subjects with higher total burdens include those with the highest weighted total burden, including but not limited to the top 10%, top 20%, top 30%, top 40%, or top 50% of the total burden of the subject population. In some implementations, the genetic variants include those associated with macular degeneration that are among the top 10%, top 20%, top 30%, top 40%, or top 50% of the relevant p-value range. In some implementations, each identified genetic variant includes a macular degeneration-associated variant with a p-value no greater than about 10. -2 Approximately 10 -3 Approximately 10 -4 Approximately 10 -5 Approximately 10 -6 Approximately 10 -7 Approximately 10 -8 Approximately 10 -9 Approximately 10 -10 Approximately 10 -11 Approximately 10 -12 Approximately 10 -13 Approximately 10 -14 Or about 10 -15 In some implementations, the identified genetic variants include those with p-values ​​less than 5 × 10⁻⁶. -8 Genetic variants associated with macular degeneration. In some embodiments, the identified genetic variants include those associated with macular degeneration in high-risk subjects, having the following odds ratios (ORs) compared to the remainder of the reference population: approximately 1.5 or greater, approximately 1.75 or greater, approximately 2.0 or greater, or approximately 2.25 or greater for the top 20% of the distribution; or approximately 1.5 or greater, approximately 1.75 or greater, approximately 2.0 or greater, approximately 2.25 or greater, approximately 2.5 or greater, or approximately 2.75 or greater. In some implementations, the odds ratio (OR) may range from about 1.0 to about 1.5, about 1.5 to about 2.0, about 2.0 to about 2.5, about 2.5 to about 3.0, about 3.0 to about 3.5, about 3.5 to about 4.0, about 4.0 to about 4.5, about 4.5 to about 5.0, about 5.0 to about 5.5, about 5.5 to about 6.0, about 6.0 to about 6.5, about 6.5 to about 7.0, or greater than 7.0. In some implementations, high-risk subjects have the lowest decile, quintile, or tertiary total load in the reference population. The threshold for total load may be determined based on the nature of the intended practical application and the risk difference that would be considered meaningful for said practical application.

[0097] In an implementation scheme for determining the total burden of CFD genetic variants associated with macular degeneration, the total burden represents the risk score of a subject developing macular degeneration. In some implementations, the total burden or risk score includes a CFD variant genomic nucleic acid molecule containing the following genetic variations: rs35186399 (19:860766:G:A; Glu69Lys), 19:860741:G:A (Trp60Ter), 19:860775:T:G, 19:863105:GC:G (Pro211ArgfsTer101), 19:860933:C:A (Tyr95Ter), 19:861912:G:T (Glu191Ter), 19:863142:CACCTCGGGCTCGCGCGT:C (Thr223LeufsTer115), 19:860714:C:A (Cys51Ter), 19:861785:C:A (Cys148Ter), 19:861926:C:A (Cys195Ter), 19:860692:AG:A (Gln44HisfsTer2), 19:861826:GCCCGGACAGC:G (Pro163CysfsTer28), 19:860752:CG:C (Ala65ArgfsTer91), 19:859745:G:T (NA), or 19:860686:C:A (Ser42Ter), or mRNA molecules generated from said genomic nucleic acid molecules, or cDNA molecules generated from said mRNA molecules. In some embodiments, the total burden of a subject may be determined by combining other genetic variants of other genes associated with macular degeneration with CFD genetic variants associated with macular degeneration to generate a polygenic risk score (PRS) for developing macular degeneration.In some implementations, the PRS includes a CFD variant genomic nucleic acid molecule containing the following genetic variations: rs35186399 (19:860766:G:A; Glu69Lys), 19:860741:G:A (Trp60Ter), 19:860775:T:G, 19:863105:GC:G (Pro211ArgfsTer101), 19:860933:C:A (Tyr95Ter), 19:861912:G:T (Glu191Ter), 19:863142:CACCTCGGGCTCGCGCGT:C (Thr223LeufsTer115), 19:860714:C:A (Cys51Ter), 19:861785:C:A (Cys148Ter), 19:861926:C:A (Cys195Ter), 19:860692:AG:A (Gln44HisfsTer2), 19:861826:GCCCGGACAGC:G (Pro163CysfsTer28), 19:860752:CG:C (Ala65ArgfsTer91), 19:859745:G:T (NA), or 19:860686:C:A (Ser42Ter), or mRNA molecules generated from said genomic nucleic acid molecules, or cDNA molecules generated from said mRNA molecules. In some embodiments, the total burden of a subject may be determined by targeting a CFD genetic variant associated with macular degeneration, such as a complement factor H (CFH) genetic variant associated with macular degeneration (e.g., Y402H), to generate a PRS for the development of macular degeneration.

[0098] This disclosure also provides a method for detecting the presence or absence of CFD variant nucleic acid molecules (i.e., genomic nucleic acid molecules, mRNA molecules, or cDNA molecules derived from mRNA molecules) in biological samples from subjects. It should be understood that gene sequences within a population and the mRNA molecules encoded by such genes can vary due to polymorphisms, such as single nucleotide polymorphisms.

[0099] Biological samples can be derived from any cells, tissues, or biological fluids from a subject. Biological samples can include any clinically relevant tissue, such as bone marrow samples, tumor biopsies, fine-needle aspiration, or bodily fluid samples, such as blood, gingival crevicular fluid, plasma, serum, lymph, ascites, cystic fluid, or urine. In some cases, samples include oral swabs. The biological samples used in the methods disclosed herein can vary based on the assay format, the nature of the detection method, and the tissue, cells, or extract used as the sample. Biological samples can be processed differently depending on the assay employed. For example, when detecting any CFD variant nucleic acid molecule, preliminary processing designed to isolate or enrich the genomic DNA of the biological sample can be employed. Various techniques can be used for this purpose. When detecting the level of any CFD variant nucleic acid molecule, different techniques can be used to enrich the biological sample with mRNA molecules. Various methods can be used to detect the presence or level of mRNA molecules or the presence of specific variant genomic DNA loci.

