Inhibitory nucleic acids for factor H family proteins

Abstract

Agents for reducing gene and / or protein expression of FH family proteins are disclosed. Articles encoding the agents and methods of using the agents, including in therapeutic and prophylactic methods, are also disclosed.

Description

[Technical Field]

[0001] This application claims priority to GB 2107586.6, filed May 27, 2021, the contents and elements of which are incorporated herein by reference for all purposes. Technical Field The present disclosure relates to the field of molecular biology, and more particularly to nucleic acid technology. The present disclosure also relates to methods of medical treatment and prophylaxis. [Background technology]

[0002] The complement system contributes to innate host immune defense by aiding in the rapid recognition and elimination of microbial invaders. However, dysregulation of complement can contribute to inflammatory, immune-related, and age-related conditions. As a result, improper regulation of the complement system has been linked to a wide variety of human diseases, including eye and kidney diseases, as well as neurological disorders and cancer (Morgan, B.P., Semin Immunopathol, 2018. 40(1): pp. 113-124; Halbgebauer, R. et al., Semin Immunol, 2018. 37: pp. 12-20; Ma, Y. et al., Aging Dis, 2019. 10(2): pp. 429-462; and Kleczko, E.K. et al., Front Immunol, 2019. 10: pp. 95).

[0003] Complement pathway activation and regulation is controlled by a complex interplay between pathway activators and inhibitors. These activators and inhibitors are generally enzymes that cleave and inactivate complement molecules on biological surfaces and / or in solution to maintain stable control of complement-activating species. The complement pathway is in a constant state of flux and balance, and disruption of this balance can lead to inappropriate activation and the consequences described above.

[0004] One activating molecule is complement component 3 (C3), a member of the alternative complement pathway and amplification loop. C3 contains a β chain and an α' chain associated through an interchain disulfide bond. During complement activation, C3 is cleaved to generate two functional fragments, C3a and C3b. C3a is a potent anaphylatoxin. Deposition of C3b on biological surfaces, such as the extracellular matrix and cell surfaces, is the central activation mechanism of the alternative pathway. C3b is a potent opsonin, targeting pathogens, antibody-antigen immune complexes, and apoptotic cells for phagocytosis by phagocytes and NK cells. Surface-bound C3b also reacts with other complement proteins to form active convertase enzymes, which can produce additional (surface-attachable) C3b molecules and act to activate and amplify the complement response (Clark, SJ et al., J Immunol, 2014. 193(10): p. 4962-70). C3b associates with factor B to form the C3bBb-type C3 convertase and with C3bBb to form the C3bBb3b-type C5 convertase. Proteolytic cleavage of C3 also produces C3a and C3b through the classical complement pathway and the lectin pathway.

[0005] Inadequate regulation of C3 convertase results in the production of large amounts of C3b and C3a molecules and a shift in the complement cascade toward the terminal lytic pathway, which generates the potent anaphylatoxin C5a and a cytolytic protein complex called the membrane attack complex; both of which provide strong inflammatory signals (Clark, SJ et al., supra). This ultimately leads to cell / tissue destruction and local immune responses.

[0006] Complement activation of C3b is controlled by the complement protein factor I (FI). FI prevents complement inactivation by cleaving C3b into a proteolytically inactive form called iC3b, which cannot participate in convertase assembly, and further cleaving it into the downstream products iC3dg and C3d. FI requires the presence of cofactors, examples of which include the blood-borne factor H (FH) protein and the membrane-bound surface cofactor "complement factor 1" (CR1:CD35). FH and CR1 also help exert decay-accelerating activity, which can assist in the deconstruction of already formed C3 convertases.

[0007] FH is encoded by the CFH gene on human chromosome 1q32 within the RCA (complement regulatory factor) gene cluster. There is a naturally occurring truncated form of FH, termed FH-like protein 1 (FHL-1), which arises from alternative splicing of the CFH gene and has FH-like cofactor activity. FH contains 20 CCP domains. FHL-1 is identical to FH for the first seven CCP domains and then terminates with a unique four-amino acid C-terminus.

[0008] Proteins encoded by the CFHR1-5 genes in the RCA locus also exert complement regulatory functions. The CFHR1-5 genes encode a group of five secreted plasma proteins (FHR1-FHR5) that are synthesized primarily by hepatocytes. The FHR proteins share some sequence homology with the C3b-binding domain of FH and are thought to enhance complement activation (Skerka et al., Mol Immunol. 2013, 56:170-180).

[0009] One complement-related disorder is macular degeneration, e.g., age-related macular degeneration (AMD). Macular degeneration is thought to be driven, in part, by complement-mediated attack on ocular tissues. The primary driver of AMD risk is genetic mutations at the RCA locus, which result in dysregulation of the complement cascade. AMD is the leading cause of blindness in developing countries; it currently accounts for 8.7% of all registered blindness cases worldwide. It is estimated that by 2020, 196 million people will be affected, increasing to 288 million by 2040 (Wong et al., Lancet Glob Heal (2014) 2:e106-16). AMD manifests as progressive destruction of the macula, the central part of the retina at the back of the eye, leading to loss of central vision. The early stages of the disease manifest as morphological changes in the retina, such as loss of blood vessels in the choriocapillaris, a highly vascularized layer found in the choroid that supplies oxygen and nutrients to the outer retina (Whitmore et al., Prog Retin Eye Res (2015) 45:1-29). The choriocapillaris is separated from the metabolically active retinal pigment epithelium (RPE) by Bruch's membrane (BrM), a thin (2-4 μm), acellular, five-layered sheet of extracellular matrix. The BrM has two primary functions: providing a substratum for the RPE and supplying blood vessel walls. The structure and function of the BrM are reviewed, for example, by Curcio and Johnson, Structure, Function and Pathology of Bruch's Membrane, in: Ryan et al. (2013), Retina, Vol. 1, Part 2: Basic Science and Translation to Therapy. 5th ed. London: Elsevier, pp. 466-481, which is incorporated herein by reference in its entirety.

[0010] The role of complement in AMD is reviewed, for example, by Zipfel et al., Chapter 2, Lambris and Adamis (eds.), Inflammation and Retinal Disease: Complement Biology and Pathology, Advances in Experimental Medicine and Biology 703, Springer Science+Business Media, LLC (2010), which is incorporated herein by reference in its entirety.The key characteristics of AMD show hyperactive complement, which includes cell / tissue destruction and local inflammatory reaction.The characteristic lesion of early AMD, called drusen, develops in the BrM adjacent to the RPE layer (Bird et al., Surv Ophthalmol 1995, 39(5):367-374). Drusen form from the accumulation of lipids, proteins, and cellular debris, and contain swathes of complement activation products (Anderson et al., Prog Retin Eye Res 2009, 29:95-112; Whitcup et al., Int J Inflam 2013, 1-10). The presence of drusen within the BrM impedes the flow of nutrients from the choroid across this extracellular matrix to RPE cells, leading to cellular dysfunction and eventual death, resulting in vision loss.

[0011] "Dry" AMD, also known as geographic atrophy, accounts for approximately 50% of late-stage AMD cases. In the remaining percentage of late-stage cases, choroidal neovascularization (CNV) develops, in which increased synthesis of vascular endothelial growth factor (VEGF) by RPE cells promotes the growth of new blood vessels from the choroid / choriocapillaris, which break through the BrM into the retina. These new blood vessels leak and eventually form scar tissue; this is referred to as "wet" (neovascular or exudative) AMD. "Wet" AMD is the most pathogenic form of late-stage AMD and has disease characteristics distinct from "dry" AMD. Treatments exist for wet AMD, such as intravitreal injection of anti-VEGF agents, which can slow or reverse the growth of these blood vessels, but cannot prevent their formation in the first place. Geographic atrophy ("dry" AMD) remains untreatable.

[0012] FHL-1 predominates in BrM, suggesting that this variant plays an important role in protecting retinal tissue from complement-mediated attack (Clark, SJ et al., supra). FH is found in the blood at higher concentrations than FHL-1. Both FH and FHL-1 protect against complement hyperactivation in the ECM of the choroid (the capillary network underlying BrM). The roles of the five FHR proteins are less well understood, but there is some evidence that they can counteract the inhibitory effects of FH and FHL-1 (Clark, SJ and PN Bishop, J Clin Med, 2015. 4(1): p. 18-31).

[0013] WO2019 / 215330 describes that FHR4 is a positive regulator of complement activation and prevents FH-mediated C3b degradation, leading to the formation of C3 convertase and progression of the complement activation loop. High levels of circulating FHR4 expressed in the liver indicate an increased risk of developing complement-related disorders.

[0014] Defining the precise molecular changes and activation states that support dysregulated complement processes in human disease tissues remains challenging, in large part because this requires measuring the protein level and understanding the relative abundance of different regulators. For effective diagnosis and treatment of complement-related disorders, it is crucial to be able to accurately measure the absolute levels of FH and related RCA locus proteins in plasma, as well as the levels of FI and C3b themselves. While assays for FH have been developed, separate measurements of FHL-1 and FHR1-5 are challenging due to the high sequence homology between all of these proteins. This sequence similarity means that, with the exception of the full-length FH protein, it has proven difficult to generate antibodies specific to only one of these family members to obtain useful immunoassays.

[0015] In another example, a recent study used mass spectrometry to quantify levels of FH and FHR1-5, but the assay failed to detect the biologically important isoform FHL-1, which is found at significant levels in the blood and at key sites in AMD pathogenesis (Zhang, P et al., Proteomics. 2017;17(6):10). [Prior art documents] [Patent documents]

[0016] [Patent Document 1] WO2019 / 215330 [Non-patent literature]

[0017] [Non-Patent Document 1] Morgan, BP, Semin Immunopathol, 2018. 40(1): p. 113-124 [Non-patent document 2] Halbgebauer, R. et al., Semin Immunol, 2018. 37: p. 12-20 [Non-patent document 3] Ma, Y. et al., Aging Dis, 2019. 10(2): p. 429-462 [Non-patent document 4] Kleczko, EK et al., Front Immunol, 2019. 10: p. 95 [Non-Patent Document 5] Clark, SJ et al., J Immunol, 2014. 193(10): pp. 4962-70 [Non-patent document 6] Skerka et al., Mol Immunol. 2013, 56:170-180). [Non-Patent Document 7] Wong et al. Lancet Glob Heal (2014) 2:e106-16 [Non-patent document 8] Whitmore et al., Prog Retin Eye Res (2015) 45:1-29 [Non-Patent Document 9] Curcio and Johnson, Structure, Function and Pathology of Bruch's Membrane, in: Ryan et al. (2013), Retina, Vol. 1, Part 2: Basic Science and Translation to Therapy. 5th ed. London: Elsevier, pp. 466-481 [Non-Patent Document 10] Zipfel et al., Chapter 2, Lambris and Adamis (eds.), Inflammation and Retinal Disease: Complementary Biology and Pathology, Advances in Experimental Medicine and Biology 703, Springer Science+Business Media, LLC (2010) [Non-Patent Document 11] Bird et al., Surv Ophthalmol 1995, 39(5):367-374 [Non-Patent Document 12] Anderson et al., Prog Retin Eye Res 2009, 29:95-112 [Non-Patent Document 13] Whitcup et al., Int J Inflam 2013, 1-10 [Non-Patent Document 14] Clark, SJ and PN Bishop, J Clin Med, 2015. 4(1): p. 18-31 [Non-Patent Document 15] Zhang, P et al., Proteomics. 2017;17(6):10 Summary of the Invention

[0018] In a first aspect, the present disclosure provides an agent for reducing gene and / or protein expression of one or more Factor H family proteins. In some embodiments, the one or more Factor H family proteins are Factor H-related proteins, optionally wherein the Factor H-related proteins are selected from FHR1, FHR2, FHR3, FHR4 and / or FHR5.

[0019] In some embodiments, the agent is an inhibitory nucleic acid. In some embodiments, the inhibitory nucleic acid comprises or encodes an antisense nucleic acid that targets a nucleotide sequence of RNA encoded by one or more genes encoding one or more Factor H family proteins.

[0020] In some embodiments, the inhibitory nucleic acid comprises or encodes an antisense nucleic acid targeting a nucleotide sequence comprising or consisting of one or more of SEQ ID NOs: 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176 and / or 177. In some embodiments, the inhibitory nucleic acid comprises or encodes an antisense nucleic acid comprising or consisting of a sequence having at least 75% sequence identity to one or more of SEQ ID NOs: 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196 and / or 197.

[0021] In some embodiments, the inhibitory nucleic acid is an siRNA, shRNA, miRNA, or an antisense oligonucleotide. In some embodiments, the agent is a gene editing tool or a gene editing system. In some embodiments, the agent is selected from: meganucleases, chemical nucleases, zinc finger nucleases (ZFNs), transcription activator-like effector-based nucleases (TALENs), or CRISPR-Cas systems (e.g., base editing systems). The CRISPR-Cas system may include a guide RNA (gRNA) and / or a tracrRNA. The CRISPR-Cas system may include a single guide RNA (sgRNA). In some embodiments, the CRISPR-Cas system includes a sequence, such as a gRNA or sgRNA, that has at least 75% sequence identity to SEQ ID NO: 224, 225, 226, or 227.

[0022] The present disclosure also provides nucleic acids, optionally isolated, encoding the agents described in this disclosure. The present disclosure also provides expression vectors comprising the nucleic acids described in this disclosure. The present disclosure also provides compositions comprising an agent, nucleic acid, or expression vector described in this disclosure and a pharmaceutically acceptable carrier, diluent, excipient, or adjuvant.

[0023] The present disclosure also provides a cell comprising an agent, nucleic acid, or expression vector described in this disclosure. The present disclosure also provides an in vitro or in vivo method for reducing gene and / or protein expression of one or more Factor H family proteins, the method comprising contacting a cell with an agent, nucleic acid, expression vector, or composition described herein.

[0024] The present disclosure also provides the use of an agent, nucleic acid, expression vector, or composition according to the present disclosure to reduce gene and / or protein expression of one or more Factor H family proteins.

[0025] The present disclosure also provides an agent, nucleic acid, expression vector, or composition according to the present disclosure for use in a method of medical treatment or prevention. The present disclosure also provides an agent, nucleic acid, expression vector, or composition according to the present disclosure for use in a method of treating or preventing a complement-associated disorder.

[0026] The present disclosure also provides the use of an agent, nucleic acid, expression vector, or composition described herein in the manufacture of a medicament for the treatment or prevention of a complement-associated disorder. The present disclosure also provides a method of treating or preventing a complement-associated disorder in a subject, the method comprising administering to the subject a therapeutically or prophylactically effective amount of an agent, nucleic acid, expression vector, or composition described herein.

[0027] The present disclosure also provides a method for selecting a subject to be administered an agent, nucleic acid, expression vector, or composition described herein, comprising: (a) determining in a blood sample obtained from the subject the level of complement proteins selected from one or more of FHR1, FHR2, FHR3, FHR4 and / or FHR5, and optionally FHL-1; (b) if the level of the complement protein determined in (a) is elevated when compared to the level of that complement protein in the blood of a control subject who does not have a complement-related disorder, select the subject as a subject to be administered an agent, nucleic acid, expression vector, or composition described herein. The method also includes the steps of:

[0028] The present disclosure also provides an agent, nucleic acid, expression vector, or composition according to the present disclosure for use in a method of treating or preventing a complement-associated disorder in a subject, the method comprising: (a) determining in a blood sample obtained from the subject the level of complement proteins selected from one or more of FHR1, FHR2, FHR3, FHR4 and / or FHR5, and optionally FHL-1; (b) determining whether the level of the complement protein(s) in (a) is elevated when compared to the level of that complement protein(s) in the blood in a control subject who does not have a complement-related disorder; and (c) administering to the subject an agent, nucleic acid, expression vector, or composition described herein. Also provided is the agent, nucleic acid, expression vector, or composition comprising the steps of:

[0029] The present disclosure also provides use of an agent, nucleic acid, expression vector, or composition described herein in the manufacture of a medicament for treating or preventing a complement-associated disorder in a subject, the method comprising: (a) determining in a blood sample obtained from the subject the level of complement proteins selected from one or more of FHR1, FHR2, FHR3, FHR4 and / or FHR5, and optionally FHL-1; (b) determining whether the level of the complement protein(s) in (a) is elevated when compared to the level of that complement protein(s) in the blood in a control subject who does not have a complement-related disorder; and (c) administering to the subject an agent, nucleic acid, expression vector, or composition described herein. The use also includes the steps of:

[0030] The present disclosure also provides a method of treating or preventing a complement-associated disorder in a subject, comprising: (a) determining in a blood sample obtained from the subject the level of complement proteins selected from one or more of FHR1, FHR2, FHR3, FHR4 and / or FHR5, and optionally FHL-1; (b) determining whether the level of the complement protein is elevated when compared to the level of that complement protein in the blood in a control subject who does not have a complement-associated disorder; and (c) administering to the subject an agent, nucleic acid, expression vector, or composition described herein. The method also includes the steps of:

[0031] In some embodiments according to various different aspects of the disclosure, the complement-associated disorder is: macular degeneration, age-related macular degeneration (AMD), geographic atrophy ("dry" (i.e., non-exudative) AMD), early AMD, early-onset macular degeneration (EOMD), intermediate AMD, late / advanced AMD, "wet" (neovascular or exudative) AMD, choroidal neovascularization (CNV), retinal dystrophies, hemolytic uremic syndrome (HUS), atypical hemolytic uremic syndrome (aHUS), DEAP HUS (FHR plasma protein deficiency and autoantibody positive form of hemolytic uremic syndrome), autoimmune uveitis, kidney injury / damage / dysfunction, glomerular disease, membranoproliferative glomerulonephritis type II (MPGN), or II), sepsis, Henoch-Schönlein purpura (HSP), IgA nephropathy, chronic kidney disease, paroxysmal nocturnal hemoglobinuria (PNH), autoimmune hemolytic anemia (AIHA), systemic lupus erythematosus (SLE), Sjögren's syndrome (SS), rheumatoid arthritis (RA), C3 glomerulopathy (C3G), dense deposit disease (DDD), C3 nephritic factor glomerulonephritis (C3 NF) The subject may be selected from the group consisting of GN, FHR5 nephropathy, hereditary angioedema (HAE), acquired angioedema (AAE), encephalomyelitis, atherosclerosis, antineutrophil cytoplasmic autoantibody (ANCA) vasculitis, neurodegenerative / neurodegenerative diseases, dementia, multiple sclerosis (MS), Lewy body disease, amyotrophic lateral sclerosis (ALS), Huntington's disease, prion disease, cancer, lung cancer, glioblastoma, e.g., glioblastoma multiforme (GBM), stroke, insulin resistance, diabetes, infectious disease, Parkinson's disease, and / or Alzheimer's disease. The subject to be treated may be or has been determined to have a complement-related disease. [Brief explanation of the drawings]