[0100] In some implementations, detecting CFD variant nucleic acid molecules in a subject includes sequence analysis of a biological sample obtained from the subject to determine the presence of CFD genomic nucleic acid molecules and / or CFD mRNA molecules and / or CFD cDNA molecules generated from mRNA molecules in the biological sample. In some implementations, the method detects genomic nucleic acid molecules containing the following genetic variations: rs35186399 (19:860766:G:A; Glu69Lys), 19:860741:G:A (Trp60Ter), 19:860775:T:G, 19:863105:GC:G (Pro211ArgfsTer101), 19:860933:C:A (Tyr95Ter), 19:861912:G:T (Glu191Ter), 19:863142:CACCTCGGGCTCGCGCGT:C (Thr223LeufsTer115), 19:860714:C:A (Cys51Ter), 19:861785:C:A (Cys148Ter), 19:861926:C:A (Cys195Ter), 19:860692:AG:A(Gln44HisfsTer2), 19:861826:GCCCGGACAGC:G (Pro163CysfsTer28), 19:860752:CG:C(Ala65ArgfsTer91), 19:859745:G:T (NA) or 19:860686:C:A (Ser42Ter), or mRNA molecules generated from said genomic nucleic acid molecules or cDNA molecules generated from said mRNA molecules.

[0101] In some implementations, methods for detecting the presence or absence of CFD variant nucleic acid molecules (such as, for example, genomic nucleic acid molecules, mRNA molecules, and / or cDNA molecules derived from mRNA molecules) in a subject include assaying a biological sample obtained from the subject. The assay determines whether the nucleic acid molecules in the biological sample contain a specific nucleotide sequence.

[0102] In some embodiments, the biological sample comprises cells or cell lysates. Such methods may further include, for example, obtaining a biological sample from a subject containing a CFD genomic nucleic acid molecule or mRNA molecule, and optionally, if it is mRNA, reverse transcribing the mRNA into cDNA. Such assays may include, for example, identifying these locations of a specific CFD nucleic acid molecule. In some embodiments, the method is an in vitro method.

[0103] In some embodiments, the determining step, detection step, or sequence analysis includes sequencing at least a portion of the nucleotide sequence of a CFD genomic nucleic acid molecule, CFD mRNA molecule, or CFD cDNA molecule in a biological sample containing genetic variations compared to a corresponding CFD reference molecule. In some embodiments, the sequencing portion includes one or more variations that cause or are predicted to cause loss of function (partial or complete).

[0104] In some implementations, the assay includes sequencing the entire nucleic acid molecule. In some implementations, only the CFD genomic nucleic acid molecule is analyzed. In some implementations, only CFD mRNA is analyzed. In some implementations, only CFD cDNA obtained from CFD mRNA is analyzed.

[0105] Modified-specific polymerase chain reaction (PCR) techniques can be used to detect mutations in nucleic acid sequences, such as SNPs. Modified-specific primers can be used because DNA polymerase will not extend when there is a mismatch with the template.

[0106] In some embodiments, the nucleic acid molecule in the sample is mRNA, and the mRNA is reverse transcribed into cDNA prior to the amplification step. In some embodiments, the nucleic acid molecule is present within cells obtained from a subject.

[0107] In some embodiments, the assay includes contacting a biological sample with primers or probes, such as modified specific primers or modified specific probes, which specifically hybridize under stringent conditions with a CFD variant genomic sequence, variant mRNA sequence, or variant cDNA sequence, rather than with a corresponding CFD reference sequence, and determining whether hybridization occurs.

[0108] In some implementations, the determining step, detection step, or sequence analysis includes: a) amplifying at least a portion of a CFD nucleic acid molecule encoding a CFD polypeptide; b) labeling the amplified nucleic acid molecule with a detectable label; c) contacting the labeled nucleic acid molecule with a support containing a probe that modifies specificity; and d) detecting the detectable label.

[0109] In some embodiments, the assay includes RNA sequencing (RNA-Seq). In some embodiments, the assay also includes, for example, reverse transcription of mRNA into cDNA via reverse transcriptase polymerase chain reaction (RT-PCR).

[0110] In some embodiments, the method utilizes probes and primers of sufficient nucleotide length to bind to the target nucleotide sequence and specifically detect and / or identify polynucleotides comprising CFD variant genomic nucleic acid molecules, variant mRNA molecules, or variant cDNA molecules. Hybridization or reaction conditions can be determined by the operator to achieve this result. The nucleotide length can be any length sufficient for the chosen detection method (including any assay described or exemplified herein). Such probes and primers can specifically hybridize to the target nucleotide sequence under highly stringent hybridization conditions. Probes and primers can have complete nucleotide sequence identity with adjacent nucleotides within the target nucleotide sequence, but can be designed using conventional methods to differ from the target nucleotide sequence while retaining the ability to specifically detect and / or identify the target nucleotide sequence. Probes and primers can have approximately 80%, approximately 85%, approximately 90%, approximately 91%, approximately 92%, approximately 93%, approximately 94%, approximately 95%, approximately 96%, approximately 97%, approximately 98%, approximately 99%, or 100% sequence identity or complementarity with the nucleotide sequence of the target nucleic acid molecule.

[0111] Illustrative examples of nucleic acid sequencing technologies include, but are not limited to, Sanger sequencing and dye-terminated sequencing. Other methods involve nucleic acid hybridization methods other than sequencing, including the use of labeled primers or probes for purified DNA, amplified DNA, and fixed cell preparations (fluorescence in situ hybridization (FISH)). In some methods, the target nucleic acid molecule can be amplified before or simultaneously with detection. Illustrative examples of nucleic acid amplification technologies include, but are not limited to, polymerase chain reaction (PCR), ligase chain reaction (LCR), strand displacement amplification (SDA), and nucleic acid sequence-based amplification (NASBA). Other methods include, but are not limited to, ligase chain reaction, strand displacement amplification, and thermophilic SDA (tSDA).

[0112] In hybridization techniques, stringent conditions can be employed to enable probes or primers to hybridize specifically with their targets. In some embodiments, under stringent conditions, polynucleotide primers or probes will hybridize with their target sequences to a detectable degree greater than hybridization with other non-target sequences, for example, at least 2, 3, 4, or more times (relative to background), including more than 10 times (relative to background). In some embodiments, under stringent conditions, polynucleotide primers or probes will hybridize with their target nucleotide sequences to a detectable degree at least 2 times greater than hybridization with other nucleotide sequences. In some embodiments, under stringent conditions, polynucleotide primers or probes will hybridize with their target nucleotide sequences to a detectable degree at least 3 times greater than hybridization with other nucleotide sequences. In some embodiments, under stringent conditions, polynucleotide primers or probes will hybridize with their target nucleotide sequences to a detectable degree at least 4 times greater than hybridization with other nucleotide sequences. In some embodiments, under stringent conditions, polynucleotide primers or probes will hybridize with their target nucleotide sequences to a detectable degree more than 10 times greater than hybridization with other nucleotide sequences (relative to background). The stringent conditions are sequence-dependent and will vary in different environments.