[0032] Embodiments and experiments illustrating the principles of the present invention will now be discussed with reference to the accompanying figures. [Figure 1]Schematic diagram showing the C3 proteolytic cascade and proteolytic events leading to the generation, degradation, and inactivation of C3b (modified from Maillard et al., J Am Soc Nephrol. 2015 Jul;26(7):1503-12). Proteoform-specific peptides for mass spectrometry analysis are underlined. [Figure 2] LC-SRM trace showing detection of heavy-labeled synthetic standard peptides of each individual RCA locus protein from plasma samples. [Figure 3-1] Linearity data for peptides derived from FH, FHL-1, and FHR1–5. [Figure 3-2] Linearity data for peptides derived from FH, FHL-1, and FHR1–5. [Figure 3-3] Linearity data for peptides derived from FH, FHL-1, and FHR1–5. [Figure 4-1] Data confirming that C3 and C3 breakdown products in human plasma can be detected by MS with sufficient specificity and sensitivity. 4A: Total ion chromatograph from SRM-MS analysis showing specific and simultaneous detection of C3b fragment-specific peptides. 4B: Linearity data for 7 of 10 peptides spiked into the plasma background. [Figure 4-2] Data confirming that C3 and C3 degradation products in human plasma can be detected by MS with sufficient specificity and sensitivity. 4C: Coomassie-stained electrophoresis gel of C3 degradation products obtained in vitro. 4D: MS quantification of key C3 fragments from the in vitro assay products shown in 4C. [Figure 5] Correlation matrix showing Pearson correlation coefficients between different variables (absolute concentration levels of the various studied proteins). [Figure 6-1] Scatter plot showing differences in protein levels between AMD subjects and control individuals, with means and p-values ​​shown (p<0.05 considered statistically significant). [Figure 6-2]Scatter plot showing differences in protein levels between AMD subjects and control individuals, with means and p-values ​​shown (p<0.05 considered statistically significant). [Figure 7] Area under the receiver operating characteristic curve for various models. [Figure 8] Receiver operating characteristic curves for models using FHR1, FHR2, FHR3, FHR4, FHR5, FHL-1 and CFH levels (two-way and three-way interactions) to predict whether an individual is an AMD case or control subject. [Figure 9-1] GWAS of circulating FHR-1 to FHR-5 protein levels reveals a strong genome-wide significant signal across the CFH locus. Local plots show genome-wide significant (P value ≤ 5x10-8) association signals from GWAS of FHR-1 to FHR-5 protein levels at the CFH locus on chromosome 1q31.3 (Panels A–E). Panel F shows the equivalent CFH region for GWAS of FHL-1 protein levels (no genome-wide significant association region was observed). The most associated variant is indicated by a purple diamond and labeled by its rs number. Other surrounding variants are indicated by colored circles reflecting the degree of linkage disequilibrium with the most associated variant (based on genotype data from the 1000 Genomes Project, November 2014). Diagrams of genes within the associated region are shown below each plot. Physical locations are based on the NCBI RefSeq hg19 human genome reference assembly. [Figure 9-2]GWAS of circulating FHR-1 to FHR-5 protein levels reveals a strong genome-wide significant signal across the CFH locus. Local plots show genome-wide significant (P value ≤ 5x10-8) association signals from GWAS of FHR-1 to FHR-5 protein levels at the CFH locus on chromosome 1q31.3 (Panels A–E). Panel F shows the equivalent CFH region for GWAS of FHL-1 protein levels (no genome-wide significant association region was observed). The most associated variant is indicated by a purple diamond and labeled by its rs number. Other surrounding variants are indicated by colored circles reflecting the degree of linkage disequilibrium with the most associated variant (based on genotype data from the 1000 Genomes Project, November 2014). Diagrams of genes within the associated region are shown below each plot. Physical locations are based on the NCBI RefSeq hg19 human genome reference assembly. [Figure 9-3] GWAS of circulating FHR-1 to FHR-5 protein levels reveals a strong genome-wide significant signal across the CFH locus. Local plots show genome-wide significant (P value ≤ 5x10-8) association signals from GWAS of FHR-1 to FHR-5 protein levels at the CFH locus on chromosome 1q31.3 (Panels A–E). Panel F shows the equivalent CFH region for GWAS of FHL-1 protein levels (no genome-wide significant association region was observed). The most associated variant is indicated by a purple diamond and labeled by its rs number. Other surrounding variants are indicated by colored circles reflecting the degree of linkage disequilibrium with the most associated variant (based on genotype data from the 1000 Genomes Project, November 2014). Diagrams of genes within the associated region are shown below each plot. Physical locations are based on the NCBI RefSeq hg19 human genome reference assembly. [Figure 10-1]Established AMD risk variants at the CFH locus are associated with circulating FHR-1, FHR-2, FHR-3, and FHR-4 protein levels at genome-wide significance levels in 252 Cambridge controls. Box plots of FHR protein levels by variant genotype for these established AMD risk variants at the CFH locus from the IAMDGC study that showed genome-wide significant (P value ≤ 5x10-8) associations in 252 controls from the Cambridge AMD cohort (Table 2). P values ​​and β values ​​from Wald tests using linear regression models adjusted for sex, age, and the first two genetic principal components (as established within the IAMDGC study) are shown in the notes below each plot. [Figure 10-2] Established AMD risk variants at the CFH locus are associated with circulating FHR-1, FHR-2, FHR-3, and FHR-4 protein levels at genome-wide significance levels in 252 Cambridge controls. Box plots of FHR protein levels by variant genotype for these established AMD risk variants at the CFH locus from the IAMDGC study that showed genome-wide significant (P value ≤ 5x10-8) associations in 252 controls from the Cambridge AMD cohort (Table 2). P values ​​and β values ​​from Wald tests using linear regression models adjusted for sex, age, and the first two genetic principal components (as established within the IAMDGC study) are shown in the notes below each plot. [Figure 11-1]Mendelian randomization analysis shows highly significant elevations in circulating FHR-1, FHR-2, FHR-3, FHR-4, and FHR-5 protein levels in advanced AMD. Mendelian randomization estimates for the association of FHR-1 (Panel A), FHR-2 (Panel B), FHR-3 (Panel C), FHR-4 (Panel D), and FHR-5 (Panel E) are presented along with corresponding traditional epidemiological odds ratio (OR) estimates obtained from logistic regression models (352 advanced AMD cases and 252 controls from the Cambridge AMD Study). Mendelian randomization estimates were obtained using Wald ratios (when only a single instrument was available; FHR1, FHR2, FHR4, FHR5) or the inverse variance weighting (IVW) method under a fixed-effects model (when multiple instruments were available; FHR3). The raw data used to calculate the Mendelian randomization estimates are provided in Table S7. The variance for each protein explained by the genetic manipulation(s) is indicated in the notes below each plot. [Figure 11-2] Mendelian randomization analysis shows highly significant elevations in circulating FHR-1, FHR-2, FHR-3, FHR-4, and FHR-5 protein levels in advanced AMD. Mendelian randomization estimates for the association of FHR-1 (Panel A), FHR-2 (Panel B), FHR-3 (Panel C), FHR-4 (Panel D), and FHR-5 (Panel E) are presented along with corresponding traditional epidemiological odds ratio (OR) estimates obtained from logistic regression models (352 advanced AMD cases and 252 controls from the Cambridge AMD Study). Mendelian randomization estimates were obtained using Wald ratios (when only a single operator was available; FHR1, FHR2, FHR4, FHR5) or the inverse variance weighting (IVW) method under a fixed-effects model (when multiple operators were available; FHR3). The raw data used to calculate the Mendelian randomization estimates are provided in Table S7. The variance for each protein explained by the genetic manipulation(s) is indicated in the notes below each plot. [Figure 12-1]Graphs showing suppression of CFH family gene expression in huH1 cells by CFHR siRNA1, 2, and 3. (A) and (B) The relative abundance of CFHR transcripts was assessed in huH1 cells after initial transfection with CFHR siRNA1, CFHR siRNA2, or CFHR siRNA3 (10 nm each) compared to 24-hour treatment with the respective scrambled / control siRNA (10 nm). *** indicates p<0.005; * indicates p<0.05. Bars show fold-change (2-ΔΔCT) ± SEM. [Figure 12-2] Graphs showing suppression of CFH family gene expression in huH1 cells by CFHR siRNA1, 2, and 3. (A) and (B) The relative abundance of CFHR transcripts was assessed in huH1 cells after initial transfection with CFHR siRNA1, CFHR siRNA2, or CFHR siRNA3 (10 nm each) compared to 24-hour treatment with the respective scrambled / control siRNA (10 nm). *** indicates p<0.005; * indicates p<0.05. Bars show fold-change (2-ΔΔCT) ± SEM. [Figure 12-3] Graphs showing suppression of CFH family gene expression in huH1 cells by CFHR siRNA1, 2, and 3. (A) and (B) The relative abundance of CFHR transcripts was assessed in huH1 cells after initial transfection with CFHR siRNA1, CFHR siRNA2, or CFHR siRNA3 (10 nm each) compared to 24-hour treatment with the respective scrambled / control siRNA (10 nm). *** indicates p<0.005; * indicates p<0.05. Bars show fold-change (2-ΔΔCT) ± SEM. [Figure 12-4]Graphs showing suppression of CFH family gene expression in huH1 cells by CFHR siRNA1, 2, and 3. (A) and (B) The relative abundance of CFHR transcripts was assessed in huH1 cells after initial transfection with CFHR siRNA1, CFHR siRNA2, or CFHR siRNA3 (10 nm each) compared to 24-hour treatment with the respective scrambled / control siRNA (10 nm). *** indicates p<0.005; * indicates p<0.05. Bars show fold-change (2-ΔΔCT) ± SEM. [Figure 12-5] Graphs showing suppression of CFH family gene expression in huH1 cells by CFHR siRNA1, 2, and 3. (A) and (B) The relative abundance of CFHR transcripts was assessed in huH1 cells after initial transfection with CFHR siRNA1, CFHR siRNA2, or CFHR siRNA3 (10 nm each) compared to 24-hour treatment with the respective scrambled / control siRNA (10 nm). *** indicates p<0.005; * indicates p<0.05. Bars show fold-change (2-ΔΔCT) ± SEM. [Figure 12-6] Graphs showing suppression of CFH family gene expression in huH1 cells by CFHR siRNA1, 2, and 3. (A) and (B) The relative abundance of CFHR transcripts was assessed in huH1 cells after initial transfection with CFHR siRNA1, CFHR siRNA2, or CFHR siRNA3 (10 nm each) compared to 24-hour treatment with the respective scrambled / control siRNA (10 nm). *** indicates p<0.005; * indicates p<0.05. Bars show fold-change (2-ΔΔCT) ± SEM. [Figure 12-7]Graph showing suppression of CFH family gene expression in huH1 cells by CFHR siRNA1, 2, and 3. (C) Secondary transfection results after 24 hours of treatment with CFHR siRNA1, CFHR siRNA2, CFHR siRNA3 (10 nm each) and their corresponding scrambled / control siRNAs (10 nm). *** indicates p<0.005; * indicates p<0.05. Bars show fold change (2-ΔΔCT) ± SEM. [Figure 12-8] Graph showing suppression of CFH family gene expression in huH1 cells by CFHR siRNA1, 2, and 3. (C) Secondary transfection results after 24 hours of treatment with CFHR siRNA1, CFHR siRNA2, CFHR siRNA3 (10 nm each) and their corresponding scrambled / control siRNAs (10 nm). *** indicates p<0.005; * indicates p<0.05. Bars show fold change (2-ΔΔCT) ± SEM. [Figure 12-9] Graph showing suppression of CFH family gene expression in huH1 cells by CFHR siRNA1, 2, and 3. (C) Secondary transfection results after 24 hours of treatment with CFHR siRNA1, CFHR siRNA2, CFHR siRNA3 (10 nm each) and their corresponding scrambled / control siRNAs (10 nm). *** indicates p<0.005; * indicates p<0.05. Bars show fold change (2-ΔΔCT) ± SEM. [Figure 12-10] Graph showing suppression of CFH family gene expression in huH1 cells by CFHR siRNA1, 2, and 3. (D) Combined results for relative quantification of CFHR transcripts from three separate transfections with CFHR siRNA1 (10 nm) treatment compared to scrambled / control siRNA (10 nm) treatment for 24 hours, in triplicates (n=9). *** indicates p<0.005; * indicates p<0.05, and bars show fold change (2-ΔΔCT) ± SEM. [Figure 13]Graph showing suppression of CFHR gene expression in huH1 cells by CFHR siRNA4, 5, and 6. Relative quantification of CFHR transcripts was assessed in huH1 cells after transfection with CFHR siRNA4, CFHR siRNA5, and CFHR siRNA6 (10 nm each) compared to treatment with the respective scrambled / control siRNA (10 nm) for 24 hours. *** indicates p<0.005; * indicates p<0.05. Bars show fold change (2-ΔΔCT) ± SEM. [Figure 14-1] Graph showing suppression of CFHR gene expression in huH1 cells by CFHR siRNAs 7, 8, and 9. Relative quantification of CFHR transcripts was assessed in huH1 cells after transfection with CFHR siRNAs 7, 8, and 9 (10 nm each) compared to treatment with the respective scrambled / control siRNAs (10 nm) for 24 hours. *** indicates p<0.005; * indicates p<0.05. Bars show fold change (2-ΔΔCT) ± SEM. [Figure 14-2] Graph showing suppression of CFHR gene expression in huH1 cells by CFHR siRNAs 7, 8, and 9. Relative quantification of CFHR transcripts was assessed in huH1 cells after transfection with CFHR siRNAs 7, 8, and 9 (10 nm each) compared to treatment with the respective scrambled / control siRNAs (10 nm) for 24 hours. *** indicates p<0.005; * indicates p<0.05. Bars show fold change (2-ΔΔCT) ± SEM. [Figure 14-3] Graph showing suppression of CFHR gene expression in huH1 cells by CFHR siRNAs 7, 8, and 9. Relative quantification of CFHR transcripts was assessed in huH1 cells after transfection with CFHR siRNAs 7, 8, and 9 (10 nm each) compared to treatment with the respective scrambled / control siRNAs (10 nm) for 24 hours. *** indicates p<0.005; * indicates p<0.05. Bars show fold change (2-ΔΔCT) ± SEM. DETAILED DESCRIPTION OF THE INVENTION

[0033] The present disclosure provides agents, e.g., inhibitory nucleic acids and gene editing systems, for reducing gene and / or protein expression of one or more Factor H family proteins.

[0034] Aspects of the present disclosure arise from the measured observation that circulating levels of all five Factor H-related (FHR) proteins are elevated in human blood in individuals suffering from several complement-associated disorders, e.g., eye disorders such as macular degeneration, kidney disorders, cancer, and CNS disorders.

[0035] Circulating levels of these FHR proteins are obtained exclusively from the liver, the only known source of expression in the human body. Expression of the proteins is largely gene-driven. The ability of FHR proteins to outcompete negative regulators of complement activation (i.e., FH and FHL-1) means that increased concentrations of these proteins may predispose a patient to more complement-activated disorders. Thus, detecting overexpression of one or more FHR proteins, for example, as described herein, predicts an individual's likelihood of developing a complement-related disorder.

[0036] By targeting the expression level of FHR protein in the liver or by directly intervening in the blood, it is possible to prevent genetically driven excess FHR protein from entering or being removed from the circulation, thus preventing its accumulation in tissues / organs and subsequent damaging complement activation, inflammation, immune cell recruitment, and ECM modeling. In this way, complement-related disorders may be treated or prevented. Currently, there are no therapies designed to reduce circulating levels of FHR protein. Therefore, described herein are agents that target each individual CFHR gene, as well as agents, such as inhibitory nucleic acids, that can simultaneously reduce the expression of multiple CFHR genes. Advantageously, the present invention also identifies agents that do not simultaneously affect the levels of FH and / or FHL-1. Some agents even increase the levels of FH and / or FHL-1 while reducing the expression of one or more FHR proteins.

[0037] The methods disclosed herein may involve, in part, the detection and quantification of complement-related proteins, particularly one or more FHR proteins, and optionally FHL-1 and / or FH. Such methods are useful for identifying and stratifying patients with disorders associated with overactivation of the complement system due to increased circulating levels of FHR proteins. Such methods may also be used to stratify patients based on their risk of developing or having a complement-related disorder. In some cases, the methods disclosed herein are used to identify appropriate treatments, such as treatments that target specific complement proteins that are overexpressed in the patient, such as the agents described herein.

[0038] Detection, differentiation, and quantification of closely related proteins can be achieved using mass spectrometry (MS). To achieve superior sensitivity with MS, for example, proteins in a sample are routinely digested into peptides using specific proteases. The industry standard protease for this purpose is trypsin. Other enzymes commonly used to digest proteins for MS analysis include elastase, chymotrypsin, or LysN.

[0039] Trypsin cleaves the C-terminus of all K and R residues unless they are followed by a proline residue, resulting in peptides that retain a basic group at their C-terminus, which subsequently aids in the ionization and gas-phase transfer of the peptide in the mass analyzer. Peptides digested by trypsin tend to ionize more efficiently during MS and therefore produce larger signals than peptides digested by nontryptic enzymes. Using MS, individual peptides in a sample digest can be detected with signals proportional to their abundance. The concentration of the parent protein can be obtained from the relative abundance (signal) of the endogenous peptide compared to an exogenous "standard" peptide, such as one containing a stable isotope.

[0040] Trypsin digestion of the complement proteins FH and FHL-1 does not yield peptides that are individually detectable using MS alone. The only FHL-1-specific tryptic peptide is a four-amino acid C-terminal sequence, which is too small to be reliably detected by MS techniques. FHR proteins also share considerable sequence identity, meaning that it is difficult to distinguish between and specifically measure them using, for example, antibody-based assays.

[0041] Also described herein is a unique targeted mass spectrometry assay that uses a non-standard proteolytic enzyme, GluC (V8 protease), to produce distinct proteotypic peptides for all FHR proteins, as well as proteotypic peptides that can be used to distinguish between FHL-1 and FH, that can be used to simultaneously detect and accurately measure in plasma all seven key regulatory proteins encoded from the CFH gene cluster: FH, FHL-1, and FHR1, FHR2, FHR3, FHR4, and FHR5, using a single MS assay.

[0042] FHL-1 is a biological entity separate from FH.This protein has similar effects to FHL-1 but is different in size, which means that its distribution in the body is likely to be different from that of FH.This is evident in the eye, where FHL-1 can cross the retinal side of Bruch's membrane, for example, where drusen forms, but where larger FH protein cannot cross.See, for example, Clark et al., J Immunol 2014, 193(10) 4962-4970 and Clark et al., Frontiers in Immunology 2017 8:1778, which are incorporated herein by reference in their entirety.In this regard, there is evidence that FHL-1 is the main driver of complement C3b turnover in the eye, which means that the level of FHL-1 may convey disease risk information better than the level of FH.

[0043] GluC can also generate proteotypic peptides for C3b and FI, allowing for the direct measurement of C3b itself as well as its proteolytic enzymes and required liquid-phase complement factors. Thus, the methods described herein mean that all of these complement proteins can be measured using a single assay.

[0044] Furthermore, C3b degradation occurs through trypsin-like cleavage at basic residues (K and R); therefore, trypsin digestion of C3b degradation products fails to produce peptides useful for analysis. In contrast, C3 turnover can be measured using the disclosed MS approach because GluC digestion also produces proteotypic neopeptides from many of the C3 inactivation and degradation products that arise during inactivation cleavage. Here, we demonstrate that the range of products resulting from C3 / C3b cleavage can be detected and quantified using the same single GluC / MS assay. This allows for accurate determination of the concentrations of all known C3 fragments, such as iC3b, C3c, C3dg, and C3d. Thus, the methods described herein are not only capable of measuring absolute levels of regulatory complement proteins, but also of tracking protein products resulting from C3 inactivation and, therefore, assessing the progression of the complement activation and amplification loop.

[0045] This is advantageous because measuring C3 breakdown products is analytically challenging. The pattern of C3 degradation is complex: first, to C3a and C3b, then C3b is cleaved to iC3b (which cannot drive membrane attack complex (MAC) formation but can still act as an opsonin), and then iC3b is subsequently inactivatingly cleaved to C3c through the release of the C3dg fragment. Attempts to detect these products with antibodies have been difficult. In this cascade, each successive cleavage step generates a new proteoform (a distinct form of the protein encoded by the same gene, including truncated forms and splice variants), which likely share sequence homology and have undergone only minor structural changes. Directing antibodies to each form is unlikely to be successful, and although methods exist that allow for the measurement of a single component, e.g., C3dg, after separation of some forms and subsequent enrichment based on polyethylene glycol, simultaneous measurement of all fragments in the same sample is currently not possible.

[0046] Thus, described herein is a single methodology for the simultaneous determination of the presence, absolute levels, and relative molar ratios of up to seven individual complement-related proteins from the CFH family, which may be referred to herein as the "complementome," as well as the C3b-inactivating enzyme FI, the central complement component C3, and seven proteins resulting from C3 degradation. The ability to detect the absolute levels of so many complement-related proteins in a single assay is critical to the successful detection, diagnosis, and treatment of complement-related diseases, for example, using the agents and systems described herein.

[0047] Complement proteins Complement is a central part of innate immunity, serving as the first line of defense against foreign and modified host cells. Complement is activated during microbial infection, induces inflammation, and promotes pathogen elimination. The complement system is composed of plasma proteins primarily produced by the liver or membrane proteins expressed on cell surfaces. Complement functions in plasma, tissues, or cells. For a review of the complement system, see, for example, Merle NS et al., Front Immunol. 2015 Jun 2;6:262, which is incorporated herein by reference in its entirety.

[0048] The complement system can be activated through three distinct pathways: the classical pathway (CP), the alternative pathway (AP), and the lectin-binding pathway (LP). In healthy individuals, the AP is persistently active at low levels to search for the presence of pathogens, while host cells are protected from complement attack and are resistant to sustained low-level activation. C3b molecules bound to host cells are rapidly inactivated by membrane-bound or plasma complement regulators.

[0049] In response to recognition of molecular components of microorganisms, complement proteins are activated sequentially in an enzymatic cascade: activation of one protein enzymatically cleaves and activates the next protein in the cascade.

[0050] The three pathways converge on the production of C3 convertase, which cleaves the central complement component C3 into a large fragment that acts as an opsonin (binding to foreign microorganisms and increasing their susceptibility to phagocytosis), the active product C3b, and C3a, an anaphylatoxin that promotes inflammation. Together with factor B (FB), C3b forms C3 convertase (C3bBb), which cleaves additional C3 molecules, generating more C3b and C3a and amplifying C3b deposition on cell surfaces. This is the complement amplification loop. C3b deposition and complement activation can occur on acellular structures (i.e., on the extracellular matrix) of the eye, such as Bruch's membrane (BrM) and the capillary septa of the choriocapillaris.

[0051] Activated C3 can trigger a lytic pathway that can damage the plasma membranes of cells and some bacteria. Another anaphylatoxin produced by this process, C5a, attracts macrophages and neutrophils and also activates mast cells.

[0052] Once activated, the complement system must be tightly regulated, because newly generated complement activation products, such as C3b, can induce severe inflammation and cell damage to the host.Several soluble and membrane-bound complement regulators ensure the control of complement activation on the host cell surface and regulate different activation phases and sites of action (Skerka et al., Mol Immunol 2013, 56:170-180).Complement regulators are further described herein.

[0053] "Complement proteins," as used herein, can be used interchangeably with "complement regulators," "regulators of complement," or "proteins of the complement system," and refer to protein components of the complement system or complement cascade, e.g., as described in Merle et al., Front. Immunol., 2015, 6:262 and Merle et al., Front. Immunol., 2015, 6:257, which are incorporated herein by reference in their entireties. "Complement proteins" as referred to herein can be involved in any of the three complement pathways and / or in the amplification loop.

[0054] In some embodiments, "complement proteins" as referred to herein are involved in the alternative pathway and / or in the complement activation loop. In some embodiments, "complement proteins" as referred to herein are involved in or are products of the degradation, turnover, and / or inactivation of C3 or C3b.

[0055] In some embodiments herein, "complement protein" as used herein may refer to one or more of FH, FHL-1, FHR1, FHR2, FHR3, FHR4, FHR5, FI, C3, C3b, C3a, iC3b, C3f, C3c, C3dg, and / or C3d.

[0056] Complement factor H (FH) family proteins Factor H (FH) controls the alternative complement pathway and amplification loop. FH inhibits C3 convertase formation by competing with FB binding to C3b, and also acts as a cofactor for C3b inactivation to iC3b by factor I (FI), thus preventing inappropriate complement activation and inflammation. FH also exerts decay-accelerating activity, which can assist in the deconstruction of already formed C3 convertase. See, for example, Clark et al., J Immunol 2014, 193(10) 4962-4970, the entire contents of which are incorporated herein by reference.

[0057] The sequence of human FH (Uniprot P08603-1) is provided herein as SEQ ID NO: 1. For a review of FH structure and function, see, e.g., Merle NS et al., Front Immunol. 2015 Jun 2;6:262, which is incorporated herein in its entirety.

[0058] Human FH contains 20 CCP domains. The CFH gene also produces a truncated form of FH called FHL-1, which contains only the first seven CCP domains and then terminates with a unique four-amino acid C-terminus (Clark et al., 2014, supra). The sequence of human FHL-1 (Uniprot:P08603-2) is provided herein as SEQ ID NO:2.

[0059] In the eye, full-length FH protein is found on the choroidal side of Bruch's membrane (BrM) and particularly accumulates in the choriocapillaris (the capillary layer in the choroid). Small amounts are also found in patches on the RPE side of BrM, but FH is not observed in the BrM itself. Meanwhile, FHL-1 has been observed throughout BrM and in other ECM structures, such as drusen (Clark et al., 2014, supra). While FHL-1 likely confers greater complement protection to BrM than FH, FH provides the primary protection of the choroidal ECM. FHL-1 is therefore thought to be the primary regulator of complement in BrM, a key site in the pathogenesis of AMD. The methods described herein allow for the individual detection and quantification of FH and FHL-1.

[0060] FH, FHL-1, and FHR1-FHR5 are described, for example, in Clark et al., J Clin Med, 2015. 4(1): 18-31, which is incorporated herein by reference in its entirety. These proteins are referred to herein as "Factor H family proteins." "FHR" stands for "Factor H related."

[0061] The CFHR genes, FHR1, FHR2, FHR3, FHR4, and FHR5, are also described in Skerka et al., Mol Immunol 2013, 56:170-180, which is incorporated herein by reference in its entirety. These proteins are highly related and share a high degree of sequence identity. The N-terminus shares 36-94% sequence identity, while the C-terminal domain is highly similar to the FH C-terminus (36-100%). The high amino acid identity between family members is evidenced by the fact that antibodies raised against FH can detect multiple FHR proteins in plasma, and antibodies raised against FHR proteins cross-react with other FHR proteins. This cross-reactivity complicates the purification of FHR proteins from plasma and the determination of their concentration.

[0062] FHR proteins are divided into two groups based on conserved domains. FHR1 (SEQ ID NO: 3), FHR2 (SEQ ID NO: 3, 4), and FHR5 (SEQ ID NO: 10) form group I and are characterized by their conserved N-terminus. They exist in plasma as homo- and heterodimers mediated by the conserved N-terminal domain. Group II contains FHR3 (SEQ ID NO: 6, 7) and FHR4 (SEQ ID NO: 8, 9), which lack the N-terminal dimerization domain but show a high degree of sequence similarity to portions of FH. All five FHR proteins function to recognize and bind C3b and contain C-terminal sequences highly similar to the C-terminus of FH.

[0063] FHR1 is known to compete with FH and FHL-1 for binding to C3b. FHR1 has also been reported to bind to the C3b component of the C5 convertase and interfere with MAC assembly (see, e.g., Heinen S et al., Blood (2009) 114 (12): 2439-2447 and Hannan JP et al., PLoS One. 2016; 11(11): e0166200, which are incorporated herein by reference in their entirety). As used herein, the term "FHR1" includes at least one of FHR1 (SEQ ID NO: 3; FHRA) and a second FHR1 isoform (FHRB) containing three point mutations, and preferably includes both FHR1 isoforms. "FHR1" refers to FHR1 from any species, and includes isoforms, fragments, variants, or homologs of FHR1 from any species. In a preferred embodiment, "FHR1" refers to human FHR1.

[0064] FHR2 can inhibit C3 convertase activity, acting to inhibit the amplification loop but also activating it. There are two FHR2 isoforms (SEQ ID NOs: 4 and 5). The protein has two glycosylated forms: a single glycosylated form (24 kDa) and a double glycosylated form (28 kDa). As used herein, the term "FHR2" includes at least one of the two isoforms or at least one glycosylated form, and preferably includes both isoforms and any glycosylated form. "FHR2" refers to FHR2 from any species and includes isoforms, fragments, variants, or homologs of FHR2 from any species. In a preferred embodiment, "FHR2" refers to human FHR2.

[0065] FHR3 binds C3b and C3d and may have low cofactor activity for FI-mediated cleavage of C3b. FHR3 may also upregulate complement. There are two FHR3 isoforms (SEQ ID NOs: 6 and 7). FHR3 is detected in plasma in multiple variants (ranging from 35 to 56 kDa), reflecting the presence of four different glycosylation variants of FHR3. As used herein, the term "FHR3" includes at least one of the two isoforms or at least one glycosylation form of FHR3, and preferably includes both isoforms and any glycosylation form. "FHR3" refers to FHR3 from any species and includes isoforms, fragments, variants, or homologs of FHR3 from any species. In a preferred embodiment, "FHR3" refers to human FHR3.

[0066] The human CFHR4 gene encodes two proteins: FHR4A (SEQ ID NO: 8) and an alternative splice variant, FHR4B (SEQ ID NO: 9). WO 2019 / 215330A1, incorporated herein in its entirety, describes FHR4 as a positive regulator of complement activation and prevents FH-mediated C3b degradation. High levels of FHR4 in tissues likely promote local inflammatory responses and cell lysis, leading to disorders associated with complement activation, and circulating FHR4 levels can be used as an indicator of risk for developing complement-related disorders. See, for example, Cipriani et al., Nat Commun 11, 778 (2020), incorporated herein in its entirety. As used herein, the term "FHR4" includes at least one of FHR4A isoform 1, FHR4A isoform 2 (G20 point deletion from isoform 1), or FHR4B, and preferably includes FHR4A isoforms 1 and 2 and FHR4B. "FHR4" refers to FHR4 from any species and includes isoforms, fragments, variants, or homologs of FHR4 from any species. In preferred embodiments, "FHR4" refers to human FHR4.

[0067] FHR5 also recognizes and binds to C3b on self-surfaces. FHR5 appears as a glycosylated protein of 62 kDa. As used herein, the term "FHR5" includes any glycosylated variant of FHR5, and preferably includes all isoforms and any glycosylated forms. As used herein, "FHR5" refers to FHR5 from any species, and includes isoforms, fragments, variants, or homologs of FHR5 from any species. In a preferred embodiment, "FHR5" refers to human FHR5.

[0068] Given the distinct roles of different CFH family members in complement activation and amplification and the pathogenesis of complement-related disorders, it is important to be able to distinguish between the presence and levels of proteins encoded by all seven CFH family members. CFH family members, particularly FHR1-5, may also be used as biomarkers for diagnosing or predicting disorders in which dysregulation of complement is pathologically implicated.

[0069] C3, C3b and degradation products C3 is a central complement component. The pathway by which C3 is processed into various downstream products can lead to complement activation, including inflammatory and immune responses, or complement inactivation and regulation. Therefore, it is important to be able to detect and measure the levels, including relative levels, of C3, C3b, and their downstream components / processing products in relation to complement pathogenesis and treatment of complement-related disorders.

[0070] C3 processing is described, for example, in Foley et al. J Thromb Haemostasis (2015) 13: 610-618, which is incorporated herein by reference in its entirety. Human C3 (UniProt:P01024; SEQ ID NO: 12) contains a 1,663 amino acid sequence (including an N-terminal 22-amino acid signal peptide). Amino acids 23-667 encode the C3 β chain (SEQ ID NO: 13), and amino acids 749-1,663 encode the C3b α' chain (SEQ ID NO: 14). The C3 β chain and C3 α' chain associate through an interchain disulfide bond (formed between cysteine ​​559 of the C3 β chain and cysteine ​​816 of the C3 α' chain) to form C3b. C3a is a 77 amino acid fragment (SEQ ID NO: 15) corresponding to amino acid positions 672-748 of C3 and is generated by proteolytic cleavage of C3 to form C3b.

[0071] Processing of C3b into inactive iC3b, which cannot itself promote further complement amplification, involves proteolytic cleavage of the C3b α' chain at amino acid positions 1303 and 1320 to form α' chain fragment 1 (corresponding to amino acid positions 749-1663 of C3; SEQ ID NO: 16) and α' chain fragment 2 (corresponding to amino acid positions 1321-1663 of C3; SEQ ID NO: 17). Thus, iC3b contains the C3 β chain, C3 α' chain fragment 1, and C3 α' chain fragment 2 (associated through a disulfide bond). Cleavage of the α' chain also liberates C3f, which corresponds to amino acid positions 1304-1320 of C3 (SEQ ID NO: 18).

[0072] iC3b is further processed into C3c, which contains the C3 β chain, C3 α' chain fragment 2, and C3c α' chain fragment 1 (corresponding to amino acid positions 749-954 of C3; SEQ ID NO: 19). This cleavage event produces fragment C3dg (corresponding to amino acid positions 955-1303 of C3; SEQ ID NO: 142), which is itself degraded into fragments C3g (corresponding to amino acid positions 955-1001 of C3; SEQ ID NO: 143) and C3d (corresponding to amino acid positions 1002-1303 of C3; SEQ ID NO: 144).

[0073] The processing of C3b into iC3b is carried out by complement factor I (FI; encoded by the gene CFI in humans). Human complement factor I (UniProt: P05156; SEQ ID NO: 11) has a 583 amino acid sequence (including an N-terminal 18-amino acid signal peptide). Amino acids 340-574 of the light chain contain the proteolytic domain of FI, a serine protease containing a catalytic triad responsible for cleaving C3b to produce iC3b (Ekdahl et al., J Immunol (1990) 144 (11): 4269-74). Proteolytic cleavage of C3b by FI is facilitated by cofactors, including FH, CR1, and possibly some FHR proteins. FI cofactors typically bind to C3b and / or FI and enhance the processing of C3b into iC3b by FI.

[0074] As used herein, any reference to a complement protein, e.g., C3, C3b, C3a, FH, FI, etc., refers to said protein from any species, and includes isoforms, fragments, variants, or homologs of said protein from any species. In some embodiments, the protein is a mammalian protein (e.g., a cynomolgus, human, and / or rodent (e.g., rat and / or murine) protein). Isoforms, fragments, variants, or homologs of the complement proteins described herein can optionally be characterized as having at least 70%, preferably 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to the amino acid sequence of the immature or mature protein from a given species, e.g., the human protein sequences provided herein. Isoforms, fragments, variants or homologs of the complement proteins described herein may optionally be functional isoforms, fragments, variants or homologs and have a functional property / activity of the reference protein, e.g., as determined by analysis with a suitable assay for that functional property / activity.

[0075] Agents capable of decreasing gene and / or protein expression Aspects and embodiments of the present disclosure relate to agents that can reduce gene and / or protein expression. As used herein, these agents can reduce or prevent the gene and / or protein expression of one or more predetermined target genes / proteins. In some embodiments, these agents can reduce or prevent the gene and / or protein expression of one or more complement proteins as described herein (e.g., one or more of FH, FHL-1, FHR1, FHR2, FHR4, FHR5, FI, C3, C3b, C3a, iC3b, C3f, C3c, C3dg, and / or C3d).

[0076] In some embodiments, the agent reduces gene and / or protein expression of one or more Factor H family proteins. In some embodiments, the agent reduces gene and / or protein expression of one or more Factor H family proteins selected from FH, FHL-1, FHR1, FHR2, FHR3, FHR4, and / or FHR5. In some embodiments, the agent reduces gene and / or protein expression of one or more Factor H family proteins selected from FHR1, FHR2, FHR3, FHR4, FHR5, FH, and / or FHL-1.

[0077] In some cases, such agents can reduce or prevent gene and / or protein expression of one or more FHR proteins (e.g., one or more of FHR1, FHR2, FHR3, FHR4, and / or FHR5). In some cases, such agents do not reduce or prevent (e.g., do not significantly reduce or prevent) gene and / or protein expression of FH and / or FHL-1. For example, such agents can reduce or prevent gene and / or protein expression of one or more FHR proteins, while not reducing or preventing gene and / or protein expression of FH and / or FHL-1. In some cases, such agents can reduce or prevent gene and / or protein expression of one or more FHR proteins, while increasing gene and / or protein expression of FH and / or FHL-1. In some cases, the agents described herein inhibit gene and / or protein expression of each of FHR1, FHR2, FHR3, FHR4, and FHR5, but do not inhibit gene and / or protein expression of FH and / or FHL-1 (e.g., the agents may increase gene and / or protein expression of FH and / or FHL-1).

[0078] An "agent capable of reducing gene and / or protein expression" or an "agent that reduces gene and / or protein expression," as used herein, may be any suitable agent that achieves said effect. The agent may be a nucleic acid, such as an inhibitory nucleic acid, as described herein below. The agent may be a nuclease-based nucleic acid editing tool, such as a meganuclease, a chemical nuclease, a zinc finger nuclease (ZFN), a transcription activator-like effector-based nuclease (TALEN), or a Cas9-based system, for example, as described herein below.

[0079] Provided herein is a method for reducing gene and / or protein expression of one or more Factor H family proteins, comprising contacting cells with an agent described herein. The method may be performed in vitro, ex vivo, or in vivo. The cells may be liver cells, such as hepatocytes.

[0080] When an agent, such as an inhibitory nucleic acid or nucleic acid editing tool, is described as reducing the expression of a protein(s), the present disclosure also contemplates reducing the gene expression of genes encoding related protein(s). That is, reference herein to inhibiting gene expression of FH and / or FHL-1 contemplates inhibiting the expression of CFH; reference herein to inhibiting gene expression of FHR1 contemplates inhibiting the expression of CFHR1; reference herein to inhibiting gene expression of FHR2 contemplates inhibiting the expression of CFHR2; reference herein to inhibiting gene expression of FHR3 contemplates inhibiting the expression of CFHR3; reference herein to inhibiting gene expression of FHR4 contemplates inhibiting the expression of CFHR4; and reference herein to inhibiting gene expression of FHR5 contemplates inhibiting the expression of CFHR5.

[0081] In some embodiments, an agent described herein, e.g., an inhibitory nucleic acid or a nucleic acid editing tool: reduce / prevent / inhibit expression of one or more genes encoding one or more Factor H family proteins (e.g., one or more of FHR1, FHR2, FHR3, FHR4, FHR5, FH, and / or FHL-1); reducing the level of RNA encoding one or more Factor H family proteins (e.g., one or more of FHR1, FHR2, FHR3, FHR4, FHR5, FH, and / or FHL-1); reduce / prevent / inhibit transcription of nucleic acid encoding one or more Factor H family proteins (e.g., one or more of FHR1, FHR2, FHR3, FHR4, FHR5, FH, and / or FHL-1) (e.g., transcription of DNA encoding one or more Factor H family proteins into RNA encoding one or more Factor H family proteins); increasing the degradation of RNA encoding one or more Factor H family proteins (e.g., one or more of FHR1, FHR2, FHR3, FHR4, FHR5, FH, and / or FHL-1); reducing / preventing / inhibiting the expression of one or more Factor H family proteins (e.g., one or more of FHR1, FHR2, FHR3, FHR4, FHR5, FH, and / or FHL-1) at the protein level; decreasing the levels of one or more Factor H family proteins (e.g., one or more of FHR1, FHR2, FHR3, FHR4, FHR5, FH, and / or FHL-1); reduce / prevent / inhibit normal post-transcriptional processing (e.g., splicing, translation and / or post-translational processing) of RNA encoding one or more Factor H family proteins (e.g., one or more of FHR1, FHR2, FHR3, FHR4, FHR5, FH and / or FHL-1); Reduce / prevent / inhibit translation of mRNA encoding one or more Factor H family proteins (e.g., one or more of FHR1, FHR2, FHR3, FHR4, FHR5, FH, and / or FHL-1). That's fine.

[0082] In combination with any one or more of the above properties, an agent according to the present disclosure may not reduce / prevent / inhibit the expression of one or more genes encoding FH and / or FHL-1, may not reduce the level of RNA encoding FH and / or FHL-1, may not reduce / prevent / inhibit the transcription of nucleic acids encoding FH and / or FHL-1, may not increase the degradation of RNA encoding FH and / or FHL-1, may not reduce / prevent / inhibit expression of FH and / or FHL-1 at the protein level, may not reduce the level of FH and / or FHL-1 protein, may not reduce / prevent / inhibit normal post-transcriptional processing of RNA encoding FH and / or FHL-1, and / or may not reduce / prevent / inhibit the translation of mRNA encoding FH and / or FHL-1.

[0083] It will be appreciated that a given agent may exhibit more than one of the properties listed in the preceding paragraph. A given agent may be evaluated for the properties listed in the preceding paragraph using an appropriate assay. The assay may be, for example, an in vitro assay, and optionally a cell-based or cell-free assay. The assay may be an ex vivo assay, i.e., performed using cells / tissues / organs obtained from a subject. The assay may be, for example, an in vivo assay, i.e., performed in a non-human animal.

[0084] If the assays are cell-based assays, they may involve treating cells with an agent to determine whether the agent exhibits one or more of the listed properties. The assays may use species labeled with a detectable entity to facilitate their detection. The assays may evaluate the listed properties after treating the cells separately with a range of amounts / concentrations (e.g., a dilution series) of a given agent. It will be appreciated that the cells used in such assays are preferably cells that express a Factor H family protein, e.g., hepatocytes. The assays may include treating the cells with, for example, an agent described herein to reduce or prevent the expression and / or activity of a Factor H family gene and / or protein.

[0085] Analyzing the results of such assays may include determining the concentration at which 50% of the maximum level of the relevant activity is achieved. The concentration of the agent at which 50% of the maximum level of the relevant activity is achieved may be referred to as the "half-maximum effective concentration" of the agent for the relevant activity, which may also be referred to as the "EC50." For example, the EC50 of a given inhibitory agent for increasing the degradation of RNA encoding one or more Factor H family proteins (e.g., one or more of FHR1, FHR2, FHR3, FHR4, FHR5, FH, and / or FHL-1) may be the concentration at which 50% of the maximum level of degradation of RNA encoding one or more Factor H family proteins (e.g., one or more of FHR1, FHR2, FHR3, FHR4, FHR5, FH, and / or FHL-1) is achieved.