[0113] Appropriate stringent conditions for promoting DNA hybridization (e.g., 6X sodium chloride / sodium citrate (SSC) at approximately 45°C, followed by washing with 2X SSC at 50°C) are known and can be found in Current Protocols in Molecular Biology, John Wiley & Sons, NY (1989), 6.3.1–6.3.6. Typically, stringent conditions for hybridization and detection will be those described below, with salt concentrations below approximately 1.5 M Na at pH 7.0 to 8.3. + Ions, typically about 0.01 to 1.0 M Na + The ion concentration (or other salt) and temperature are at least about 30°C for short probes (e.g., 10 to 50 nucleotides) and at least about 60°C for longer probes (e.g., greater than 50 nucleotides). Tight conditions can also be achieved by adding a destabilizing agent (e.g., formamide). Optionally, the wash buffer may contain about 0.1% to about 1% SDS. The duration of hybridization is typically less than about 24 hours, typically about 4 to about 12 hours. The washing time will be at least long enough to reach equilibration.

[0114] In some implementations, the isolated nucleic acid molecules contain at least about 5, at least about 8, at least about 10, at least about 11, at least about 12, at least about 13, at least about 14, at least about 15, at least about 16, at least about 17, at least about 18, at least about 19, at least about 20, at least about 21, at least about 22, at least about 23, at least about 24, at least about 25, at least about 30, at least about 35, at least about 40, at least about 45, at least about 50, at least about 5 5, at least about 60, at least about 65, at least about 70, at least about 75, at least about 80, at least about 85, at least about 90, at least about 95, at least about 100, at least about 200, at least about 300, at least about 400, at least about 500, at least about 600, at least about 700, at least about 800, at least about 900, at least about 1000, at least about 2000, at least about 3000, at least about 4000, or at least about 5000 nucleotides or composed thereof. In some embodiments, the isolated nucleic acid molecule comprises or consists of at least about 5, at least about 8, at least about 10, at least about 11, at least about 12, at least about 13, at least about 14, at least about 15, at least about 16, at least about 17, at least about 18, at least about 19, at least about 20, at least about 21, at least about 22, at least about 23, at least about 24, or at least about 25 nucleotides. In some embodiments, the isolated nucleic acid molecule comprises or consists of at least about 18 nucleotides. In some embodiments, the isolated nucleic acid molecule comprises or consists of at least about 15 nucleotides. In some embodiments, the isolated nucleic acid molecule comprises or consists of about 10 to about 35, about 10 to about 30, about 10 to about 25, about 12 to about 30, about 12 to about 28, about 12 to about 24, about 15 to about 30, about 15 to about 25, about 18 to about 30, about 18 to about 25, about 18 to about 24, or about 18 to about 22 nucleotides. In some embodiments, the isolated nucleic acid molecule comprises or consists of about 18 to about 30 nucleotides. In some embodiments, the isolated nucleic acid molecule comprises or consists of at least about 15 nucleotides to at least about 35 nucleotides.

[0115] In some implementations, such isolated nucleic acid molecules hybridize under stringent conditions with CFD variant nucleic acid molecules, such as genomic nucleic acid molecules, mRNA molecules, and / or cDNA molecules. These nucleic acid molecules can be used as probes, primers, modified-specific probes, or modified-specific primers, as described or illustrated herein, and include, but are not limited to, primers, probes, antisense RNA, shRNA, and siRNA, each described in more detail elsewhere herein, and can be used in any of the methods described herein.

[0116] In some embodiments, the isolated nucleic acid molecule hybridizes with at least about 15 consecutive nucleotides of a nucleic acid molecule having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identity with a CFD variant nucleic acid molecule. In some embodiments, the isolated nucleic acid molecule comprises or consists of about 15 to about 100 nucleotides or about 15 to about 35 nucleotides. In some embodiments, the isolated nucleic acid molecule comprises or consists of about 15 to about 100 nucleotides. In some embodiments, the isolated nucleic acid molecule comprises or consists of about 15 to about 35 nucleotides.

[0117] In some embodiments, the specific probe and specific primer are modified to contain DNA. In some embodiments, the specific probe and specific primer are modified to contain RNA.

[0118] In some embodiments, the probes and primers described herein (including modified-specific probes and modified-specific primers) have nucleotide sequences that specifically hybridize with any nucleic acid molecule disclosed herein or its complementary sequence. In some embodiments, the probes and primers specifically hybridize with any nucleic acid molecule disclosed herein under stringent conditions.

[0119] In some implementations, primers (including modified-specific primers) can be used in next-generation sequencing or high-throughput sequencing. In some cases, primers can be modified, including modified-specific primers. In particular, primers can contain various modifications used in different steps of, for example, massively parallel signature sequencing (MPSS), polymerase cloning sequencing (Polony sequencing), and 454 pyrosequencing. Modified primers can be used in several steps of the process, including the use of biotinylated primers in the cloning step and fluorescently labeled primers in the bead loading and detection steps. Polymerase cloning sequencing is typically performed using paired-end tagged libraries, where each DNA template molecule is approximately 135 bp in length. Biotinylated primers are used in the bead loading step and emulsion PCR. Fluorescently labeled degenerate nonameric oligonucleotides are used in the detection step. The adaptor may contain a 5'-biotin tag for immobilizing the DNA library onto streptavidin-coated beads.

[0120] The probes and primers described herein can be used to detect nucleotide variations within any of the CFD variant nucleic acid molecules disclosed herein. The primers described herein can be used to amplify any CFD variant nucleic acid molecule or fragment thereof.

[0121] In the context of this disclosure, "specific hybridization" means that a probe or primer (such as, for example, by altering a specific probe or primer) does not hybridize with the nucleic acid sequence encoding a CFD reference genome nucleic acid molecule, a CFD reference mRNA molecule, and / or a CFD reference cDNA molecule.

[0122] In some embodiments, the probe (such as, for example, a modified specific probe) contains a label. In some embodiments, the label is a fluorescent label, a radioactive label, or biotin.

[0123] This disclosure also provides supports for substrates to which one or more of the probes disclosed herein are attached. A solid support is a solid substrate or support to which molecules (such as any of the probes disclosed herein) can associate. One form of a solid support is an array. Another form of a solid support is an array of detectors. An array of detectors is a solid support to which multiple different probes are coupled in an array, grid, or other organized pattern. One form of a solid substrate is a microtiter dish, such as a standard 96-well type. In some embodiments, a porous glass slide, typically containing one array per well, may be used.

[0124] Genomic nucleic acid molecules, mRNA molecules, and cDNA molecules can originate from any organism. For example, genomic nucleic acid molecules, mRNA molecules, and cDNA molecules can be human or orthologs of another organism (e.g., non-human mammals, rodents, mice, or rats). It should be understood that gene sequences within a population can vary due to polymorphisms such as single nucleotide polymorphisms.

[0125] This article also provides functional polynucleotides that can interact with the disclosed nucleic acid molecules. Examples of functional polynucleotides include, but are not limited to, antisense molecules, aptamers, ribozymes, triplet-forming molecules, and external guide sequences. Functional polynucleotides can act as influencers, inhibitors, regulators, and stimulators of the specific activities of target molecules, or they can possess novel activities independent of any other molecule.

[0126] The isolated nucleic acid molecules disclosed herein may include RNA, DNA, or both RNA and DNA. The isolated nucleic acid molecules may also be ligated or fused to heterologous nucleic acid sequences (such as in a vector) or heterologous tags. For example, the isolated nucleic acid molecules disclosed herein may be in a vector or as an exogenous donor sequence comprising the isolated nucleic acid molecule and a heterologous nucleic acid sequence. The isolated nucleic acid molecules may also be ligated or fused to heterologous tags. Tags may be directly detectable (such as fluorophores) or indirectly detectable (such as haptens, enzymes, or fluorophore quenchers). Such tags can be detected by spectroscopic, photochemical, biochemical, immunochemical, or chemical means. Such tags include, for example, radioactive tags, pigments, dyes, chromogens, spin tags, and fluorescent tags. Tags may also be, for example, chemiluminescent substances; metal-containing substances; or enzymes, wherein enzyme-dependent secondary signal generation occurs. The term “tag” may also refer to a “label” or hapten that selectively binds to a conjugated molecule such that the conjugated molecule is used to generate a detectable signal when subsequently added with a substrate. For example, biotin can be used as a tag, along with avidin or streptavidin conjugates of horseradish peroxidase (HRP), to bind to the tag and be checked using a calorimetric substrate (such as, for example, tetramethylbenzidine (TMB)) or a fluorescent substrate to detect the presence of HRP. Exemplary tags that can be used as tags to facilitate purification include, but are not limited to, myc, HA, FLAG or 3XFLAG, 6Xhis or polyhistidine, glutathione S-transferase (GST), maltose-binding proteins, epitope tags, or the Fc portion of immunoglobulins. Many tags include, for example, particles, fluorophores, haptens, enzymes and their calorimetric, fluorescent, and chemiluminescent substrates, and other tags.

[0127] The percentage of identity (or complementarity) between specific elongations of nucleotide sequences within nucleic acid molecules or amino acid sequences within polypeptides can be determined using the BLAST program (Basic Local Alignment Search Tool) and the PowerBLAST program (Altschul et al., J. Mol. Biol., 1990, 215, 403-410; Zhang and Madden, Genome Res., 1997, 7, 649-656) or routinely using the Gap program (Wisconsin Sequence Analysis Package for Unix, version 8, Genetics Computer Group, University Research Park, Madison Wis.) with default settings (which uses the algorithm of Smith and Waterman (Adv. Appl. Math., 1981, 2, 482-489)). In this document, a higher percentage of sequence identity is preferred over a lower percentage when referring to sequence identity percentages.

[0128] This disclosure also provides a macular degeneration therapeutic agent for treating, preventing, or inhibiting macular degeneration, for use in treating or preventing macular degeneration in subjects with CFD variant nucleic acid molecules. Any of the macular degeneration therapeutic agents for treating, preventing, or inhibiting macular degeneration described herein may be used herein. Any of the CFD variant nucleic acid molecules disclosed herein may be used herein. In some implementations, the CFD variant nucleic acid molecule is a CFD variant genomic nucleic acid molecule containing the following genetic variations: rs35186399 (19:860766:G:A; Glu69Lys), 19:860741:G:A (Trp60Ter), 19:860775:T:G, 19:863105:GC:G (Pro211ArgfsTer101), 19:860933:C:A (Tyr95Ter), 19:861912:G:T (Glu191Ter), 19:863142:CACCTCGGGCTCGCGCGT:C (Thr223LeufsTer115), 19:860714:C:A (Cys51Ter), 19:861785:C:A (Cys148Ter), 19:861926:C:A (Cys195Ter), 19:860692:AG:A (Gln44HisfsTer2), 19:861826:GCCCGGACAGC:G (Pro163CysfsTer28), 19:860752:CG:C (Ala65ArgfsTer91), 19:859745:G:T (NA) or 19:860686:C:A (Ser42Ter), or an mRNA molecule produced from the said genomic nucleic acid molecule or a cDNA molecule produced from the said mRNA molecule.

[0129] This disclosure also provides the use of macular degeneration therapeutic agents for treating, preventing, or inhibiting macular degeneration in the preparation of medicaments for treating or preventing macular degeneration in subjects with CFD variant nucleic acid molecules. Any of the macular degeneration therapeutic agents for treating, preventing, or inhibiting macular degeneration described herein may be used herein. Any of the CFD variant nucleic acid molecules disclosed herein may be used herein. In some implementations, the CFD variant nucleic acid molecule is a CFD variant genomic nucleic acid molecule containing the following genetic variations: rs35186399 (19:860766:G:A; Glu69Lys), 19:860741:G:A (Trp60Ter), 19:860775:T:G, 19:863105:GC:G (Pro211ArgfsTer101), 19:860933:C:A (Tyr95Ter), 19:861912:G:T (Glu191Ter), 19:860714:C:A (Cys51Ter), 19:863142:CACCTCGGGCTCGCGCGT:C (Thr223LeufsTer115), 19:861785:C:A (Cys148Ter), 19:861926:C:A (Cys195Ter), 19:860692:AG:A (Gln44HisfsTer2), 19:861826:GCCCGGACAGC:G (Pro163CysfsTer28), 19:860752:CG:C (Ala65ArgfsTer91), 19:859745:G:T (NA) or 19:860686:C:A (Ser42Ter), or an mRNA molecule produced from the said genomic nucleic acid molecule or a cDNA molecule produced from the said mRNA molecule.