[0086] Depending on the property, the EC50 may also be referred to as the "half-maximal inhibitory concentration" or "IC50," which is the concentration of an agent at which 50% of the maximal level of inhibition of a given property is observed. For example, the IC50 for a given inhibitory agent to reduce expression of a gene encoding one or more Factor H family proteins (e.g., one or more of FHR1, FHR2, FHR3, FHR4, FHR5, FH, and / or FHL-1) may be the concentration at which 50% of the maximal level of inhibition of expression of the gene is achieved.

[0087] Assays comprising detecting and / or quantifying the levels of RNA encoding one or more Factor H family proteins (e.g., one or more of FHR1, FHR2, FHR3, FHR4, FHR5, FH and / or FHL-1) may be used to identify agents capable of reducing / preventing gene expression of one or more Factor H family proteins, and / or reducing / preventing transcription of nucleic acids encoding one or more Factor H family proteins, and / or reducing the levels of RNA encoding one or more Factor H family proteins, and / or increasing the degradation of RNA encoding one or more Factor H family proteins.

[0088] Such assays may quantify RNA encoding one or more factor H family proteins by RT-qPCR (a technique well known to those skilled in the art). The method may use primers and / or probes for the detection and / or quantification of RNA encoding one or more factor H family proteins. Such assays involve introducing (e.g., by transfection) into cells expressing one or more factor H family proteins in in vitro culture (i) a putative agent, such as an inhibitory nucleic acid, or (ii) a control agent (e.g., a nucleic acid known not to affect the level of RNA encoding one or more factor H family proteins), and subsequently (e.g., for a suitable period of time, i.e., a decrease in the level of gene expression of one or more factor H family proteins / transcription of nucleic acids encoding one or more factor H family proteins / levels of RNA encoding one or more factor H family proteins, or levels of RNA encoding one or more factor H family proteins, or levels of RNA encoding one or more factor H family proteins, which are to be observed). (i) and (ii) measuring the level of RNA encoding one or more Factor H family proteins in the cells described in (i) and (ii), (after a period of time sufficient for an increase in the level of degradation of the RNA encoding one or more Factor H family proteins), and (iii) comparing the detected levels of RNA encoding one or more Factor H family proteins to determine whether the putative agent reduces / prevents gene expression / transcription of one or more Factor H family proteins and / or reduces the level of RNA encoding one or more Factor H family proteins and / or increases the degradation of RNA encoding one or more Factor H family proteins.

[0089] An assay comprising detecting and / or quantifying the level of RNA (e.g., mature mRNA) encoding one or more isoforms of one or more Factor H family proteins may be used to identify an agent, such as an inhibitory nucleic acid or a nucleic acid editing tool, that can reduce / prevent the normal splicing of pre-mRNA encoding one or more Factor H family proteins. Such an assay may comprise quantifying RNA (e.g., mature mRNA) encoding one or more isoforms of one or more Factor H family proteins by RT-qPCR. The method may use primers and / or probes for detecting and / or quantifying mature mRNA produced by classical splicing of pre-mRNA transcribed from a gene encoding one or more Factor H family proteins, and / or primers and / or probes for detecting and / or quantifying mature mRNA produced by alternative splicing of pre-mRNA transcribed from a gene encoding one or more Factor H family proteins.

[0090] The mature mRNA produced by classical splicing of a pre-mRNA transcribed from a gene encoding one or more Factor H family proteins may be a mature mRNA encoding a major isoform produced by expression of a gene encoding one or more Factor H family proteins. The major isoform is the most commonly produced / detected isoform. For example, the mature mRNA produced by classical splicing of a pre-mRNA transcribed from human FHR2 may be a mature mRNA encoding human FHR2 isoform 1 (i.e., having the amino acid sequence set forth in SEQ ID NO: 4).

[0091] A mature mRNA produced by alternative splicing of a pre-mRNA transcribed from a gene encoding one or more Factor H family proteins may be a mature mRNA encoding an isoform that is different from the major isoform produced by expression of a gene encoding one or more Factor H family proteins. For example, a mature mRNA produced by alternative splicing of a pre-mRNA transcribed from human FHR2 may be a mature mRNA encoding an isoform of human FHR2 other than isoform 1 (i.e., having an amino acid sequence that is not identical to SEQ ID NO:4); for example, a mature mRNA encoding human FHR2 isoform 2 (i.e., having an amino acid sequence that is not identical to SEQ ID NO:5).

[0092] Such an assay may comprise introducing (e.g., by transfection) into cells expressing one or more Factor H family proteins in in vitro culture (i) a putative agent, e.g., an inhibitory nucleic acid, or (ii) a control agent (e.g., a nucleic acid known not to affect splicing of pre-mRNA encoding one or more Factor H family proteins), and subsequently (e.g., after a suitable period of time, i.e., a period sufficient for the effect on splicing of pre-mRNA encoding one or more Factor H family proteins to be observed), measuring the levels of mature mRNA encoding one or more isoforms of one or more Factor H family proteins in the cells described in (i) and (ii), and (iii) comparing the levels of mature mRNA encoding the isoform(s) to determine whether the putative agent reduces / prevents normal splicing of pre-mRNA encoding one or more Factor H family proteins.

[0093] Assays that involve detecting the level of the relevant protein(s) using techniques well known to those of skill in the art, such as antibody / reporter-based methods (Western blot, ELISA, immunohisto / cytochemistry, etc.), may be used to identify agents that are capable of decreasing the level of one or more Factor H family proteins and / or decreasing / preventing translation of mRNA encoding one or more Factor H family proteins (e.g., one or more of FHR1, FHR2, FHR3, FHR4, FHR5, FH, and / or FHL-1). The methods may use antibodies specific for one or more Factor H family proteins. Such assays involve introducing (e.g., by transfection) into cells expressing one or more factor H family proteins in in vitro culture (i) a putative agent, such as an inhibitory nucleic acid, or (ii) a control agent (e.g., a nucleic acid known not to affect the levels of RNA encoding one or more factor H family proteins), and subsequently (e.g., after a suitable period of time, i.e., a period sufficient for the level of one or more factor H family proteins to be observed to be reduced), measuring the levels of one or more factor H family proteins in the cells described in (i) and (ii), and (iii) comparing the detected levels of one or more factor H family proteins to determine whether the putative agent reduces the levels of the associated protein and / or reduces / prevents translation of mRNA encoding one or more factor H family proteins.

[0094] In some embodiments, an agent according to the present disclosure reduces, in a given assay, the expression of one or more genes encoding one or more Factor H family proteins (e.g., one or more of FHR1, FHR2, FHR3, FHR4, FHR5, FH, and / or FHL-1) by more than the amount of the agent in the absence or the amount of an equal amount of a control agent known not to be an inhibitor of the relevant gene(s). It may be possible to reduce the expression level observed in the presence of the agent by less than 1-fold, e.g., to one of: <0.99-fold, <0.95-fold, <0.9-fold, <0.85-fold, <0.8-fold, <0.75-fold, <0.7-fold, <0.65-fold, <0.6-fold, <0.55-fold, <0.5-fold, <0.45-fold, <0.4-fold, <0.35-fold, <0.3-fold, <0.25-fold, <0.2-fold, <0.15-fold, <0.1-fold, <0.05-fold, or <0.01-fold. In some embodiments, expression of the FH and / or FHL-1 genes may be unaffected by the agent.

[0095] In some embodiments, an agent described herein may be capable of reducing expression of one or more genes encoding one or more Factor H family proteins (e.g., one or more of FHR1, FHR2, FHR3, FHR4, FHR5, FH, and / or FHL-1) in a given assay by less than 100%, e.g., one of: < 99%, < 95%, < 90%, < 85%, < 80%, < 75%, < 70%, < 65%, < 60%, < 55%, < 50%, < 45%, < 40%, < 35%, < 30%, < 25%, < 20%, < 15%, < 10%, < 5%, or < 1% of the level of expression observed in the absence of the agent or in the presence of an equal amount of a control agent known not to be an inhibitor of the relevant gene(s). In some embodiments, gene expression of FH and / or FHL-1 may be unaffected by the agent.

[0096] In some embodiments, an agent described herein is known to reduce the level of expression of a gene encoding one or more Factor H family proteins (e.g., one or more of FHR1, FHR2, FHR3, FHR4, FHR5, FH, and / or FHL-1) in a given assay relative to the absence of the agent, or to not reduce the expression of a gene encoding one or more Factor H family proteins. In some embodiments, the level of FH and / or FHL-1 gene expression may be reduced by less than 1-fold, e.g., to one of: <0.99-fold, <0.95-fold, <0.9-fold, <0.85-fold, <0.8-fold, <0.75-fold, <0.7-fold, <0.65-fold, <0.6-fold, <0.55-fold, <0.5-fold, <0.45-fold, <0.4-fold, <0.35-fold, <0.3-fold, <0.25-fold, <0.2-fold, <0.15-fold, <0.1-fold, <0.05-fold, or <0.01-fold, that observed in the presence of the same amount of a control agent. In some embodiments, the level of FH and / or FHL-1 gene expression may be unaffected by the agent.

[0097] In some embodiments, an agent described herein may be capable of reducing the level of expression of a gene encoding one or more Factor H family proteins (e.g., one or more of FHR1, FHR2, FHR3, FHR4, FHR5, FH, and / or FHL-1) in a given assay by less than 100%, e.g., by one of: < 99%, < 95%, < 90%, < 85%, < 80%, < 75%, < 70%, < 65%, < 60%, < 55%, < 50%, < 45%, < 40%, < 35%, < 30%, < 25%, < 20%, < 15%, < 10%, < 5%, or < 1% of the level observed in the absence of the agent or in the presence of the same amount of a control agent known not to reduce expression of genes encoding one or more Factor H family proteins. In some embodiments, the level of gene expression of FH and / or FHL-1 may be unaffected by the agent.

[0098] In some embodiments, an agent described herein is known to decrease the level of RNA encoding one or more Factor H family proteins (e.g., one or more of FHR1, FHR2, FHR3, FHR4, FHR5, FH, and / or FHL-1) in a given assay in the absence of the agent, or to not decrease the level of RNA encoding one or more Factor H family proteins. In some embodiments, the level of RNA encoding FH and / or FHL-1 may be reduced by less than 1-fold, e.g., <0.99-fold, <0.95-fold, <0.9-fold, <0.85-fold, <0.8-fold, <0.75-fold, <0.7-fold, <0.65-fold, <0.6-fold, <0.55-fold, <0.5-fold, <0.45-fold, <0.4-fold, <0.35-fold, <0.3-fold, <0.25-fold, <0.2-fold, <0.15-fold, <0.1-fold, <0.05-fold, or <0.01-fold, of the level observed in the presence of the same amount of a control agent. In some embodiments, the level of RNA encoding FH and / or FHL-1 may be unaffected by the agent.

[0099] In some embodiments, an agent described herein is capable of reducing the level of RNA encoding one or more Factor H family proteins (e.g., one or more of FHR1, FHR2, FHR3, FHR4, FHR5, FH, and / or FHL-1) in a given assay by less than 100%, e.g., <99%, <95%, <90%, <85%, <80%, <75%, <70%, <65%, <60%, <55%, <50%, <45%, <40%, <35%, <30%, <25%, <20%, <15%, <10%, <5%, or <1%, of the level observed in the absence of the agent or in the presence of the same amount of a control agent known not to reduce the level of RNA encoding one or more Factor H family proteins. In some embodiments, the level of RNA encoding FH and / or FHL-1 may be unaffected by the agent.

[0100] In some embodiments, an agent described herein is known to decrease the level of transcription of a nucleic acid encoding one or more Factor H family proteins (e.g., one or more of FHR1, FHR2, FHR3, FHR4, FHR5, FH, and / or FHL-1) in a given assay in the absence of the agent, or does not decrease the transcription of a nucleic acid encoding one or more Factor H family proteins. It may be possible to reduce the level of transcription of a nucleic acid encoding FH and / or FHL-1 by less than 1-fold, e.g., to one of: <0.99-fold, <0.95-fold, <0.9-fold, <0.85-fold, <0.8-fold, <0.75-fold, <0.7-fold, <0.65-fold, <0.6-fold, <0.55-fold, <0.5-fold, <0.45-fold, <0.4-fold, <0.35-fold, <0.3-fold, <0.25-fold, <0.2-fold, <0.15-fold, <0.1-fold, <0.05-fold, or <0.01-fold, of the level observed in the presence of the same amount of a control agent. In some embodiments, the level of transcription of a nucleic acid encoding FH and / or FHL-1 may be unaffected by the agent.

[0101] In some embodiments, an agent described herein may be capable of reducing the level of transcription of a nucleic acid encoding one or more Factor H family proteins (e.g., one or more of FHR1, FHR2, FHR3, FHR4, FHR5, FH, and / or FHL-1) in a given assay by less than 100%, e.g., by one of: < 99%, < 95%, < 90%, < 85%, < 80%, < 75%, < 70%, < 65%, < 60%, < 55%, < 50%, < 45%, < 40%, < 35%, < 30%, < 25%, < 20%, < 15%, < 10%, < 5%, or < 1% of the expression level observed in the absence of the agent or in the presence of the same amount of a control agent known not to reduce transcription of nucleic acids encoding one or more Factor H family proteins. In some embodiments, the level of transcription of nucleic acid encoding FH and / or FHL-1 may be unaffected by the agent.

[0102] In some embodiments, the agents described herein enhance the levels of one or more Factor H family proteins (e.g., one or more of FHR1, FHR2, FHR3, FHR4, FHR5, FH, and / or FHL-1) in a given assay, as observed in the absence of the agent or in the presence of an equal amount of a control agent known not to be an inhibitor of the relevant protein(s). In some embodiments, the level of FH and / or FHL-1 protein(s) may be reduced by less than 1-fold the expected level, e.g., to one of <0.99-fold, <0.95-fold, <0.9-fold, <0.85-fold, <0.8-fold, <0.75-fold, <0.7-fold, <0.65-fold, <0.6-fold, <0.55-fold, <0.5-fold, <0.45-fold, <0.4-fold, <0.35-fold, <0.3-fold, <0.25-fold, <0.2-fold, <0.15-fold, <0.1-fold, <0.05-fold, or <0.01-fold. In some embodiments, the level of FH and / or FHL-1 protein(s) may be unaffected by the agent.

[0103] In some embodiments, an agent according to the present disclosure may be capable of reducing the level of one or more Factor H family proteins (e.g., one or more of FHR1, FHR2, FHR3, FHR4, FHR5, FH, and / or FHL-1) in a given assay by less than 100%, e.g., <99%, <95%, <90%, <85%, <80%, <75%, <70%, <65%, <60%, <55%, <50%, <45%, <40%, <35%, <30%, <25%, <20%, <15%, <10%, <5%, or <1% of the level observed in the absence of the agent or in the presence of an equal amount of a control agent known not to be an inhibitor of the relevant protein(s). In some embodiments, the level of FH and / or FHL-1 protein(s) may be unaffected by the agent.

[0104] In some embodiments, an agent according to the present disclosure reduces normal splicing of pre-mRNA encoding one or more Factor H family proteins (e.g., one or more of FHR1, FHR2, FHR3, FHR4, FHR5, FH, and / or FHL-1) in a given assay in the absence of the agent or reduces normal splicing of pre-mRNA encoding one or more Factor H family proteins. It may be possible to reduce the level of FH and / or FHL-1 expression by less than 1-fold, e.g., to one of the following: <0.99-fold, <0.95-fold, <0.9-fold, <0.85-fold, <0.8-fold, <0.75-fold, <0.7-fold, <0.65-fold, <0.6-fold, <0.55-fold, <0.5-fold, <0.45-fold, <0.4-fold, <0.35-fold, <0.3-fold, <0.25-fold, <0.2-fold, <0.15-fold, <0.1-fold, <0.05-fold, or <0.01-fold, of the level observed in the presence of the same amount of a control agent known to be absent. In some embodiments, normal splicing of pre-mRNA encoding FH and / or FHL-1 may be unaffected by the agent.

[0105] In some embodiments, an agent described herein may be capable of reducing the level of normal splicing of pre-mRNA encoding one or more Factor H family proteins (e.g., one or more of FHR1, FHR2, FHR3, FHR4, FHR5, FH, and / or FHL-1) in a given assay by less than 100%, e.g., by one of: <99%, <95%, <90%, <85%, <80%, <75%, <70%, <65%, <60%, <55%, <50%, <45%, <40%, <35%, <30%, <25%, <20%, <15%, <10%, <5%, or <1% of the level observed in the absence of the agent or in the presence of the same amount of a control agent known not to reduce normal splicing of pre-mRNA encoding one or more Factor H family proteins. In some embodiments, normal splicing of the pre-mRNA encoding FH and / or FHL-1 may not be affected by the agent.

[0106] In some embodiments, an agent described in the present disclosure is known to decrease the translation of mRNA encoding one or more Factor H family proteins (e.g., one or more of FHR1, FHR2, FHR3, FHR4, FHR5, FH, and / or FHL-1) in a given assay in the absence of the agent or does not decrease the translation of mRNA encoding one or more Factor H family proteins. In some embodiments, the level of FH and / or FHL-1 may be reduced by one of the following: less than 1-fold, e.g., <0.99-fold, <0.95-fold, <0.9-fold, <0.85-fold, <0.8-fold, <0.75-fold, <0.7-fold, <0.65-fold, <0.6-fold, <0.55-fold, <0.5-fold, <0.45-fold, <0.4-fold, <0.35-fold, <0.3-fold, <0.25-fold, <0.2-fold, <0.15-fold, <0.1-fold, <0.05-fold, or <0.01-fold, of the level observed in the presence of the same amount of a control agent. In some embodiments, translation of mRNA encoding FH and / or FHL-1 may be unaffected by the agent.

[0107] In some embodiments, an agent described herein may be capable of reducing translation of mRNA encoding one or more Factor H family proteins (e.g., one or more of FHR1, FHR2, FHR3, FHR4, FHR5, FH, and / or FHL-1) in a given assay by less than 100%, e.g., one of: <99%, <95%, <90%, <85%, <80%, <75%, <70%, <65%, <60%, <55%, <50%, <45%, <40%, <35%, <30%, <25%, <20%, <15%, <10%, <5%, or <1% of the level observed in the absence of the agent or in the presence of the same amount of a control agent known not to reduce translation of mRNA encoding one or more Factor H family proteins. In some embodiments, translation of mRNA encoding FH and / or FHL-1 may be unaffected by the agent.

[0108] Preferred levels of reduction as described in the preceding paragraph are reductions of less than 0.5-fold / ≦50%, for example, to one of less than 0.4-fold / ≦40%, less than 0.3-fold / ≦30%, less than 0.2-fold / ≦20%, less than 0.15-fold / ≦15%, or less than 0.1-fold / ≦10%.

[0109] In some embodiments, an agent described herein is known to not increase the degradation of RNA encoding one or more Factor H family proteins (e.g., one or more of FHR1, FHR2, FHR3, FHR4, FHR5, FH, and / or FHL-1) in the absence of the agent or the degradation of RNA encoding one or more Factor H family proteins in a given assay. In some embodiments, the FH and / or FHL-1 encoding RNA may be increased by more than 1-fold, e.g., one of >1.01-fold, >1.02-fold, >1.03-fold, >1.04-fold, >1.05-fold, >1.1-fold, >1.2-fold, >1.3-fold, >1.4-fold, >1.5-fold, >1.6-fold, >1.7-fold, >1.8-fold, >1.9-fold, >2-fold, >3-fold, >4-fold, >5-fold, >6-fold, >7-fold, >8-fold, >9-fold, or >10-fold the expression level observed in the presence of the same amount of a control agent being administered. In some embodiments, the RNA encoding FH and / or FHL-1 may be unaffected, e.g., not degraded, by the agent.

[0110] In some embodiments, the agent described herein prevents or silences the expression of one or more genes encoding one or more Factor H family proteins (e.g., one or more of FHR1, FHR2, FHR3, FHR4, FHR5, FH, and / or FHL-1). In some embodiments, the agent described herein prevents or silences the expression of one or more Factor H family proteins (e.g., one or more of FHR1, FHR2, FHR3, FHR4, FHR5, FH, and / or FHL-1) at the protein level. As used herein, the expression of a given gene(s) / protein(s) can be considered "prevented" or "silenced" if the expression level is less than 0.1-fold / ≦10% of the level observed in the absence of the agent or in the presence of the same amount of a control agent that is known not to be an inhibitor of the relevant gene(s) / protein(s). In some embodiments, expression of FH and / or FHL-1 may be unaffected, ie, not silenced, by the agent.

[0111] In any of the above embodiments, gene and / or protein expression of one or both of FH and / or FHL-1 may be increased by the agent in combination with the agent's effect on gene and / or protein expression of, for example, FHR1, FHR2, FHR3, FHR4, and / or FHR5 (e.g., through increased transcription and / or translation or decreased degradation).