[0130] This disclosure also provides CFD inhibitors for the treatment or prevention of macular degeneration in subjects who are CFD-referenced or heterozygous for CFD variant nucleic acid molecules. Any of the CFD inhibitors described herein may be used herein. Any of the CFD variant nucleic acid molecules disclosed herein may be used herein. In some implementations, the CFD variant nucleic acid molecule is a CFD variant genomic nucleic acid molecule containing the following genetic variations: rs35186399 (19:860766:G:A; Glu69Lys), 19:860741:G:A (Trp60Ter), 19:860775:T:G, 19:863105:GC:G (Pro211ArgfsTer101), 19:860933:C:A (Tyr95Ter), 19:861912:G:T (Glu191Ter), 19:860714:C:A (Cys51Ter), 19:863142:CACCTCGGGCTCGCGCGT:C (Thr223LeufsTer115), 19:861785:C:A (Cys148Ter), 19:861926:C:A (Cys195Ter), 19:860692:AG:A(Gln44HisfsTer2), 19:861826:GCCCGGACAGC:G (Pro163CysfsTer28), 19:860752:CG:C(Ala65ArgfsTer91), 19:859745:G:T (NA) or 19:860686:C:A (Ser42Ter), or an mRNA molecule produced from the said genomic nucleic acid molecule or a cDNA molecule produced from the said mRNA molecule.

[0131] This disclosure also provides the use of CFD inhibitors in the preparation of medicaments for the treatment or prevention of macular degeneration in subjects who are CFD-referenced or heterozygous for CFD variant nucleic acid molecules. Any of the CFD inhibitors described herein may be used herein. Any of the CFD variant nucleic acid molecules disclosed herein may be used herein. In some implementations, the CFD variant nucleic acid molecule is a CFD variant genomic nucleic acid molecule containing the following genetic variations: rs35186399 (19:860766:G:A; Glu69Lys), 19:860741:G:A (Trp60Ter), 19:860775:T:G, 19:863105:GC:G (Pro211ArgfsTer101), 19:860933:C:A (Tyr95Ter), 19:861912:G:T (Glu191Ter), 19:863142:CACCTCGGGCTCGCGCGT:C (Thr223LeufsTer115), 19:860714:C:A (Cys51Ter), 19:861785:C:A (Cys148Ter), 19:861926:C:A (Cys195Ter), 19:860692:AG:A(Gln44HisfsTer2), 19:861826:GCCCGGACAGC:G (Pro163CysfsTer28), 19:860752:CG:C(Ala65ArgfsTer91), 19:859745:G:T (NA) or 19:860686:C:A (Ser42Ter), or an mRNA molecule produced from the said genomic nucleic acid molecule or a cDNA molecule produced from the said mRNA molecule.

[0132] In some embodiments, a CFD inhibitor and a macular degeneration treatment agent are contained within a pharmaceutical composition. In some embodiments, the CFD inhibitor is contained within a first pharmaceutical composition and the macular degeneration treatment agent is contained within a second pharmaceutical composition. In some embodiments, the first pharmaceutical composition and the second pharmaceutical composition are administered simultaneously. In some embodiments, the first pharmaceutical composition is administered before the second pharmaceutical composition. In some embodiments, the first pharmaceutical composition is administered after the second pharmaceutical composition.

[0133] All patent documents, websites, other publications, registration numbers, etc., cited above or below are incorporated herein by reference in their entirety for all purposes, to the extent that each individual item is specifically and individually indicated as being incorporated by reference. If different versions of a sequence are associated with registration numbers at different times, the version associated with the registration number on the effective filing date of this application is indicated. The effective filing date is the earlier of the actual filing date or the filing date of the priority application referencing the registration number (if applicable). Similarly, if different versions of publications, websites, etc., are published at different times, the version most recently published on the effective filing date of this application is indicated, unless otherwise stated. Unless otherwise specifically stated, any feature, step, element, embodiment, or aspect of this disclosure may be used in combination with any other feature, step, element, embodiment, or aspect. Although this disclosure has been described in detail by illustration and example for clarity and understanding purposes, it will be apparent that certain changes and modifications may be implemented within the scope of the appended claims.

[0134] The following examples are provided to describe the embodiments in more detail. They are intended to illustrate, but not limit, the claimed embodiments. The following examples provide a disclosure and description, for those skilled in the art, of how the compounds, compositions, articles, apparatuses, and / or methods described herein are prepared and evaluated, and are intended to be exemplary only and not to limit the scope of any claim. Efforts have been made to ensure accuracy regarding figures (such as, for example, amounts, temperatures, etc.), but some errors and deviations may be expected. Unless otherwise stated, parts are parts by weight, temperatures are in °C or at ambient temperature, and pressures are at or near atmospheric pressure.

[0135] Example 1: General Methods Whole-exome association analysis was performed to identify genetic variants influencing the risk of age-related macular degeneration. DNA was extracted from whole blood and / or saliva samples from participants recruited by collaborators at the Regeneron Genetics Center biobanks and electronic health record cohorts. These cohorts included the United Kingdom Biobank (UKB) cohort, the Geisinger Health System (GHS) MyCode Community Health Advocacy DiscoverEHR cohort, the Mount Sinai BioMe Biobank cohort (SINAI), the University of Pennsylvania Penn Medicine Biobank (UPENN-PMBB), the Malmo Diet and Cancer Study (MDCS), and biobanks at the University of Colorado, UCLA, and Indiana University. DNA was cut and hybridized with an exome capture kit to remove most of the non-protein-coding DNA. DNA libraries were prepared for sequencing on a standard Illumina DNA sequencer. Sequence data were mapped to a reference genome, and variant sequences for all participants were identified. Using Regenie software, these variant sequences were used for standard genome-wide and exome-wide statistical analyses to identify associations with phenotypes.

[0136] Phenotypes are defined using International Classification of Diseases (ICD) 10 codes associated with age-related macular degeneration diagnosed by clinicians. Table 1 shows associations among a set of rare variants (<5%) predicted to result in loss of function of CFD transcripts or proteins (as described in this paper). Table 2 shows these associations categorized into top variants and residual signals from other variants. Table 3 shows the signals from the top missense variants and all predicted loss-of-function (pLoF) variants.

[0137] Example 2: Gene-level associations of CFD protection against macular degeneration. Table 1 shows CFD variants grouped by function that have a protective effect against macular degeneration. GENE P used the aggregated Cauchy combination test (ACAT) to combine evidence from multiple frequency thresholds and burden / gene tests. pLoF= Probable loss-of-function variants were categorized by grouping predicted stop codon gain, frameshift, splice acceptor, and splice donor variants onto transcript ENST00000327726.11.

[0138] Table 1: CFD load test (Gene P=9.4E-8)

[0139] Table 2 shows the residual protective signal after removing the top missense variant.

[0140] Table 2

[0141] Table 3 shows the top missense and all evidence supporting possible loss of function (pLoF) variants.

[0142] Table 3

[0143] Figure 1 shows the analysis of wet and dry AMD.

[0144] Figure 2 illustrates how CFD missense variants alter the risk of individuals with a high genetic risk of AMD based on genetic risk score stratification. See Figure 2, quintiles = CFD missense variant carriers (case / control) (1=29 / 1573; 2=37 / 1571; 3=27 / 1592; 4=31 / 1582; ​​and 5=56 / 1551); and CFD missense variant non-carriers (case / control) (1=3653 / 162376; 2=3902 / 162135; 3=4231 / 161892; 4=5114 / 161101; and 5=7896 / 158095). In Gorman et al., medRxiv 2022.08.16.22278855 (World Wide Web "doi.org / 10.1101 / 2022.08.16.22278855"), after distinguishing between carriers and non-carriers of CFD protective missense variants, the stratified prevalence of GRS for all variants was compared. The results showed that the protective effect of CFD was strongest in the 60% with the highest overall genetic risk.

[0145] Figure 3 shows that the CFD protective signal remains consistent across the burden mask and top variant in the maximum queue. It has a protective effect on both wet and dry AMD.

[0146] Example 3: Lower Risk of AMD in Individuals Carrying CFD E69K Mutations. Genome-wide association analyses of common and rare exon variants were performed on individuals diagnosed with AMD and controls in nine cohorts. Four cohorts were diagnosed with advanced AMD and were evaluable. Association analyses were performed using Regenie, a software that takes into account population structure and is capable of summative tests (burden tests) on mutations in genes. Meta-analyses were performed on the different cohorts. The results are shown in Table 4.

[0147] Table 4

[0148] *pLoF = Possible loss-of-function mutations, including stop codon gain, frameshift, splice donor, and splice acceptor.

[0149] The top variant 19:860766:G:A (E69K) shows consistent protection for AMD and AMD subtypes (see Figure 4).

[0150] Example 4: E69K mutations can reduce the risk of the most common cause of AMD.

[0151] CFH is an inhibitor of the alternative pathway of complement signaling. Common missense variants result in the CFH Y402H mutation, which is associated with decreased CFH activity, increased alternative pathway activity, and an increased risk of AMD. GWAS signaling near CFH accounts for approximately 10% of all AMD risk.

[0152] The lack of protection from the CFD wild-type (GG) indicated an increased risk of additional copies of CFH Y402H: the odds ratio (OR) changed from 1 to 1.4 to 1.9. A protective variant of CFD (E69K, GA) reduced the risk of CFH Y402H: the OR changed from 0.95 to 1.1 to 1.1. The “leveling” of risk suggests that this mutation is sufficient to block AP signaling. The two protective variants indicate a larger effect.

[0153] The interaction between genes was tested by stratifying the data on the Y402H mutation and calculating the protective effect of CFD E69K in the stratification, thus calculating the P-values: 1) No Y402H: OR=0.97; P-value=0.52; 2) One Y402H: OR=0.76; P-value=0.0013; and 3) Two Y402H: OR=0.58; P-value=7.8e-7.

[0154] The results are shown in Figure 5.

[0155] Example 5: CFD mRNA and protein expression in the human eye. Single-cell RNA sequencing of the human eye showed expression in choroidal cells. Cfd in situ hybridization confirmed expression in the neuroretina (microglia) and choroid (Schwann cells and fibroblasts) (see Figure 6). CFD mRNA in the choroid was significantly upregulated compared to that in the retina and RPE. Furthermore, in situ hybridization showed similar expression patterns in normal and AMD-affected eyes, expressing CFD in neuroretinal and choroidal cells, but not in the RPE. Since CFD is known to be expressed in adipose tissue, adipose tissue was provided as a positive control.

[0156] CFD proteins were detected in retinal pigment epithelial cells and choroidal tissue by immunohistochemistry (see Figure 7). Human eye sections showed CFD proteins in RPE, choroid, and sclera. No differences were found between AMD and non-AMD eyes. Human adipose tissue was used as a positive control. CFD proteins were identified not only in the choroid and choroidal capillary layer but also in RPE cells. These data suggest that CFD in the eye may originate from local and distal expression sites; however, no significant increase in CFD deposition was observed in post-symptomatic AMD eyes.

[0157] Example 6: Variant Characterization Using an In Vitro C3 Convertase Assay To understand the direction of effect against a protective CFD variant (E69K), a series of CFD mutant recombinant proteins were generated, and their catalytic activity was tested using an in vitro C3b / CFB convertase assay. Although the CFD E69K mutation was not located at the active site, outside the factor B binding site, or within a known repressive loop, this variant was found to reduce, but not completely eliminate, the catalytic activity of the CFD. This identified a novel CFD functional site and demonstrated that even partial loss of function could provide significant protection against AMD. Specifically, the CFD variant was generated and purified from transfected cells (see Figure 8). The C3b / CFB convertase assay used the purified proteins to test the function of the CFD (see Figure 9). The assay validation results are shown in Figure 10. The dose-response and kinetics are shown in Figures 11 and 12, respectively. Figure 11 shows the CFBa cleavage bands after an in vitro cleavage assay of an indicator CFD (WT, E69K mutant, or S208A mutant) containing C3b, Fb, and an indicator dose. Figure 11 also shows the in vitro cleavage assays of indicator CFDs (WT, E69K mutant, or S208A mutant) containing C3b, CFB, and indicator concentrations. CFBa cleavage band intensities were quantified and normalized to C3b band intensities. Multiple unpaired t-tests were performed, with Sidak-Bonferroni correction (*p<.05, **p<.005). The results indicate that: 1) the generated CFDs were functionally validated in the C3b / CFB convertase assay, and 2) the CFD E69K variant exhibited significantly reduced alternative pathway activity. Human genetics and these experimental data together demonstrate that inhibiting AP function by targeting CFDs may be sufficient to reduce overall complement activity.

[0158] The results of Examples 3-6 indicate that: 1) CFD protein is mainly produced by adipose tissue, although CFD has also been found in the choroid and RPE cells of the eye; 2) the CFD missense variant (19:860766:G:A, E69K) was identified as protective against AMD; 3) the CFD E69K mutation is not found in the active site, factor B binding site, or known repressive loop; 4) this mutation may stabilize the inactive conformation or negatively interact with the cleavage localization of C3bB within the catalytic site; and 5) this variant reduces but does not completely eliminate the catalytic activity of CFD.