[0112] In any of the above embodiments, the agents described herein are: a)FHR1; b) FHR2; c)FHR3; d)FHR4; e)FHR5; f) FHR1 and FHR2; g) FHR1 and FHR3; h) FHR1 and FHR4; i) FHR1 and FHR5; j) FHR2 and FHR3; k) FHR2 and FHR4; l)FHR2 and FHR5; m) FHR3 and FHR4; n)FHR3 and FHR5; o)FHR4 and FHR5; p)FHR1, FHR2 and FHR3; q) FHR1, FHR2 and FHR4; r) FHR1, FHR2 and FHR5; s)FHR1, FHR3 and FHR4; t)FHR1, FHR3 and FHR5; u) FHR1, FHR4 and FHR5; v) FHR2, FHR3 and FHR4; w) FHR2, FHR3 and FHR5; x) FHR2, FHR4 and FHR5; y) FHR3, FHR4 and FHR5; z) FHR1, FHR2, FHR3 and FHR4; aa) FHR1, FHR2, FHR3 and FHR5; bb) FHR1, FHR2, FHR4 and FHR5; cc) FHR2, FHR3, FHR4 and FHR5; dd) FHR1, FHR3, FHR4 and FHR5; or ee) FHR1, FHR2, FHR3, FHR4 and FHR5 may exert the disclosed effects on gene and / or protein expression.

[0113] In some embodiments, an agent described herein has a concentration of ≦1 μM, e.g., ≦500 nM, ≦100 nM, ≦75 nM, ≦50 nM, ≦40 nM, ≦30 nM, ≦20 nM, ≦15 nM, ≦12.5 nM, ≦10 nM, ≦9 nM, ≦8 nM, ≦7 nM, ≦6 nM, ≦5 nM, ≦4 nM ≦3 nM, ≦2 nM, ≦1 nM, ≦900 pM, ≦800 pM, ≦700 pM, ≦600 pM, ≦500 pM, ≦4 and an IC for inhibition of gene and / or protein expression of one or more Factor H family proteins (e.g., one or more of FHR1, FHR2, FHR3, FHR4, FHR5, FH, and / or FHL-1) of one of 00 pM, ≦300 pM, ≦200 pM, ≦100 pM, ≦50 pM, ≦40 pM, ≦30 pM, ≦20 pM, ≦10 pM, or ≦1 pM. 50 It may have a value.

[0114] In preferred embodiments, an agent (e.g., an siRNA) according to the present disclosure has an IC for inhibiting gene and / or protein expression of one or more Factor H family proteins (e.g., one or more of FHR1, FHR2, FHR3, FHR4, FHR5, FH, and / or FHL-1) of ≦1 nM, ≦900 pM, ≦800 pM, ≦700 pM, ≦600 pM, ≦500 pM, ≦400 pM, ≦300 pM, ≦200 pM, ≦100 pM, ≦50 pM, ≦40 pM, ≦30 pM, ≦20 pM, ≦10 pM, or ≦1 pM. 50 It may have a value.

[0115] Inhibitory nucleic acids and related articles Aspects and embodiments of the present disclosure relate to agents that are or contain inhibitory nucleic acids. As used herein, "inhibitory nucleic acid" refers to a nucleic acid that can reduce or prevent the gene and / or protein expression of one or more predetermined genes / proteins. The term "agent" as used herein can refer to one or more inhibitory nucleic acids.

[0116] The inhibitory nucleic acids described herein are suitable for reducing gene and / or protein expression of complement proteins (e.g., one or more Factor H family proteins) as described herein. More specifically, aspects and embodiments of the present disclosure relate to inhibitory nucleic acids that target one or more complement proteins (e.g., one or more of FH, FHL-1, FHR1, FHR2, FHR3, FHR4, FHR5, FI, C3, C3b, C3a, iC3b, C3f, C3c, C3dg, and / or C3d) as described herein. In some embodiments, the present disclosure relates to inhibitory nucleic acids that target one or more of FHR1, FHR2, FHR3, FHR4, FHR5, FH, and / or FHL-1. In some embodiments, the present disclosure relates to inhibitory nucleic acids that target one or more of FHR1, FHR2, FHR3, FHR4, and / or FHR5.

[0117] The inhibitory nucleic acids described herein may comprise or consist of DNA and / or RNA. Inhibitory nucleic acids may be single-stranded (e.g., in the case of antisense oligonucleotides) or may be double-stranded or contain double-stranded regions (e.g., in the case of siRNA, miRNA, shRNA, etc.). Inhibitory nucleic acids may contain both double-stranded and single-stranded regions (e.g., in the case of shRNA and pre-miRNA molecules, which are double-stranded in the stem region of the hairpin structure and single-stranded in the loop region of the hairpin structure).

[0118] In some embodiments, the inhibitory nucleic acid according to the present disclosure may be an antisense nucleic acid as described herein. In some embodiments, the inhibitory nucleic acid may comprise an antisense nucleic acid as described herein. In some embodiments, the inhibitory nucleic acid may encode an antisense nucleic acid as described herein.

[0119] As used herein, "antisense nucleic acid" refers to a nucleic acid (e.g., DNA or RNA) that is complementary to at least a portion of a target nucleotide sequence (e.g., an RNA encoding a Factor H family protein). Antisense nucleic acids described herein are preferably single-stranded nucleic acids and bind to the target nucleotide sequence through Watson-Crick base pairing. Complementary base pairing may involve hydrogen bonding between complementary base pairs. Antisense nucleic acids may be provided in the form of single-stranded molecules, such as antisense oligonucleotides, or may be included in double-stranded molecular species, such as siRNA, miRNA, and shRNA molecules.

[0120] The complementary base pairing between the antisense nucleic acid and its target nucleotide sequence may be complete. In such embodiments, the antisense nucleic acid comprises or consists of the reverse complement of its target nucleotide sequence, and complementary base pairing occurs between each nucleotide of the target nucleotide sequence and the complementary nucleotide in the antisense nucleic acid. Alternatively, the complementary base pairing between the antisense nucleic acid and its target nucleotide sequence may be incomplete / partial. In such embodiments, complementary base pairing occurs between some but not all nucleotides of the target nucleotide sequence and the complementary nucleotide in the antisense nucleic acid.

[0121] Such binding between nucleic acids through complementary base pairing may be referred to as “hybridization.” Through binding to its target nucleotide sequence, the antisense nucleic acid may form a nucleic acid complex comprising (i) the antisense nucleic acid and (ii) a target nucleic acid comprising the target nucleotide sequence.

[0122] The nucleotide sequence of antisense nucleic acid is sufficiently complementary to its target nucleotide sequence so as to bind or hybridize with the target nucleotide sequence.It will be appreciated that antisense nucleic acid preferably has a high degree of sequence identity with the reverse complement of its target nucleotide sequence.In some embodiments, antisense nucleic acid comprises or consists of the nucleotide sequence with the reverse complement of its target nucleotide sequence that has at least 75% sequence identity (for example, at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity).

[0123] In some embodiments, an antisense nucleic acid according to the present disclosure comprises: the reverse complement of its target nucleotide sequence, or a nucleotide sequence that contains 1 to 10 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) substitutions relative to the reverse complement of its target nucleotide sequence.

[0124] In some embodiments, the target nucleotide sequence of an antisense nucleic acid according to the present disclosure comprises or consists of one of 5 to 100 nucleotides, for example, 10 to 80, 12 to 50, or 15 to 30 nucleotides.

[0125] In some embodiments, antisense nucleic acids reduce / prevent transcription of a nucleic acid containing its target nucleotide sequence, hi some embodiments, antisense nucleic acids reduce / prevent the association of a nucleic acid containing its target nucleotide sequence with factors necessary for normal transcription (e.g., enhancers, RNA polymerase).

[0126] In some embodiments, an antisense nucleic acid increases / enhances degradation of a nucleic acid containing its target nucleotide sequence, e.g., through RNA interference. In some embodiments, an antisense nucleic acid decreases / prevents translation of a nucleic acid containing its target nucleotide sequence, e.g., through RNA interference or antisense degradation by RNase H.

[0127] RNA interference is described, for example, in Agrawal et al., Microbiol. Mol. Bio. Rev. (2003) 67(4): 657-685 and Hu et al., Sig. Transduc. Tar. Ther. (2020) 5(101), both of which are incorporated herein by reference in their entirety. Briefly, double-stranded RNA molecules are recognized by the Argonaute component of the RNA-induced silencing complex (RISC). The double-stranded RNA is separated into single strands and integrated into active RISC by the RISC loading complex (RLC). The RISC-integrated strand binds to its target RNA through complementary base pairing, and depending on the identity of the RISC-integrated RNA and the degree of complementarity to the target RNA, the RISC then either cleaves the target RNA, causing its degradation, or otherwise blocks ribosome access, preventing its translation. RNAi-based therapies have been approved for several indications (Kim, Chonnam Med J. (2020) 56(2): 87-93).

[0128] In some embodiments, an antisense nucleic acid reduces / prevents normal post-transcriptional processing (e.g., splicing and / or translation) of a nucleic acid containing its target nucleotide sequence. In some embodiments, an antisense nucleic acid reduces or alters the splicing of a pre-mRNA containing its target nucleotide sequence into a mature mRNA. In some embodiments, an antisense nucleic acid reduces the translation of an mRNA containing its target nucleotide sequence into a protein.

[0129] In some embodiments, an antisense nucleic acid reduces or prevents the association of a nucleic acid containing its target nucleotide sequence with factors required for normal post-transcriptional processing (e.g., components of the spliceosome). In such instances, the antisense nucleic acid may be referred to as a "splice-switching" nucleic acid.

[0130] Splice-switching nucleic acids are reviewed, for example, in Haves and Hastings, Nucleic Acids Res. (2016) 44(14): 6549-6563, which is incorporated herein by reference in its entirety. Splice-switching nucleic acids include, for example, splice-switching oligonucleotides (SSOs). These disrupt normal splicing of target RNA transcripts by blocking RNA:RNA base pairing and / or protein:RNA binding interactions between components of the splicing machinery and pre-mRNA. Splice-switching nucleic acids may be used to alter the number / ratio of mature mRNA transcripts encoding one or more Factor H family proteins (e.g., one or more of FHR1, FHR2, FHR3, FHR4, FHR5, FH, and / or FHL-1). Splice-switching nucleic acids may also be designed to target specific regions of target transcripts, for example, to achieve skipping of exon(s) of interest, e.g., exons encoding domains / regions of interest. SSOs often include modifications to the oligonucleotide sugar-phosphate backbone to reduce / prevent RNase H degradation, such as phosphorothioate linkages, phosphorodiamidate linkages, e.g., phosphorodiamidate morpholinos (PMOs), and may include, for example, peptide nucleic acids (PNAs), locked nucleic acids (LNAs), methoxyethyl nucleotide modifications, e.g., 2'O-methyl (2'OMe) and 2'-O-methoxyethyl (MOE) ribose modifications, and / or 5'-methylcytosine modifications.

[0131] In some embodiments, an antisense nucleic acid inhibits / reduces translation of a nucleic acid containing its target nucleotide sequence, hi some embodiments, an antisense nucleic acid reduces / prevents the association of a nucleic acid containing its target nucleotide sequence with factors necessary for translation (e.g., ribosomes).

[0132] It will be appreciated that the target nucleotide sequence to which the antisense nucleic acid binds is a nucleotide sequence encoding a protein whose expression is desired to be inhibited. Thus, in aspects and embodiments of the present disclosure, the target nucleotide sequence of the antisense nucleic acid is the nucleotide sequence of a gene(s) encoding one or more Factor H family proteins (e.g., FHR1, FHR2, FHR3, FHR4, FHR5, FH, and / or FHL-1). For example, the target nucleotide sequence may be a nucleotide sequence contained in one or more of SEQ ID NOs: 218-223.

[0133] The following paragraphs describe target nucleotide sequences that can be targeted by agents described in this disclosure, such as inhibitory nucleic acids and / or gene editing systems, such as CRISPR / Cas systems, including guide RNAs.

[0134] In some embodiments, the target nucleotide sequence is the nucleotide sequence of DNA encoding one or more Factor H family proteins (e.g., one or more of CFHR1, CFHR2, CFHR3, CFHR4, CFHR5, and / or CFH). In some embodiments, the target nucleotide sequence is the nucleotide sequence of RNA encoded by one or more genes encoding one or more Factor H family proteins (e.g., one or more of CFHR1, CFHR2, CFHR3, CFHR4, CFHR5, and / or CFH). In some embodiments, the target nucleotide sequence is the nucleotide sequence of RNA encoding one or more Factor H family proteins (e.g., one or more of FHR1, FHR2, FHR3, FHR4, FHR5, FH, and / or FHL-1). In some embodiments, the target nucleotide sequence comprises one or more nucleotides of an exon of an RNA encoding one or more Factor H family proteins (e.g., one or more of FHR1, FHR2, FHR3, FHR4, FHR5, FH, and / or FHL-1). In some embodiments, the target nucleotide sequence is the nucleotide sequence of an exon of an RNA encoding one or more Factor H family proteins (e.g., one or more of FHR1, FHR2, FHR3, FHR4, FHR5, FH, and / or FHL-1).

[0135] In some embodiments, the target nucleotide sequence is an RNA (e.g., mRNA) encoded by one or more genes encoding one or more Factor H-related proteins, or the nucleotide sequence of an RNA encoding one or more Factor H-related proteins (e.g., one or more of FHR1, FHR2, FHR3, FHR4, and / or FHR5).

[0136] In some embodiments, the target nucleotide sequence is the nucleotide sequence of RNA encoded by a gene encoding FHR1. In some embodiments, the target nucleotide sequence is the nucleotide sequence of RNA encoding FHR1. In some embodiments, the target nucleotide sequence comprises one or more nucleotides of an exon of RNA encoding FHR1. In some embodiments, the target nucleotide sequence is the nucleotide sequence of an exon of RNA encoding FHR1. In some embodiments, the target nucleotide sequence is the nucleotide sequence of an exon of RNA encoding FHR1. In some embodiments, the target nucleotide sequence is the nucleotide sequence of RNA encoded by a gene encoding FHR2. In some embodiments, the target nucleotide sequence is the nucleotide sequence of RNA encoding FHR2. In some embodiments, the target nucleotide sequence comprises one or more nucleotides of an exon of RNA encoding FHR2. In some embodiments, the target nucleotide sequence is the nucleotide sequence of an exon of RNA encoding FHR2. In some embodiments, the target nucleotide sequence is the nucleotide sequence of RNA encoded by a gene encoding FHR3. In some embodiments, the target nucleotide sequence is the nucleotide sequence of RNA encoding FHR3. In some embodiments, the target nucleotide sequence comprises one or more nucleotides of an exon of RNA encoding FHR3. In some embodiments, the target nucleotide sequence is the nucleotide sequence of an exon of RNA encoding FHR3. In some embodiments, the target nucleotide sequence is the nucleotide sequence of RNA encoded by a gene encoding FHR4. In some embodiments, the target nucleotide sequence is the nucleotide sequence of RNA encoding FHR4. In some embodiments, the target nucleotide sequence comprises one or more nucleotides of an exon of RNA encoding FHR4. In some embodiments, the target nucleotide sequence is the nucleotide sequence of an exon of RNA encoding FHR4.In some embodiments, the target nucleotide sequence is the nucleotide sequence of RNA encoded by a gene encoding FHR5. In some embodiments, the target nucleotide sequence is the nucleotide sequence of RNA encoding FHR5. In some embodiments, the target nucleotide sequence comprises one or more nucleotides of an exon of RNA encoding FHR5. In some embodiments, the target nucleotide sequence is the nucleotide sequence of an exon of RNA encoding FHR5. In some embodiments, the target nucleotide sequence is the nucleotide sequence of RNA encoded by a gene encoding FH. In some embodiments, the target nucleotide sequence is the nucleotide sequence of RNA encoding FH. In some embodiments, the target nucleotide sequence comprises one or more nucleotides of an exon of RNA encoding FH. In some embodiments, the target nucleotide sequence is the nucleotide sequence of an exon of RNA encoding FH.

[0137] In some embodiments, the target nucleotide sequence is a nucleotide sequence found in RNA encoding more than one Factor H family protein (e.g., selected from FHR1, FHR2, FHR3, FHR4, FHR5, FH, and / or FHL-1, e.g., selected from FHR1, FHR2, FHR3, FHR4, and / or FHR5). By targeting such nucleotide sequences, a given agent, such as an inhibitory nucleic acid described herein, may be able to reduce the gene and / or protein expression of different Factor H family proteins encoded by RNAs containing the relevant target sequences. Any combination of nucleotide sequences found in one or more Factor H family proteins is contemplated.

[0138] The "antisense nucleic acid" in the following paragraphs may be present in any agent, e.g., an inhibitory nucleic acid, or in a nucleic acid for targeting a gene editing system, e.g., a gRNA or sgRNA.

[0139] For example, SEQ ID NO: 158 of the present disclosure is found in the sequence of RNA encoding FHR1 and also in the sequence of RNA encoding FHR2. Thus, an antisense nucleic acid having a nucleotide sequence with at least 70% sequence identity to SEQ ID NO: 178 reduces gene and / or protein expression of at least FHR1 and FHR2. In some cases, an antisense nucleic acid having a nucleotide sequence with at least 70% sequence identity to SEQ ID NO: 178 reduces gene and / or protein expression of FHR1, FHR2, FHR3, FHR4, and FHR5. In some cases, an antisense nucleic acid having a nucleotide sequence with at least 70% sequence identity to SEQ ID NO: 178 has no effect on or increases gene and / or protein expression of FH and / or FHL-1.

[0140] SEQ ID NO: 159 of the present disclosure is found in the sequence of an RNA encoding FHR1. Thus, an antisense nucleic acid having a nucleotide sequence with at least 70% sequence identity to SEQ ID NO: 179 reduces gene and / or protein expression of at least FHR1. In some cases, an antisense nucleic acid having a nucleotide sequence with at least 70% sequence identity to SEQ ID NO: 179 reduces gene and / or protein expression of FHR1, FHR3, FHR4, and FHR5. In some cases, an antisense nucleic acid having a nucleotide sequence with at least 70% sequence identity to SEQ ID NO: 179 has no effect on or increases gene and / or protein expression of FHR2, FH, and / or FHL-1.

[0141] SEQ ID NO: 160 of the present disclosure is found in the sequence of an RNA encoding FHR1. Thus, an antisense nucleic acid having a nucleotide sequence with at least 70% sequence identity to SEQ ID NO: 180 reduces gene and / or protein expression of at least FHR1. In some cases, an antisense nucleic acid having a nucleotide sequence with at least 70% sequence identity to SEQ ID NO: 180 reduces gene and / or protein expression of FHR1, FHR3, FHR4, and FHR5. In some cases, an antisense nucleic acid having a nucleotide sequence with at least 70% sequence identity to SEQ ID NO: 180 has no effect on or increases gene and / or protein expression of FHR2, FH, and / or FHL-1.

[0142] SEQ ID NO: 161 of the present disclosure is found in the sequence of an RNA encoding FHR3. Thus, an antisense nucleic acid having a nucleotide sequence with at least 70% sequence identity to SEQ ID NO: 181 reduces gene and / or protein expression of at least FHR3. In some cases, an antisense nucleic acid having a nucleotide sequence with at least 70% sequence identity to SEQ ID NO: 181 reduces gene and / or protein expression of FHR3, FHR4, and optionally FHR5. In some cases, an antisense nucleic acid having a nucleotide sequence with at least 70% sequence identity to SEQ ID NO: 181 has no effect on or increases gene and / or protein expression of FHR1, FHR2, FHR5, FH, and / or FHL-1.

[0143] Similarly, SEQ ID NO: 162 is found in the sequence of RNA encoding FHR3 and also in the sequence of RNA encoding FHR4, and therefore, antisense nucleic acids having a nucleotide sequence with at least 70% sequence identity to SEQ ID NO: 182 are expected to reduce gene and / or protein expression of at least FHR3 and FHR4. In some cases, antisense nucleic acids having a nucleotide sequence with at least 70% sequence identity to SEQ ID NO: 181 have no effect on or increase gene and / or protein expression of FHR1, FHR2, FHR3, FHR4, FHR5, FH, and / or FHL-1.

[0144] SEQ ID NO: 163 of the present disclosure is found in the sequence of an RNA encoding FHR3. Thus, an antisense nucleic acid having a nucleotide sequence with at least 70% sequence identity to SEQ ID NO: 183 reduces gene and / or protein expression of at least FHR3. In some cases, an antisense nucleic acid having a nucleotide sequence with at least 70% sequence identity to SEQ ID NO: 183 reduces gene and / or protein expression of FHR3, FHR4, and optionally FHR5. In some cases, an antisense nucleic acid having a nucleotide sequence with at least 70% sequence identity to SEQ ID NO: 183 has no effect on or increases gene and / or protein expression of FHR1, FHR2, FHR5, FH, and / or FHL-1.

[0145] SEQ ID NO: 164 of the present disclosure is found in the sequence of an RNA encoding FHR4. Thus, an antisense nucleic acid having a nucleotide sequence with at least 70% sequence identity to SEQ ID NO: 184 reduces gene and / or protein expression of at least FHR4. In some cases, an antisense nucleic acid having a nucleotide sequence with at least 70% sequence identity to SEQ ID NO: 184 reduces gene and / or protein expression of FHR3, FHR4, and optionally FHR5. In some cases, an antisense nucleic acid having a nucleotide sequence with at least 70% sequence identity to SEQ ID NO: 184 has no effect on or increases gene and / or protein expression of FHR1, FHR2, FHR5, FH, and / or FHL-1.

[0146] SEQ ID NO: 165 of the present disclosure is found in the sequence of an RNA encoding FHR4. Thus, an antisense nucleic acid having a nucleotide sequence with at least 70% sequence identity to SEQ ID NO: 185 reduces gene and / or protein expression of at least FHR4. In some cases, an antisense nucleic acid having a nucleotide sequence with at least 70% sequence identity to SEQ ID NO: 185 reduces gene and / or protein expression of FHR2, FHR3, and / or FHR4. In some cases, an antisense nucleic acid having a nucleotide sequence with at least 70% sequence identity to SEQ ID NO: 184 has no effect on or increases gene and / or protein expression of FHR1, FHR5, FH, and / or FHL-1.

[0147] SEQ ID NO: 166 is found in the sequence of RNA encoding FHR3 and also in the sequence of RNA encoding FHR4, and therefore, antisense nucleic acids having a nucleotide sequence with at least 70% sequence identity to SEQ ID NO: 186 will reduce gene and / or protein expression of at least FHR3 and FHR4. In some cases, antisense nucleic acids having a nucleotide sequence with at least 70% sequence identity to SEQ ID NO: 184 will have no effect on or increase gene and / or protein expression of FHR1, FHR2, FHR3, FHR4, FHR5, FH, and / or FHL-1.

[0148] Similarly, SEQ ID NO: 169 is found in the sequence of RNA encoding FH and is also found in the sequence of RNA encoding FHR1 and FHR2, and therefore, an antisense nucleic acid having a nucleotide sequence having at least 70% sequence identity to SEQ ID NO: 189 reduces gene and / or protein expression of at least FH, CFHR1, and CFHR2. Similarly, SEQ ID NO: 170 is found in the sequence of RNA encoding FH and is also found in the sequence of RNA encoding FHR1, and therefore, an antisense nucleic acid having a nucleotide sequence having at least 70% sequence identity to SEQ ID NO: 190 reduces gene and / or protein expression of at least FH and FHR1. Similarly, SEQ ID NO: 171 is found in the sequence of RNA encoding FH and is also found in the sequence of RNA encoding FHR1, and therefore, an antisense nucleic acid having a nucleotide sequence having at least 70% sequence identity to SEQ ID NO: 191 reduces gene and / or protein expression of at least FH and FHR1. Similarly, SEQ ID NO: 172 is found in the sequence of RNA encoding FH and also in the sequence of RNA encoding FHR2, and therefore, an antisense nucleic acid having a nucleotide sequence with at least 70% sequence identity to SEQ ID NO: 192 reduces gene and / or protein expression of at least FH and FHR2.

[0149] In some embodiments, the target nucleotide sequence is the nucleotide sequence of RNA encoded by a gene encoding FHR1 and / or FHR2. In some embodiments, the target nucleotide sequence is the nucleotide sequence of RNA encoding FHR1 and / or FHR2. In some embodiments, the target nucleotide sequence comprises one or more nucleotides of an exon of RNA encoding FHR1 and / or FHR2. In some embodiments, the target nucleotide sequence is the nucleotide sequence of an exon of RNA encoding FHR1 and / or FHR2. In some embodiments, the target nucleotide sequence is the nucleotide sequence of RNA encoded by a gene encoding FHR3 and / or FHR4. In some embodiments, the target nucleotide sequence is the nucleotide sequence of RNA encoding FHR3 and / or FHR4. In some embodiments, the target nucleotide sequence comprises one or more nucleotides of an exon of RNA encoding FHR3 and / or FHR4. In some embodiments, the target nucleotide sequence is the nucleotide sequence of an exon of RNA encoding FHR3 and / or FHR4. In some embodiments, the target nucleotide sequence is a nucleotide sequence of RNA encoded by a gene encoding FHR1, FHR2, and / or FH. In some embodiments, the target nucleotide sequence is a nucleotide sequence of RNA encoding FHR1, FHR2, and / or FH. In some embodiments, the target nucleotide sequence comprises one or more nucleotides of an exon of RNA encoding FHR1, FHR2, and / or FH. In some embodiments, the target nucleotide sequence is a nucleotide sequence of an exon of RNA encoding FHR1, FHR2, and / or FH. In some embodiments, the target nucleotide sequence is a nucleotide sequence of RNA encoded by a gene encoding FHR1 and / or FH.In some embodiments, the target nucleotide sequence is a nucleotide sequence of RNA encoding FHR1 and / or FH. In some embodiments, the target nucleotide sequence comprises one or more nucleotides of an exon of RNA encoding FHR1 and / or FH. In some embodiments, the target nucleotide sequence is a nucleotide sequence of an exon of RNA encoding FHR1 and / or FH. In some embodiments, the target nucleotide sequence is a nucleotide sequence of RNA encoded by a gene encoding FHR2 and / or FH. In some embodiments, the target nucleotide sequence is a nucleotide sequence of RNA encoding FHR2 and / or FH. In some embodiments, the target nucleotide sequence comprises one or more nucleotides of an exon of RNA encoding FHR2 and / or FH. In some embodiments, the target nucleotide sequence is a nucleotide sequence of an exon of RNA encoding FHR2 and / or FH.

[0150] In some embodiments, the target nucleotide sequence is the nucleotide sequence of SEQ ID NO: 218, which is the mRNA sequence encoding FHR1 (thymine (T) is replaced with uracil (U)). In some embodiments, the target nucleotide sequence is the nucleotide sequence between positions 1 and 164, inclusive, of SEQ ID NO: 218. In some embodiments, the target nucleotide sequence is the nucleotide sequence between positions 531 and 721, inclusive, of SEQ ID NO: 218. In some embodiments, the target nucleotide sequence is the nucleotide sequence between positions 722 and 904, inclusive, of SEQ ID NO: 218.

[0151] In some embodiments, the target nucleotide sequence is the nucleotide sequence of SEQ ID NO: 219, which is the mRNA sequence encoding FHR2 (thymine (T) is replaced with uracil (U)). In some embodiments, the target nucleotide sequence is the nucleotide sequence between positions 1 and 201, inclusive, of SEQ ID NO: 219. In some embodiments, the target nucleotide sequence is the nucleotide sequence between positions 757 and 1498, inclusive, of SEQ ID NO: 219.

[0152] In some embodiments, the target nucleotide sequence is the nucleotide sequence of SEQ ID NO: 220, which is the mRNA sequence encoding FHR3 (thymine (T) is replaced with uracil (U)). In some embodiments, the target nucleotide sequence is the nucleotide sequence between positions 661 and 2934, inclusive, of SEQ ID NO: 220. In some embodiments, the target nucleotide sequence is the nucleotide sequence between positions 661 and 843, inclusive, of SEQ ID NO: 220. In some embodiments, the target nucleotide sequence is the nucleotide sequence between positions 844 and 2394, inclusive, of SEQ ID NO: 220.

[0153] In some embodiments, the target nucleotide sequence is the nucleotide sequence of SEQ ID NO:221, which is the mRNA sequence encoding FHR4 (thymine (T) is replaced with uracil (U)). In some embodiments, the target nucleotide sequence is the nucleotide sequence between positions 1 and 157, inclusive, of SEQ ID NO:221. In some embodiments, the target nucleotide sequence is the nucleotide sequence between positions 716 and 898, inclusive, of SEQ ID NO:221. In some embodiments, the target nucleotide sequence is the nucleotide sequence between positions 1280 and 1456, inclusive, of SEQ ID NO:221. In some embodiments, the target nucleotide sequence is the nucleotide sequence between positions 1457 and 2063, inclusive, of SEQ ID NO:221. In some embodiments, the target nucleotide sequence is the nucleotide sequence between positions 1457 and 1639, inclusive, of SEQ ID NO:221.

[0154] In some embodiments, the target nucleotide sequence is the nucleotide sequence of SEQ ID NO: 222, which is the mRNA sequence encoding FHR5 (thymine (T) is replaced with uracil (U)). In some embodiments, the target nucleotide sequence is the nucleotide sequence between positions 363 and 539, inclusive, of SEQ ID NO: 222. In some embodiments, the target nucleotide sequence is the nucleotide sequence between positions 1623 and 2814, inclusive, of SEQ ID NO: 222.

[0155] In some embodiments, the target nucleotide sequence is the nucleotide sequence of SEQ ID NO: 223, which is the mRNA sequence encoding FH (thymine (T) is replaced with uracil (U)). In some embodiments, the target nucleotide sequence is the nucleotide sequence between positions 3386 and 3568 of SEQ ID NO: 223, inclusive.

[0156] In some embodiments, the target nucleotide sequence is or comprises the nucleotide sequence of SEQ ID NO: 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, or 177. In some embodiments, the target nucleotide sequence is or comprises the nucleotide sequence of SEQ ID NO: 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, or 168. In some embodiments, the target nucleotide sequence is or comprises the nucleotide sequence of SEQ ID NO: 158. In some embodiments, the target nucleotide sequence is or comprises the nucleotide sequence of SEQ ID NO: 159. In some embodiments, the target nucleotide sequence is or comprises the nucleotide sequence of SEQ ID NO: 160. In some embodiments, the target nucleotide sequence is or comprises the nucleotide sequence of SEQ ID NO: 161. In some embodiments, the target nucleotide sequence is or comprises the nucleotide sequence of SEQ ID NO: 162. In some embodiments, the target nucleotide sequence is or comprises the nucleotide sequence of SEQ ID NO: 163. In some embodiments, the target nucleotide sequence is or comprises the nucleotide sequence of SEQ ID NO: 164. In some embodiments, the target nucleotide sequence is or comprises the nucleotide sequence of SEQ ID NO: 165. In some embodiments, the target nucleotide sequence is or comprises the nucleotide sequence of SEQ ID NO: 166. In some embodiments, the target nucleotide sequence is or comprises the nucleotide sequence of SEQ ID NO: 167. In some embodiments, the target nucleotide sequence is or comprises the nucleotide sequence of SEQ ID NO: 168.In some embodiments, the target nucleotide sequence is or comprises the nucleotide sequence of SEQ ID NO: 169. In some embodiments, the target nucleotide sequence is or comprises the nucleotide sequence of SEQ ID NO: 170. In some embodiments, the target nucleotide sequence is or comprises the nucleotide sequence of SEQ ID NO: 171. In some embodiments, the target nucleotide sequence is or comprises the nucleotide sequence of SEQ ID NO: 172. In some embodiments, the target nucleotide sequence is or comprises the nucleotide sequence of SEQ ID NO: 173. In some embodiments, the target nucleotide sequence is or comprises the nucleotide sequence of SEQ ID NO: 174. In some embodiments, the target nucleotide sequence is or comprises the nucleotide sequence of SEQ ID NO: 175. In some embodiments, the target nucleotide sequence is or comprises the nucleotide sequence of SEQ ID NO: 176. In some embodiments, the target nucleotide sequence is or comprises the nucleotide sequence of SEQ ID NO: 177.

[0157] In some embodiments, the antisense nucleic acid comprises or consists of a sequence having at least 75% sequence identity (e.g., at least one of 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher sequence identity) to the reverse complement of SEQ ID NO: 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, or 177. In some embodiments, the antisense nucleic acid comprises or consists of a sequence having at least 75% sequence identity (e.g., at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher sequence identity) to the reverse complement of SEQ ID NO: 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, or 168. In some embodiments, the antisense nucleic acid comprises or consists of a sequence having at least 75% sequence identity (e.g., at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher sequence identity) to the reverse complement of SEQ ID NO: 158. In some embodiments, the antisense nucleic acid comprises or consists of a sequence having at least 75% sequence identity (e.g., at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher sequence identity) to the reverse complement of SEQ ID NO: 159.In some embodiments, the antisense nucleic acid comprises or consists of a sequence having at least 75% sequence identity (e.g., at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher sequence identity) to the reverse complement of SEQ ID NO: 160. In some embodiments, the antisense nucleic acid comprises or consists of a sequence having at least 75% sequence identity (e.g., at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher sequence identity) to the reverse complement of SEQ ID NO: 161. In some embodiments, the antisense nucleic acid comprises or consists of a sequence having at least 75% sequence identity (e.g., at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher sequence identity) to the reverse complement of SEQ ID NO: 162. In some embodiments, the antisense nucleic acid comprises or consists of a sequence having at least 75% sequence identity (e.g., at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher sequence identity) to the reverse complement of SEQ ID NO: 163.In some embodiments, the antisense nucleic acid comprises or consists of a sequence having at least 75% sequence identity (e.g., at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher sequence identity) to the reverse complement of SEQ ID NO: 164. In some embodiments, the antisense nucleic acid comprises or consists of a sequence having at least 75% sequence identity (e.g., at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher sequence identity) to the reverse complement of SEQ ID NO: 165. In some embodiments, the antisense nucleic acid comprises or consists of a sequence having at least 75% sequence identity (e.g., at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher sequence identity) to the reverse complement of SEQ ID NO: 166. In some embodiments, the antisense nucleic acid comprises or consists of a sequence having at least 75% sequence identity (e.g., at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher sequence identity) to the reverse complement of SEQ ID NO: 167.In some embodiments, the antisense nucleic acid comprises or consists of a sequence having at least 75% sequence identity (e.g., at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher sequence identity) to the reverse complement of SEQ ID NO: 168. In some embodiments, the antisense nucleic acid comprises or consists of a sequence having at least 75% sequence identity (e.g., at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher sequence identity) to the reverse complement of SEQ ID NO: 169. In some embodiments, the antisense nucleic acid comprises or consists of a sequence having at least 75% sequence identity (e.g., at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher sequence identity) to the reverse complement of SEQ ID NO: 170. In some embodiments, the antisense nucleic acid comprises or consists of a sequence having at least 75% sequence identity (e.g., at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher sequence identity) to the reverse complement of SEQ ID NO: 171.In some embodiments, the antisense nucleic acid comprises or consists of a sequence having at least 75% sequence identity (e.g., at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher sequence identity) to the reverse complement of SEQ ID NO: 172. In some embodiments, the antisense nucleic acid comprises or consists of a sequence having at least 75% sequence identity (e.g., at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher sequence identity) to the reverse complement of SEQ ID NO: 173. In some embodiments, the antisense nucleic acid comprises or consists of a sequence having at least 75% sequence identity (e.g., at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher sequence identity) to the reverse complement of SEQ ID NO: 174. In some embodiments, the antisense nucleic acid comprises or consists of a sequence having at least 75% sequence identity (e.g., at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher sequence identity) to the reverse complement of SEQ ID NO: 175.In some embodiments, the antisense nucleic acid comprises or consists of a sequence having at least 75% sequence identity (e.g., at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher sequence identity) to the reverse complement of SEQ ID NO: 176. In some embodiments, the antisense nucleic acid comprises or consists of a sequence having at least 75% sequence identity (e.g., at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher sequence identity) to the reverse complement of SEQ ID NO: 177.

[0158] In some embodiments, the antisense nucleic acid comprises or consists of a sequence having at least 75% sequence identity (e.g., at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity) to SEQ ID NO: 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196 or 197. In some embodiments, the antisense nucleic acid comprises or consists of a sequence having at least 75% sequence identity (e.g., at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher sequence identity) to SEQ ID NO: 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, or 188. In some embodiments, the antisense nucleic acid comprises or consists of a sequence having at least 75% sequence identity (e.g., at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher sequence identity) to SEQ ID NO: 178. In some embodiments, the antisense nucleic acid comprises or consists of a sequence having at least 75% sequence identity (e.g., at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher sequence identity) to SEQ ID NO: 179.In some embodiments, the antisense nucleic acid comprises or consists of a sequence having at least 75% sequence identity (e.g., at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher sequence identity) to SEQ ID NO: 180. In some embodiments, the antisense nucleic acid comprises or consists of a sequence having at least 75% sequence identity to SEQ ID NO: 181 (e.g., at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher sequence identity). In some embodiments, the antisense nucleic acid comprises or consists of a sequence having at least 75% sequence identity (e.g., at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher sequence identity) to SEQ ID NO: 182. In some embodiments, the antisense nucleic acid comprises or consists of a sequence having at least 75% sequence identity (e.g., at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher sequence identity) to SEQ ID NO: 183. In some embodiments, the antisense nucleic acid comprises or consists of a sequence having at least 75% sequence identity (e.g., at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher sequence identity) to SEQ ID NO: 184.In some embodiments, the antisense nucleic acid comprises or consists of a sequence having at least 75% sequence identity (e.g., at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher sequence identity) to SEQ ID NO: 185. In some embodiments, the antisense nucleic acid comprises or consists of a sequence having at least 75% sequence identity to SEQ ID NO: 186 (e.g., at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher sequence identity). In some embodiments, the antisense nucleic acid comprises or consists of a sequence having at least 75% sequence identity (e.g., at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher sequence identity) to SEQ ID NO: 187. In some embodiments, the antisense nucleic acid comprises or consists of a sequence having at least 75% sequence identity (e.g., at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher sequence identity) to SEQ ID NO: 188. In some embodiments, the antisense nucleic acid comprises or consists of a sequence having at least 75% sequence identity to SEQ ID NO: 189 (e.g., at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher sequence identity).In some embodiments, the antisense nucleic acid comprises or consists of a sequence having at least 75% sequence identity to SEQ ID NO: 190 (e.g., at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher sequence identity). In some embodiments, the antisense nucleic acid comprises or consists of a sequence having at least 75% sequence identity to SEQ ID NO: 191 (e.g., at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher sequence identity). In some embodiments, the antisense nucleic acid comprises or consists of a sequence having at least 75% sequence identity (e.g., at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher sequence identity) to SEQ ID NO: 192. In some embodiments, the antisense nucleic acid comprises or consists of a sequence having at least 75% sequence identity (e.g., at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher sequence identity) to SEQ ID NO: 193. In some embodiments, the antisense nucleic acid comprises or consists of a sequence having at least 75% sequence identity to SEQ ID NO: 194 (e.g., at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher sequence identity).In some embodiments, the antisense nucleic acid comprises or consists of a sequence having at least 75% sequence identity to SEQ ID NO: 195 (e.g., at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher sequence identity). In some embodiments, the antisense nucleic acid comprises or consists of a sequence having at least 75% sequence identity to SEQ ID NO: 196 (e.g., at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher sequence identity). In some embodiments, the antisense nucleic acid comprises or consists of a sequence having at least 75% sequence identity to SEQ ID NO: 197 (e.g., at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher sequence identity).

[0159] One of skill in the art, in view of the present disclosure, will be readily able to select appropriate inhibitory nucleic acids for reducing gene and / or protein expression of a given Factor H family protein.

[0160] In embodiments in which it is desirable to inhibit gene and / or protein expression of FHR1, the inhibitory nucleic acid may comprise or encode an antisense nucleic acid having a target nucleotide sequence that is a nucleotide sequence encoding FHR1 (e.g., the nucleotide sequence of an RNA encoded by a gene encoding FHR1, or the nucleotide sequence of an RNA encoding FHR1). In some embodiments, the target nucleotide sequence comprises one or more nucleotides of an exon of the RNA encoding FHR1 (e.g., is the nucleotide sequence of an exon of the RNA encoding FHR1). In some embodiments, the target nucleotide sequence is the nucleotide sequence of SEQ ID NO:218 (e.g., the nucleotide sequence between positions 1 and 164 (inclusive) of SEQ ID NO:218, the nucleotide sequence between positions 531 and 721 (inclusive) of SEQ ID NO:218, or the nucleotide sequence between positions 722 and 904 (inclusive) of SEQ ID NO:218). In some embodiments, the target nucleotide sequence is or comprises SEQ ID NO:158, 159, 160, 169, 170, or 171. In some embodiments, the inhibitory nucleic acid may comprise or encode an antisense nucleic acid comprising or consisting of a sequence having at least 75% sequence identity (e.g., at least one of 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher sequence identity) to the reverse complement of SEQ ID NO: 158, 159, 160, 169, 170, or 171.In some embodiments, the inhibitory nucleic acid may comprise or encode an antisense nucleic acid comprising or consisting of a sequence having at least 75% sequence identity (e.g., at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher sequence identity) to SEQ ID NO: 178, 179, 180, 189, 190, or 191.