[0159] In addition to the various modifications described herein, various modifications to the subject matter will be apparent to those skilled in the art from the foregoing description. Such modifications are also intended to fall within the scope of the appended claims. Every reference cited in this application (including, but not limited to, journal articles, U.S. and non-U.S. patents, patent application publications, international patent application publications, gene bank accession numbers, etc.) is incorporated herein by reference in its entirety and for all purposes.

Claims

1. A method for treating a subject with macular degeneration or at risk of developing macular degeneration, the method comprising administering a complement factor D (CFD) inhibitor to the subject.

2. The method of claim 1, wherein the macular degeneration is wet macular degeneration.

3. The method of claim 1, wherein the macular degeneration is dry macular degeneration.

4. The method of claim 1, wherein the macular degeneration is intermediate macular degeneration.

5. The method of any one of claims 1 to 4, wherein the CFD inhibitor comprises an inhibitory nucleic acid molecule that hybridizes with a CFD nucleic acid molecule.

6. The method of claim 5, wherein the inhibitory nucleic acid molecule comprises an antisense nucleic acid molecule, small interfering RNA (siRNA), and / or short hairpin RNA (shRNA).

7. The method of claim 6, wherein the inhibitory nucleic acid molecule comprises siRNA.

8. The method of claim 6, wherein the inhibitory nucleic acid molecule comprises an antisense nucleic acid molecule.

9. The method of any one of claims 1 to 8, wherein the subject is further administered a macular degeneration treatment agent or macular degeneration therapy.

10. The method of any one of claims 1 to 9, further comprising detecting the presence or absence of CFD variant nucleic acid molecules in a biological sample from the subject.

11. The method of claim 10, further comprising administering to the subject an amount of macular degeneration treatment agent or macular degeneration therapy equal to or less than a standard dose when the CFD variant nucleic acid molecule is not present in the biological sample.

12. The method of claim 10, further comprising administering to the subject an amount of macular degeneration treatment agent or macular degeneration therapy equal to or less than a standard dose when the subject is heterozygous for the CFD variant nucleic acid molecule.

13. The method of any one of claims 10 to 12, wherein the CFD variant nucleic acid molecule comprises a splice site variant, a stop codon gain variant, a start codon deletion variant, a stop codon deletion variant, a frameshift variant, a missense variant, an in-frame insertion deletion variant, and / or a variant encoding a truncated CFD variant polypeptide.

14. The method of any one of claims 10 to 13, wherein the CFD variant nucleic acid molecule comprises the following genetic variations: rs35186399 (19:860766:G:A; Glu69Lys), 19:860741:G:A (Trp60Ter), 19:860775:T:G, 19:863105:GC:G (Pro211ArgfsTer101), 19:860933:C:A (Tyr95Ter), 19:861912:G:T (Glu191Ter), 19:860714:C:A (Cys51Ter), 19:863142:CACCTCGGGCTCGCGCGT:C (Thr223LeufsTer115), 19:861785:C:A (Cys148Ter), 19:861926:C:A (Cys195Ter), 19:860692:AG:A (Gln44HisfsTer2), 19:861826:GCCCGGACAGC:G (Pro163CysfsTer28), 19:860752:CG:C (Ala65ArgfsTer91), 19:859745:G:T (NA) or 19:860686:C:A (Ser42Ter).

15. A method of treating a subject with macular degeneration or at risk of developing macular degeneration by administering a macular degeneration treatment agent or macular degeneration therapy, the method comprising: Whether the subject has a complement factor D (CFD) variant nucleic acid molecule has been determined or has been determined by obtaining or having obtained a biological sample from the subject; And the biological sample is subjected to or has been subjected to sequence analysis to determine whether the subject has a genotype containing a CFD variant nucleic acid molecule; and the macular degeneration treatment agent or macular degeneration therapy and / or CFD inhibitor is administered or continued to be administered to a subject serving as a CFD reference in an amount equal to or less than the standard dose; the macular degeneration treatment agent or macular degeneration therapy and / or CFD inhibitor is administered or continued to be administered to a subject heterozygous for the CFD variant nucleic acid molecule in an amount equal to or less than the standard dose; or the macular degeneration treatment agent or macular degeneration therapy is administered or continued to be administered to a subject homozygous for the CFD variant nucleic acid molecule in a standard dose; wherein the presence of the CFD variant nucleic acid molecule indicates a reduced risk of the subject developing macular degeneration.

16. The method of claim 15, wherein the CFD inhibitor comprises an inhibitory nucleic acid molecule that hybridizes with a CFD nucleic acid molecule.

17. The method of claim 16, wherein the inhibitory nucleic acid molecule comprises an antisense nucleic acid molecule, a small interfering RNA (siRNA), and / or a short hairpin RNA (shRNA).

18. The method of claim 16, wherein the inhibitory nucleic acid molecule comprises siRNA.

19. The method of claim 16, wherein the inhibitory nucleic acid molecule comprises an antisense nucleic acid molecule.

20. The method of any one of claims 15 to 19, wherein the subject is heterozygous for the CFD variant nucleic acid molecule, and the subject is given or continues to be given an amount of the macular degeneration treatment agent or macular degeneration therapy and the CFD inhibitor that is the same as or less than the standard dose.

21. The method of any one of claims 15 to 19, wherein the subject is a CFD reference, and the subject is given or continues to be given an amount of the macular degeneration treatment agent or macular degeneration therapy and the CFD inhibitor that is the same as or less than a standard dose.

22. The method of any one of claims 15 to 21, wherein the CFD variant nucleic acid molecule comprises a splice site variant, a stop codon gain variant, a start codon deletion variant, a stop codon deletion variant, a frameshift variant, a missense variant, an in-frame insertion deletion variant, and / or a variant encoding a truncated CFD variant polypeptide.

23. The method of any one of claims 15 to 22, wherein the CFD variant nucleic acid molecule comprises the following genetic variations: rs35186399 (19:860766:G:A; Glu69Lys), 19:860741:G:A (Trp60Ter), 19:860775:T:G, 19:863105:GC:G (Pro211ArgfsTer101), 19:860933:C:A (Tyr95Ter), 19:861912:G:T (Glu191Ter), 19:860714:C:A (Cys51Ter), 19:863142:CACCTCGGGCTCGCGCGT:C (Thr223LeufsTer115), 19:861785:C:A (Cys148Ter), 19:861926:C:A (Cys195Ter), 19:860692:AG:A (Gln44HisfsTer2), 19:861826:GCCCGGACAGC:G (Pro163CysfsTer28), 19:860752:CG:C (Ala65ArgfsTer91), 19:859745:G:T (NA) or 19:860686:C:A (Ser42Ter).

24. A method for identifying subjects at increased risk of developing macular degeneration, the method comprising: The presence or absence of a complement factor D (CFD) variant nucleic acid molecule in a biological sample obtained from the subject was determined or has been determined; wherein: when the subject is a CFD reference, the risk of the subject developing macular degeneration is increased; and when the subject is heterozygous or homozygous for the CFD variant nucleic acid molecule, the risk of the subject developing macular degeneration is reduced.

25. The method of claim 24, wherein the CFD variant nucleic acid molecule is a splice site variant, a stop codon gain variant, a start codon deletion variant, a stop codon deletion variant, a frameshift variant, a missense variant, an in-frame insertion deletion variant, or a variant encoding a truncated CFD variant polypeptide.

26. The method of claim 24 or claim 25, wherein the CFD variant nucleic acid molecule comprises the following genetic variations: rs35186399 (19:860766:G:A; Glu69Lys), 19:860741:G:A (Trp60Ter), 19:860775:T:G, 19:863105:GC:G (Pro211ArgfsTer101), 19:860933:C:A (Tyr95Ter), 19:861912:G:T (Glu191Ter), 19:863142:CACCTCGGGCTCGCGCGT:C (Thr223LeufsTer115), 19:860714:C:A (Cys51Ter), 19:861785:C:A (Cys148Ter), 19:861926:C:A (Cys195Ter), 19:860692:AG:A (Gln44HisfsTer2), 19:861826:GCCCGGACAGC:G (Pro163CysfsTer28), 19:860752:CG:C (Ala65ArgfsTer91), 19:859745:G:T (NA) or 19:860686:C:A (Ser42Ter).

27. The method of any one of claims 24 to 26, further comprising administering to a subject serving as a CFD reference an amount of macular degeneration treatment agent or macular degeneration therapy and / or CFD inhibitor equal to or less than a standard dose.

28. The method of claim 27, wherein the subject is a CFD reference, and the subject is given or continues to be given an amount of the macular degeneration treatment agent or macular degeneration therapy and the CFD inhibitor that is the same as or less than the standard dose.

29. The method of any one of claims 24 to 26, further comprising administering to a subject heterozygous for a CFD variant nucleic acid molecule an amount equal to or less than a standard dose of a macular degeneration treatment agent or macular degeneration therapy and / or a CFD inhibitor.

30. The method of claim 29, wherein the subject is heterozygous for the CFD variant nucleic acid molecule, and the subject is given or continues to be given an amount of the macular degeneration treatment agent or macular degeneration therapy and the CFD inhibitor that is the same as or less than the standard dose.

31. The method of any one of claims 27 to 30, wherein the CFD inhibitor comprises an inhibitory nucleic acid molecule that hybridizes with a CFD nucleic acid molecule.

32. The method of claim 31, wherein the inhibitory nucleic acid molecule comprises an antisense nucleic acid molecule, small interfering RNA (siRNA), and / or short hairpin RNA (shRNA).

33. The method of claim 32, wherein the inhibitory nucleic acid molecule comprises siRNA.

34. The method of claim 32, wherein the inhibitory nucleic acid molecule comprises an antisense nucleic acid molecule.

35. A macular degeneration treatment agent for treating or preventing macular degeneration in subjects with a complement factor D (CFD) variant nucleic acid molecule.

36. The macular degeneration treatment of claim 35, wherein the CFD variant nucleic acid molecule is a splice site variant, a stop codon gain variant, a start codon deletion variant, a stop codon deletion variant, a frameshift variant, a missense variant, an in-frame insertion deletion variant, or a variant encoding a truncated CFD variant polypeptide.

37. The macular degeneration treatment agent of claim 35 or claim 36, wherein the CFD variant nucleic acid molecule comprises the following genetic variations: rs35186399 (19:860766:G:A; Glu69Lys), 19:860741:G:A (Trp60Ter), 19:860775:T:G, 19:863105:GC:G (Pro211ArgfsTer101), 19:860933:C:A (Tyr95Ter), 19:861912:G:T (Glu191Ter), 19:863142:CACCTCGGGCTCGCGCGT:C (Thr223LeufsTer115), 19:860714:C:A (Cys51Ter), 19:861785:C:A (Cys148Ter), 19:861926:C:A (Cys195Ter), 19:860692:AG:A (Gln44HisfsTer2), 19:861826:GCCCGGACAGC:G (Pro163CysfsTer28), 19:860752:CG:C (Ala65ArgfsTer91), 19:859745:G:T (NA) or 19:860686:C:A (Ser42Ter).

38. A complement factor D (CFD) inhibitor for the treatment or prevention of macular degeneration in subjects who are CFD references or heterozygous for CFD variant nucleic acid molecules.

39. The CFD inhibitor of claim 38, wherein the CFD variant nucleic acid molecule is a splice site variant, a stop codon gain variant, a start codon deletion variant, a stop codon deletion variant, a frameshift variant, a missense variant, an in-frame insertion deletion variant, or a variant encoding a truncated CFD variant polypeptide.

40. The CFD inhibitor of claim 38 or claim 39, wherein the CFD variant nucleic acid molecule comprises the following genetic variations: rs35186399 (19:860766:G:A; Glu69Lys), 19:860741:G:A (Trp60Ter), 19:860775:T:G, 19:863105:GC:G (Pro211ArgfsTer101), 19:860933:C:A (Tyr95Ter), 19:861912:G:T (Glu191Ter), 19:860714:C:A (Cys51Ter), 19:863142:CACCTCGGGCTCGCGCGT:C (Thr223LeufsTer115), 19:861785:C:A (Cys148Ter), 19:861926:C:A (Cys195Ter), 19:860692:AG:A (Gln44HisfsTer2), 19:861826:GCCCGGACAGC:G (Pro163CysfsTer28), 19:860752:CG:C (Ala65ArgfsTer91), 19:859745:G:T (NA) or 19:860686:C:A (Ser42Ter).

41. The CFD inhibitor of any one of claims 38 to 40, wherein the CFD inhibitor comprises an inhibitory nucleic acid molecule that hybridizes with a CFD nucleic acid molecule.

42. The CFD inhibitor of claim 41, wherein the inhibitory nucleic acid molecule comprises an antisense nucleic acid molecule, small interfering RNA (siRNA), and / or short hairpin RNA (shRNA).

43. The CFD inhibitor of claim 42, wherein the inhibitory nucleic acid molecule comprises siRNA.

44. The CFD inhibitor of claim 42, wherein the inhibitory nucleic acid molecule comprises an antisense nucleic acid molecule.

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