[0161] In embodiments in which it is desirable to inhibit gene and / or protein expression of FHR2, the inhibitory nucleic acid may comprise or encode an antisense nucleic acid having a target nucleotide sequence that is a nucleotide sequence encoding FHR2 (e.g., the nucleotide sequence of an RNA encoded by a gene encoding FHR2, or the nucleotide sequence of an RNA encoding FHR2). In some embodiments, the target nucleotide sequence comprises one or more nucleotides of an exon of the RNA encoding FHR2 (e.g., is the nucleotide sequence of an exon of the RNA encoding FHR2). In some embodiments, the target nucleotide sequence is the nucleotide sequence of SEQ ID NO:219 (e.g., the nucleotide sequence between positions 1 and 201, inclusive, of SEQ ID NO:219 or the nucleotide sequence between positions 757 and 1498, inclusive, of SEQ ID NO:219). In some embodiments, the target nucleotide sequence is or comprises SEQ ID NO:158, 169, 172, or 173. In some embodiments, the inhibitory nucleic acid may comprise or encode an antisense nucleic acid comprising or consisting of a sequence having at least 75% sequence identity (e.g., at least one of 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher sequence identity) to the reverse complement of SEQ ID NO: 158, 169, 172, or 173. In some embodiments, the inhibitory nucleic acid may comprise or encode an antisense nucleic acid comprising or consisting of a sequence having at least 75% sequence identity (e.g., at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher sequence identity) to SEQ ID NO: 178, 189, 192, or 193.

[0162] In embodiments in which it is desirable to inhibit gene and / or protein expression of FHR3, the inhibitory nucleic acid may comprise or encode an antisense nucleic acid having a target nucleotide sequence that is a nucleotide sequence encoding FHR3 (e.g., the nucleotide sequence of RNA encoded by a gene encoding FHR3, or the nucleotide sequence of RNA encoding FHR3). In some embodiments, the target nucleotide sequence comprises one or more nucleotides of an exon of the RNA encoding FHR3 (e.g., is the nucleotide sequence of an exon of the RNA encoding FHR3). In some embodiments, the target nucleotide sequence is the nucleotide sequence of SEQ ID NO:220 (e.g., the nucleotide sequence between positions 661 and 2934 (inclusive) of SEQ ID NO:220, the nucleotide sequence between positions 661 and 843 (inclusive) of SEQ ID NO:220, or the nucleotide sequence between positions 844 and 2934 (inclusive) of SEQ ID NO:220). In some embodiments, the target nucleotide sequence is or comprises SEQ ID NO:161, 162, 163, 166, 174, 175, or 176. In some embodiments, the inhibitory nucleic acid may comprise or encode an antisense nucleic acid comprising or consisting of a sequence having at least 75% sequence identity (e.g., at least one of 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher sequence identity) to the reverse complement of SEQ ID NO: 161, 162, 163, 166, 174, 175, or 176.In some embodiments, the inhibitory nucleic acid may comprise or encode an antisense nucleic acid comprising or consisting of a sequence having at least 75% sequence identity (e.g., at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher sequence identity) to SEQ ID NO: 181, 182, 183, 186, 194, 195, or 196.

[0163] In embodiments in which it is desirable to inhibit gene and / or protein expression of FHR4, the inhibitory nucleic acid may comprise or encode an antisense nucleic acid having a target nucleotide sequence that is a nucleotide sequence encoding FHR4 (e.g., the nucleotide sequence of an RNA encoded by a gene encoding FHR4, or the nucleotide sequence of an RNA encoding FHR4). In some embodiments, the target nucleotide sequence comprises one or more nucleotides of an exon of the RNA encoding FHR4 (e.g., is the nucleotide sequence of an exon of the RNA encoding FHR4). In some embodiments, the target nucleotide sequence is the nucleotide sequence of SEQ ID NO:221 (e.g., the nucleotide sequence between positions 1 and 157 (inclusive) of SEQ ID NO:221, the nucleotide sequence between positions 716 and 898 (inclusive) of SEQ ID NO:221, the nucleotide sequence between positions 1280 and 1456 (inclusive) of SEQ ID NO:221, the nucleotide sequence between positions 1457 and 2063 (inclusive) of SEQ ID NO:221, or the nucleotide sequence between positions 1457 and 1639 (inclusive) of SEQ ID NO:221). In some embodiments, the target nucleotide sequence is or comprises SEQ ID NO:162, 164, 165, 166, or 177. In some embodiments, the inhibitory nucleic acid may comprise or encode an antisense nucleic acid comprising or consisting of a sequence having at least 75% sequence identity (e.g., at least one of 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher sequence identity) to the reverse complement of SEQ ID NO: 162, 164, 165, 166, or 177.In some embodiments, the inhibitory nucleic acid may comprise or encode an antisense nucleic acid comprising or consisting of a sequence having at least 75% sequence identity (e.g., at least one of 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher sequence identity) to SEQ ID NO: 182, 184, 185, 186, or 197.

[0164] In embodiments in which it is desirable to inhibit gene and / or protein expression of FHR5, the inhibitory nucleic acid may comprise or encode an antisense nucleic acid having a target nucleotide sequence that is a nucleotide sequence encoding FHR5 (e.g., the nucleotide sequence of an RNA encoded by a gene encoding FHR5, or the nucleotide sequence of an RNA encoding FHR5). In some embodiments, the target nucleotide sequence comprises one or more nucleotides of an exon of the RNA encoding FHR5 (e.g., is the nucleotide sequence of an exon of the RNA encoding FHR5). In some embodiments, the target nucleotide sequence is the nucleotide sequence of SEQ ID NO:222 (e.g., the nucleotide sequence between positions 363 and 539, inclusive, of SEQ ID NO:222, or the nucleotide sequence between positions 1623 and 2814, inclusive, of SEQ ID NO:222). In some embodiments, the target nucleotide sequence is or comprises SEQ ID NO:167 or 168. In some embodiments, the inhibitory nucleic acid may comprise or encode an antisense nucleic acid comprising or consisting of a sequence having at least 75% sequence identity (e.g., at least one of 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher sequence identity) to the reverse complement of SEQ ID NO: 167 or 168. In some embodiments, the inhibitory nucleic acid may comprise or encode an antisense nucleic acid comprising or consisting of a sequence having at least 75% sequence identity (e.g., at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher sequence identity) to SEQ ID NO: 187 or 188.

[0165] In embodiments in which it is desirable to inhibit gene and / or protein expression of FH and / or FHL-1, the inhibitory nucleic acid may comprise or encode an antisense nucleic acid having a target nucleotide sequence that is a nucleotide sequence encoding FH and / or FHL-1 (e.g., the nucleotide sequence of an RNA encoded by a gene encoding FH and / or FHL-1, or the nucleotide sequence of an RNA encoding FH and / or FHL-1). In some embodiments, the target nucleotide sequence comprises one or more nucleotides of an exon of an RNA encoding FH and / or FHL-1 (e.g., is the nucleotide sequence of an exon of an RNA encoding FH and / or FHL-1). In some embodiments, the target nucleotide sequence is the nucleotide sequence of SEQ ID NO:223 (e.g., the nucleotide sequence between positions 3386 and 3568, inclusive, of SEQ ID NO:223). In some embodiments, the target nucleotide sequence is or comprises SEQ ID NO:169, 170, 171, or 172. In some embodiments, the inhibitory nucleic acid may comprise or encode an antisense nucleic acid comprising or consisting of a sequence having at least 75% sequence identity (e.g., at least one of 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher sequence identity) to the reverse complement of SEQ ID NO: 169, 170, 171, or 172. In some embodiments, the inhibitory nucleic acid may comprise or encode an antisense nucleic acid comprising or consisting of a sequence having at least 75% sequence identity (e.g., at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher sequence identity) to SEQ ID NO: 189, 190, 191, or 192.

[0166] In some embodiments, an agent described herein, e.g., an inhibitory nucleic acid, inhibits gene and / or protein expression of only FHR1, i.e., the agent does not inhibit gene and / or protein expression of FHR2, FHR3, FHR4, FHR5, FH, or FHL-1. In some embodiments, an agent described herein, e.g., an inhibitory nucleic acid, inhibits gene and / or protein expression of only FHR2, i.e., the agent does not inhibit gene and / or protein expression of FHR1, FHR3, FHR4, FHR5, FH, or FHL-1. In some embodiments, an agent described herein, e.g., an inhibitory nucleic acid, inhibits gene and / or protein expression of only FHR3, i.e., the agent does not inhibit gene and / or protein expression of FHR1, FHR2, FHR4, FHR5, FH, or FHL-1. In some embodiments, an agent described herein, e.g., an inhibitory nucleic acid, inhibits gene and / or protein expression of only FHR4, i.e., the agent does not inhibit gene and / or protein expression of FHR1, FHR2, FHR3, FHR5, FH, or FHL-1. In some embodiments, an agent described herein, e.g., an inhibitory nucleic acid, inhibits gene and / or protein expression of only FHR5, i.e., the agent does not inhibit gene and / or protein expression of FHR1, FHR2, FHR3, FHR4, FH, or FHL-1. In some embodiments, an agent described herein, e.g., an inhibitory nucleic acid, inhibits gene and / or protein expression of only FH, i.e., the agent does not inhibit gene and / or protein expression of FHR1, FHR2, FHR3, FHR4, FHR5, or FHL-1. In some embodiments, an agent described herein, e.g., an inhibitory nucleic acid, inhibits gene and / or protein expression of only FHL-1, i.e., the agent does not inhibit gene and / or protein expression of FHR1, FHR2, FHR3, FHR4, FHR5, or FH. In some embodiments, an agent described herein, e.g., an inhibitory nucleic acid, inhibits gene and / or protein expression of only FH and FHL-1, i.e., the agent does not inhibit gene and / or protein expression of FHR1, FHR2, FHR3, FHR4, or FHR5.

[0167] In some embodiments, the agents described herein, e.g., inhibitory nucleic acids, inhibit gene and / or protein expression of one or more Factor H family proteins, and the inhibited proteins are not FH and / or FHL-1. That is, the agents described herein, e.g., inhibitory nucleic acids, may inhibit gene and / or protein expression of one or more Factor H family proteins selected from FHR1, FHR2, FHR3, FHR4, and / or FHR5, but do not inhibit gene and / or protein expression of FH and / or FHL-1. In some cases, the agents described herein, e.g., inhibitory nucleic acids, inhibit gene and / or protein expression of each of FHR1, FHR2, FHR3, FHR4, and FHR5, but do not inhibit gene and / or protein expression of FH and / or FHL-1.

[0168] In some embodiments, an agent described herein, e.g., an inhibitory nucleic acid, inhibits gene and / or protein expression of one or more Factor H family proteins (e.g., FHR2, FHR3, FHR4, FHR5, FH, and / or FHL-1), but does not inhibit gene and / or protein expression of FHR1. In some embodiments, an agent described herein, e.g., an inhibitory nucleic acid, inhibits gene and / or protein expression of one or more Factor H family proteins (e.g., FHR1, FHR3, FHR4, FHR5, FH, and / or FHL-1), but does not inhibit gene and / or protein expression of FHR2. In some embodiments, an agent described herein, e.g., an inhibitory nucleic acid, inhibits gene and / or protein expression of one or more Factor H family proteins (e.g., FHR1, FHR2, FHR4, FHR5, FH, and / or FHL-1), but does not inhibit gene and / or protein expression of FHR3. In some embodiments, an agent described herein, e.g., an inhibitory nucleic acid, inhibits gene and / or protein expression of one or more Factor H family proteins (e.g., FHR1, FHR2, FHR3, FHR5, FH, and / or FHL-1), but does not inhibit gene and / or protein expression of FHR4. In some embodiments, an agent described herein, e.g., an inhibitory nucleic acid, inhibits gene and / or protein expression of one or more Factor H family proteins (e.g., FHR1, FHR2, FHR3, FHR4, FH, and / or FHL-1), but does not inhibit gene and / or protein expression of FHR5.

[0169] In some embodiments, an agent described herein, e.g., an inhibitory nucleic acid, inhibits gene and / or protein expression of one or more Factor H family proteins (e.g., FHR2, FHR3, FHR4, FHR5, FH, and / or FHL-1), but does not inhibit gene and / or protein expression of FHR1 or FHR3. In some embodiments, an agent described herein, e.g., an inhibitory nucleic acid, inhibits gene and / or protein expression of one or more Factor H family proteins (e.g., FHR2, FHR3, FHR4, FHR5, FH, and / or FHL-1), but does not inhibit gene and / or protein expression of FHR3 or FH.

[0170] In some embodiments, the target nucleotide sequence is not the nucleotide sequence of SEQ ID NO: 221, which is the mRNA sequence encoding FHR4 (thymine (T) replaced with uracil (U)).

[0171] In some embodiments, the target nucleotide sequence is not the nucleotide sequence of SEQ ID NO: 218, which is an mRNA sequence encoding FHR1 (thymine (T) is replaced by uracil (U)). In some embodiments, the target nucleotide sequence is not the nucleotide sequence of SEQ ID NO: 219, which is an mRNA sequence encoding FHR2 (thymine (T) is replaced by uracil (U)). In some embodiments, the target nucleotide sequence is not the nucleotide sequence of SEQ ID NO: 220, which is an mRNA sequence encoding FHR3 (thymine (T) is replaced by uracil (U)). In some embodiments, the target nucleotide sequence is not the nucleotide sequence of SEQ ID NO: 222, which is an mRNA sequence encoding FHR5 (thymine (T) is replaced by uracil (U)). In some embodiments, the target nucleotide sequence is not the nucleotide sequence of SEQ ID NO: 223, which is an mRNA sequence encoding FH (thymine (T) is replaced by uracil (U)).

[0172] In some embodiments, the agents described herein, e.g., inhibitory nucleic acids, do not bear substantial sequence identity to the sequences disclosed in any one of WO 2007 / 144621, WO 2019 / 051443, WO 2006 / 088950, or WO 2012 / 112955, the entireties of which are incorporated herein by reference.

[0173] In some embodiments, an agent described herein, e.g., an inhibitory nucleic acid, comprises or consists of a sequence having less than 97%, less than 95%, less than 90%, less than 85%, less than 80%, less than 75%, less than 70%, less than 65%, less than 60%, less than 55%, less than 50%, less than 45%, less than 40%, less than 35%, less than 30%, less than 25%, or less than 20% sequence identity to a sequence disclosed in any one of WO 2007 / 144621, WO 2019 / 051443, WO 2006 / 088950, or WO 2012 / 112955 (e.g., SEQ ID NO: 3, 4, or 5 disclosed in WO 2007 / 144621, or SEQ ID NO: 1, 2, 16, 17, or 18 disclosed in WO 2019 / 051443).

[0174] In some embodiments, the inhibitory nucleic acid is selected from: an siRNA, miRNA, shRNA, pri-miRNA, pre-miRNA, saRNA, snRNA, or antisense oligonucleotide (e.g., a gapmer), or a nucleic acid encoding any of these. In some embodiments, the inhibitory nucleic acid is selected from: an siRNA, miRNA, shRNA. In some embodiments, the inhibitory nucleic acid is an siRNA.

[0175] In some embodiments, an inhibitory nucleic acid may comprise an antisense nucleic acid described herein as part of a larger nucleic acid species. For example, in some embodiments, an inhibitory nucleic acid may be an siRNA, miRNA, shRNA, pri-miRNA, pre-miRNA, saRNA, or snRNA that comprises an antisense nucleic acid described herein.

[0176] In some embodiments, the inhibitory nucleic acid is a small interfering RNA (siRNA). As used herein, "siRNA" refers to a double-stranded RNA molecule having a length of between 17 and 30 (e.g., 20 and 27) base pairs that can participate in the RNA interference (RNAi) pathway for targeted degradation of a target RNA. A double-stranded siRNA molecule may be formed as a nucleic acid complex of highly complementary RNA strands. In some embodiments, the siRNA molecule comprises a symmetric 3' overhang (e.g., a "UU" 3' overhang), e.g., containing one or two nucleotides. The strand of the siRNA molecule that is complementary to the target nucleotide sequence (i.e., the antisense nucleic acid) is referred to as the guide RNA, and the other strand is referred to as the passenger strand. The structure and function of siRNA are described, for example, in Kim and Rossi, Biotechniques. 2008 Apr; 44(5): 613-616. In some embodiments, the inhibitory nucleic acid comprises the guide and passenger strands of an siRNA.

[0177] In some embodiments, an inhibitory nucleic acid of the disclosure has at least 75% sequence identity (e.g., at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) to a SEQ ID NO: in column A of Table 12. and sequences having at least 75% sequence identity (e.g., at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity) to a SEQ ID NO: in column B of Table 12. In some cases, a sequence from column A and a sequence from column B are provided in the same row of Table 12.

[0178] In some embodiments, the guide strand of an siRNA described herein may comprise or consist of an antisense nucleic acid described in the embodiments of the antisense nucleic acids described herein (e.g., a sequence having at least 70% sequence identity to one or more of SEQ ID NOs: 178-197).

[0179] In some embodiments, the inhibitory nucleic acid is a microRNA (miRNA) or its precursor (e.g., pri-miRNA or pre-miRNA). miRNA molecules have a similar structure but are endogenously encoded and derived from the processing of small hairpin RNA molecules. They are originally expressed as long primary transcripts (pri-miRNAs), which are processed in the nucleus into 60-70 nucleotide hairpins (pre-miRNAs) and further processed in the cytoplasm into small double-stranded nucleic acids that interact with RISC and target mRNAs. miRNAs contain a "seed sequence" that is essential for binding to target mRNAs. The "seed sequence" usually contains 6 nucleotides and is located at positions 2-7 of the 5' end of the miRNA.

[0180] In some embodiments, the inhibitory nucleic acid is a small hairpin RNA (shRNA). The shRNA molecule comprises a sequence of nucleotides with a high degree of complementarity that associate with each other through complementary base pairing to form a stem region in the hairpin. The sequences of nucleotides with a high degree of complementarity may be linked by one or more nucleotides that form a loop region in the hairpin. The shRNA molecule may be processed (e.g., through catalytic cleavage by DICER) to form an siRNA or miRNA molecule. The shRNA may have a length of between 35 and 100 (e.g., 40 and 70) nucleotides. The stem region of the hairpin may have a length of between 17 and 30 (e.g., 20 and 27) base pairs. The stem region may contain GU pairings that stabilize the hairpin structure.

[0181] In some embodiments, the inhibitory nucleic acid is a Dicer small interfering RNA (dsiRNA). As used herein, "dsiRNA" refers to a double-stranded RNA molecule having a length of about 27 base pairs, which is processed by Dicer into siRNA for RNAi-mediated degradation of the target RNA. dsiRNA is described, for example, in Raja et al., Asian J Pharm Sci. (2019) 14(5): 497-510, the entire contents of which are incorporated herein by reference. dsiRNA is optimized for Dicer processing and may have increased potency compared to 21-mer siRNA (see, for example, Kim et al., Nat Biotechnol. (2005) 23(2):222-226), which may relate to the link between Dicer-mediated nuclease activity and RISC loading.

[0182] siRNA, dsiRNA, miRNA and shRNA for targeting inhibition of gene and / or protein expression of one or more predetermined target genes / proteins can be identified / designed according to principles and / or using tools known to those skilled in the art.The parameters and tools for designing siRNA and shRNA molecules are described, for example, in Fakhr et al., Cancer Gene Therapy (2016) 23:73-82 (incorporated herein in its entirety).Software that can be used by those skilled in the art to design such molecules is summarized in Table 1 of Fakhr et al., Cancer Gene Therapy (2016) 23:73-82, and includes, for example, siRNA Wizard (InvivoGen).Details for creating such molecules can be found on the websites of commercial suppliers such as Ambion, Dharmacon, GenScript, Invitrogen and OligoEngine.

[0183] In some embodiments, the inhibitory nucleic acid is an antisense oligonucleotide (ASO). ASOs are single-stranded nucleic acid molecules that contain or consist of an antisense nucleic acid against a target nucleotide sequence. Antisense oligonucleotides described herein may contain or consist of an antisense nucleic acid as described herein. ASOs may modify the expression of an RNA molecule containing a target nucleotide sequence by altering splicing or by recruiting RNase H to degrade the RNA containing the target nucleotide sequence. RNase H recognizes the nucleic acid complex molecule formed when an ASO binds to an RNA containing the target nucleotide sequence. ASOs described herein may contain or consist of an antisense nucleic acid as described herein. ASOs may be 17-30 nucleotides in length. Many ASOs are designed as chimeras, containing a mixture of bases with different chemistries, or as gapmers, containing a central DNA segment surrounded by "wings" of modified bases. ASOs are described, for example, in Scoles et al., Neurol Genet. 2019 Apr;5(2):e323. ASOs sometimes contain modifications to the sugar-phosphate backbone, such as phosphorothioate linkages, phosphorodiamidate linkages, e.g., phosphorodiamidate morpholinos (PMOs), to reduce / prevent RNase H degradation, and may contain peptide nucleic acids (PNAs), locked nucleic acids (LNAs), methoxyethyl nucleotide modifications, e.g., 2'O-methyl (2'OMe) and 2'-O-methoxyethyl (MOE) ribose modifications, and / or 5'-methylcytosine modifications.

[0184] The inhibitory nucleic acids described herein may contain chemically modified nucleotide acids, e.g., where the phosphonate and / or ribose and / or base are chemically modified. Such modifications may affect the activity, specificity, and / or stability of the nucleic acid. One or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or all one) nucleotides of the inhibitory nucleic acid may contain such chemical modifications.

[0185] Modifications contemplated in accordance with the inhibitory nucleic acids described herein include those described in Hu et al., Sig. Transduc. Tar. Ther. (2020) 5(101), which is incorporated herein by reference in its entirety, particularly those shown in Figure 2 of Hu et al., Sig. Transduc. Tar. Ther. (2020) 5(101). Further modifications contemplated in accordance with the inhibitory nucleic acids described herein include those described in Selvam et al., Chem Biol Drug Des. (2017) 90(5): 665-678, which is incorporated herein by reference in its entirety.

[0186] In some embodiments, the inhibitory nucleic acids according to the present disclosure comprise a phosphonate modification selected from: phosphorothioate (e.g., Rp isomers, Sp isomers), phosphorodithioate, methyl phosphonate, methoxypropyl phosphonate, 5'-(E)-vinyl phosphonate, 5'-methyl phosphonate, (S)-5'-C-methyl with phosphate, 5'-phosphorothioate, and peptide nucleic acid.

[0187] In some embodiments, the inhibitory nucleic acids described herein comprise a ribose modification selected from: 2'-O-methyl, 2'-O-methoxyethyl, 2'-fluoro, 2'-deoxy-2'-fluoro, 2'-methoxyethyl, 2'-O-alkyl, 2'-O-allyl, 2'-C-allyl, 2'-deoxy, 2'-hydroxyl, 2'-arabino-fluoro, 2'-O-benzyl, 2'-O-methyl-4-pyridine, locked nucleic acid, (S)-cEt-BNA, tricyclo-DNA, PMO, unlocked nucleic acid, hexitol nucleic acid, and glycol nucleic acid.

[0188] In some embodiments, the inhibitory nucleic acids described herein comprise a base modification selected from: pseudouridine, 2'-thiouridine, N6'-methyladenosine, 5'-methylcytidine, 5'-fluoro-2'-deoxyuridine, N-ethylpiperidine 7'-EAA triazole modified adenosine, N-ethylpiperidine 6'-triazole modified adenosine, 6'-phenylpyrrolo-cytosine, 2',4'-difluorotoluylribonucleoside, and 5'-nitroindole.

[0189] In some embodiments, the inhibitory nucleic acids described herein comprise modifications to incorporate moieties that facilitate delivery to and / or uptake by a cell type or tissue of interest. Modifications to nucleic acids that facilitate targeted delivery to a cell type and / or tissue of interest are described, for example, in Lorenzer et al., J Control Release (2015) 203:1-15, which is incorporated herein by reference in its entirety.

[0190] In some embodiments, the inhibitory nucleic acids described in this disclosure include modifications to incorporate moieties that facilitate delivery to and / or uptake by liver cells or tissue. In some embodiments, the inhibitory nucleic acid is conjugated to N-acetylgalactosamine (GalNAc). GalNAc interacts with the asialoglycoprotein receptor (ASGPR) expressed by hepatocytes. After GalNAc binds to ASGPR, the nucleic acid conjugated to GalNAc is efficiently internalized by hepatocytes through receptor-mediated endocytosis (see, for example, Nair et al., J. Am. Chem. Soc. (2014) 136(49): 16958-16961). In some embodiments, the inhibitory nucleic acid is conjugated to one or more (e.g., 1, 2, 3, 4 or more) GalNAc moieties. In some embodiments, one or more GalNAc moieties may be covalently linked to the 5' or 3' end of the chain of the inhibitory nucleic acid.

[0191] In some embodiments, the inhibitory nucleic acid is conjugated to α-tocopherol (i.e., vitamin E). Nucleic acid-α-tocopherol conjugates have been used for targeted delivery of inhibitory nucleic acids to the liver (see, e.g., Nishina et al., Mol Ther. (2008) 16(4):734-740). In some embodiments, the inhibitory nucleic acid is conjugated to one or more (e.g., 1, 2, 3, 4, or more) α-tocopherol moieties. In some embodiments, one or more α-tocopherol moieties may be covalently attached to the 5' or 3' end of the inhibitory nucleic acid strand.

[0192] In embodiments in which an inhibitory nucleic acid contains nucleotides containing chemical modifications as described herein, the nucleotide sequence is nevertheless evaluated for purposes of sequence comparison according to the present disclosure as if the equivalent unmodified nucleotides were present instead. Nucleic acids containing nucleotides containing modified phosphate groups are evaluated for purposes of nucleotide sequence comparison as if the nucleic acid contained only nucleotides containing unmodified phosphate groups. Nucleic acids containing nucleotides containing modified ribose groups are evaluated for purposes of nucleotide sequence comparison as if the nucleic acid contained only nucleotides containing unmodified ribose groups.

[0193] Nucleic acids containing nucleotides containing modified base groups are evaluated for nucleotide sequence comparison as if each modified base were unmodified. For example, nucleic acids containing nucleotides containing pseudouridine, 2-thiouridine, and / or 5'-fluoro-2'-deoxyuridine are evaluated for nucleotide sequence comparison as if a uridine-containing nucleotide were present at each position instead. For example, nucleic acids containing nucleotides containing N6'-methyladenosine, N-ethylpiperidine 7'-EAA triazole-modified adenine, and / or N-ethylpiperidine 6'-triazole-modified adenine are evaluated for nucleotide sequence comparison as if an adenine-containing nucleotide were present at each position instead. For example, nucleic acids containing nucleotides containing 5'-methylcytidine and / or 6'-phenylpyrrolo-cytosine are evaluated for nucleotide sequence comparison as if a cytosine-containing nucleotide were present at each position instead.

[0194] The inhibitory nucleic acid may be produced recombinantly, for example, by transcription of a nucleic acid sequence contained in a vector. Transcription may be performed in a cell-free transcription reaction or in a cell containing a nucleic acid encoding the inhibitory nucleic acid. In some embodiments, the inhibitory nucleic acid is produced intracellularly, for example, by transcription from a vector. A vector encoding such a molecule may be introduced into a cell by any method known in the art. Optionally, expression of the nucleic acid may be controlled using a cell-specific (e.g., liver cell) promoter.

[0195] For example, shRNA molecules can be produced intracellularly by transcription from a vector. shRNA can be produced intracellularly by transfecting cells with a vector encoding the shRNA sequence under the control of an RNA polymerase promoter.

[0196] Inhibitory nucleic acids can also be synthesized using standard solid-phase or solution-phase synthesis, which is well known in the art. Solid-phase synthesis can use phosphoramidite chemistry. Briefly, a solid-supported nucleotide can be detritylated and then coupled with an appropriately activated nucleoside phosphoramidite to form a phosphite triester linkage. Capping can then be performed, followed by oxidation of the phosphite triester with an oxidant, typically iodine. The cycle can then be repeated to obtain a polynucleotide.

[0197] Genome editing tools and related items Aspects and embodiments of the present disclosure relate to agents that are or include known gene editing tools or systems. As used herein, a "genome / gene editing tool" or a "genome / gene editing system" refers to a tool or system that can reduce or prevent gene and / or protein expression of one or more predetermined target genes / proteins.

[0198] Genome editing tools / systems can comprise nucleases, i.e., polypeptides that have nuclease activity.Nucleases are, for example, generally described in Yang, Q Rev Biophys. 2011 Feb;44(1):1-93, the entirety of which is incorporated herein by reference.Nucleases are broadly divided into endonucleases and exonucleases according to the region of target nucleic acid they act on; endonucleases act on regions within target nucleic acid, while exonucleases digest nucleic acid from one or both of the 5' and 3' ends of target nucleic acid.

[0199] Target sequences for gene editing tools / systems are described herein above. The nucleases described herein may act on DNA, RNA, or both DNA and RNA. Nucleases that act on DNA may be referred to as having deoxyribonuclease (DNase) activity. Such nucleases may also be referred to as DNases. Nucleases that act on RNA may be referred to as having ribonuclease (RNase) activity. Such nucleases may also be referred to as RNases.

[0200] Target sequences for the agents described herein may include all or part of SEQ ID NOs: 218, 219, 220, 221, 222 and / or 223. The nucleases described herein may be capable of cleaving single-stranded or double-stranded nucleic acid substrates, or both. Nucleases that act on single-stranded nucleic acid molecules may be referred to as having single-stranded nuclease activity. Such nucleases may also be referred to as single-stranded nucleases. Nucleases that act on double-stranded nucleic acid molecules may be referred to as having double-stranded nuclease activity. Such nucleases may also be referred to as double-stranded nucleases.

[0201] Some nucleases act on their substrate nucleic acids in a non-specific manner. Some nucleases are site-specific and have nuclease activity targeted to specific regions of the substrate nucleic acid based on the recognition of a predetermined structure(s) (structure-specific) or nucleic acid sequence(s) (sequence-specific). Some nucleases may be targeted to specific structures / sequence(s) of the substrate nucleic acid. Such nucleases may be referred to as "targetable," "programmable," or "site-specific nucleases" (SSNs). Targetable nucleases are, for example, reviewed in Carroll, Annu Rev Biochem. (2014) 83:409-39, the entire contents of which are incorporated herein by reference.

[0202] Guided nucleases use guide nucleic acid molecules that direct the guided nuclease to a specific structure formed by the substrate nucleic acid and / or the sequence of the substrate nucleic acid. The guide nucleic acid can target the nuclease to a specific region or regions of the substrate nucleic acid through complementary base pairing between the nucleotides of the guide nucleic acid and the nucleotides of the substrate nucleic acid. Nucleic acid-guided nucleases can use RNA guides (RNA-guided nucleases), DNA (DNA-guided nucleases), or both RNA and DNA guides.

[0203] Gene editing using site-specific nucleases (SSNs) is reviewed, for example, in Eid and Mahfouz, Exp Mol Med. 2016 Oct; 48(10): e265, which is incorporated herein by reference in its entirety. Enzymes capable of generating site-specific double-strand breaks (DSBs) can be engineered to introduce DSBs into a target nucleic acid sequence(s) of interest. DSBs can be repaired either by error-prone non-homologous end joining (NHEJ), which often involves the insertion or deletion of nucleotides and religates the two ends of the break. Alternatively, DSBs can be repaired by high-homology-directed repair (HDR), in which a DNA template with ends homologous to the break site is provided and introduced at the site of the DSB.

[0204] SSNs that can be engineered to generate target nucleic acid sequence-specific DSBs include zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and the clustered regularly interspaced short palindromic repeats / CRISPR-associated 9 (CRISPR / Cas9) system.

[0205] The ZFN system is generally described, for example, in Umov et al., Nat Rev Genet. (2010) 11(9):636-46, which is incorporated herein by reference in its entirety. ZFNs contain a programmable zinc finger DNA binding domain and a DNA cleavage domain (e.g., a FokI endonuclease domain). The DNA binding domain may be identified by screening a zinc finger array that allows binding to a target nucleic acid sequence.

[0206] The TALEN system is generally described, for example, in Mahfouz et al., Plant Biotechnol J. (2014) 12(8):1006-14, which is incorporated herein by reference in its entirety. TALENs contain a programmable DNA-binding TALE domain and a DNA-cleavage domain (e.g., a FokI endonuclease domain). TALEs contain repeat domains consisting of 33-39 amino acid repeats, which are identical except for two residues at positions 12 and 13 of each repeat, which are repeat variable dinucleotides (RVDs). Each RVD determines the binding of the repeat to a nucleotide in the target DNA sequence according to the following relationship: "HD" binds to C, "NI" binds to A, "NG" binds to T, and "NN" or "NK" bind to G (Moscou and Bogdanove, Science (2009) 326(5959):1501).

[0207] CRISPR / Cas9 and related systems, such as CRISPR / Cpf1, CRISPR / C2c1, CRISPR / C2c2, and CRISPR / C2c3, are reviewed, for example, in Nakade et al., Bioengineered (2017) 8(3):265-273, the entire contents of which are incorporated herein by reference. These systems include an endonuclease (e.g., Cas9, Cpf1, etc.) and a single-stranded guide RNA (sgRNA) molecule. The sgRNA may be engineered to target the endonuclease activity to a nucleic acid sequence of interest.

[0208] Other targeting approaches for genome engineering, such as meganucleases, are reviewed in Silva et al., Curr Gene Ther. 2011 Feb; 11(1): 11-27, which is incorporated herein in its entirety.

[0209] In some embodiments, the genome editing system for reducing expression of one or more Factor H family genes / proteins described herein is selected from: a ZFN system, a TALEN system, a CRISPR / Cas system, a CRISPR / Cas9 system, a CRISPR / Cpf1 system, a CRISPR / C2c1 system, a CRISPR / C2c2 system, a CRISPR / C2c3 system, or a meganuclease.

[0210] In some embodiments, the CRISPR / Cas system for use as the agent described herein comprises a Cas nuclease, a crisprRNA (crRNA or guide RNA (gRNA)), and a transactivating crRNA (trRNA or tracrRNA). In this system, the crRNA comprises a sequence complementary to the target DNA and acts to guide the Cas nuclease to the target site in the genome, and the tracrRNA acts as a binding scaffold for the Cas nuclease required for Cas activity. In some embodiments, the CRISPR / Cas system comprises a guide RNA (gRNA).

[0211] In some embodiments, the CRISPR / Cas genome editing systems described herein comprise a Cas nuclease and a single guide RNA (sgRNA) that guides the Cas nuclease to a target site in a target gene. The sgRNA comprises a target-specific crRNA fused to a scaffold tracrRNA in a single nucleic acid.

[0212] In some embodiments, the crRNA, gRNA, or sgRNA described herein comprises or consists of a sequence set forth in SEQ ID NOs: 224 to 227. In some embodiments, the crRNA, gRNA, or sgRNA described herein comprises or consists of a sequence having at least 60%, e.g., at least 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NOs: 224 to 227.

[0213] In some embodiments, the crRNA, gRNA, or sgRNA described herein comprises or consists of a sequence having at least 60%, e.g., at least 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 224, and, e.g., when used in a CRISPR / Cas system, reduces or prevents gene expression of one or more of FHR1, FHR2, FHR3, FHR4, and / or FHR5. In some cases, the CRISPR / Cas system does not reduce or prevent gene expression of FH and / or FHL-1.

[0214] In some embodiments, the crRNA, gRNA, or sgRNA described herein comprises or consists of a sequence having at least 60%, e.g., at least 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 225, 226, or 227, and, e.g., when used in a CRISPR / Cas system, reduces or prevents gene expression of one or more of FHR1, FHR2, FHR3, FHR4, and / or FHR5. In some cases, the CRISPR / Cas system does not reduce or prevent gene expression of FH and / or FHL-1.

[0215] Design of guide RNAs suitable for use in CRISPR / Cas systems is well within the routine practice of one of ordinary skill in the art using software platforms publicly available, for example, from Harvard University (http: / / chopchop.cbu.uib.no / ), Synthego (https: / / design.synthego.com / # / ), or Integrated DNA Technologies (https: / / eu.idtdna.com / site / order / designtool / index / CRISPR_PREDESIGN).

[0216] The CRISPR / Cas system has been adapted for use in gene silencing.For example, in some embodiments, the nucleic acid may encode a fusion protein, comprising a Cas protein or peptide fused to a transcriptional repressor.In some embodiments, the Cas protein is catalytically inactive (dead).The fusion protein may be guided to the site of interest in the genome by either sgRNA or crRNA.When the fusion protein binds to the site of interest, the transcriptional repressor can reduce the expression of the gene of interest.

[0217] CRISPR / Cas systems can also be used to introduce targeted modifications into target nucleic acid sequences, such as genomic DNA, using, for example, base editing or prime editing techniques. Base editing and base editors (i.e., polypeptides possessing "base editing" activity) are described, for example, in Rees and Liu Nat Rev Genet (2018) 19(12): 770-788 (revised according to Rees and Liu, Nat Rev Genet (2018) 19:801), which is incorporated herein by reference in its entirety.

[0218] This may be achieved using a Cas protein fused to a base editor, such as an adenine base editor or a cytidine base editor, such as a cytidine deaminase, as disclosed, for example, in WO2017070633A2, the entire contents of which are incorporated herein by reference. Cytidine deaminases include, for example, CDA, APOBEC1, APOBEC1(W90Y / R126E), APOBEC1(W90Y / R132E), APOBEC1(R126E / R132E), APOBEC1(W90Y / R126E / R132E), CDA1, APOBEC3A, APOBEC3A(N37G), AID, AID(P182X), and AIDΔ. Adenine base editors include, for example, adenine deaminases, which catalyze the conversion of adenosine to inosine. Adenosine deaminases include, for example, TadA and mutants thereof.

[0219] For example, the CRISPR / Cas system may be a prime editing system. Such a prime editing system may use a fusion protein. For example, the fusion protein may include a catalytically impaired Cas domain (e.g., a "nickase") and a reverse transcriptase. The catalytically impaired Cas domain may be capable of cleaving a single strand of DNA to produce a nicked DNA duplex. The prime editing system may include a prime editing guide RNA (pegRNA) that includes an extended sgRNA that includes a primer binding site and a reverse transcriptase template sequence. When the DNA duplex is nicked by the catalytically impaired Cas, the primer binding site allows the 3' end of the nicked DNA strand to hybridize to the pegRNA, while the RT template serves as a template for synthesizing edited genetic information.

[0220] Articles encoding and / or delivering agents of the invention The present disclosure provides nucleic acids that comprise or encode the agents described herein, for example, inhibitory nucleic acids or at least part of gene editing systems / complexes. In some embodiments, the nucleic acids that comprise or encode the agents comprise or consist of DNA and / or RNA.

[0221] The nucleic acid may comprise a DNA or mRNA sequence encoding a Cas protein or peptide, such as a Cas9 protein or peptide. In some embodiments, the nucleic acid comprises an sgRNA (e.g., as described herein). In some embodiments, the nucleic acid comprises a crRNA (gRNA) and / or a tracrRNA. In some embodiments, the nucleic acid comprises DNA or mRNA encoding a Cas protein or peptide, a crRNA, and a tracrRNA. In some embodiments, the nucleic acid comprises DNA or mRNA encoding a Cas protein or peptide and a crRNA (gRNA) and / or a tracrRNA. In some embodiments, the nucleic acid comprises DNA or mRNA encoding a Cas protein or peptide and a sgRNA.

[0222] The present disclosure also provides vectors comprising nucleic acids that contain or encode the agents described in this disclosure. The nucleic acids and vectors described in this disclosure may be provided in purified or isolated form, i.e., purified or isolated from other nucleic acids or in naturally occurring biological materials.

[0223] The nucleotide sequence of a nucleic acid comprising or encoding an agent described in the present disclosure may be contained in a vector, such as an expression vector. As used herein, a "vector" refers to a nucleic acid molecule used as a vehicle for transferring an exogenous nucleic acid into a cell. The vector may be a vector for expressing a nucleic acid in a cell. Such a vector may include a promoter sequence operably linked to a nucleotide sequence encoding the sequence to be expressed. The vector may also include a termination codon and an expression enhancer. The vector may also include a control element, such as a polyadenylation site. The nucleic acid may be expressed from the vector described in the present disclosure using any suitable vector, promoter, enhancer, and termination codon known in the art.

[0224] The term "operably linked" includes situations where a selected nucleic acid sequence and a regulatory nucleic acid sequence (e.g., a promoter and / or enhancer) are covalently linked in a manner that places expression of the nucleic acid sequence under the influence or control of the regulatory sequences (thereby forming an expression cassette). Thus, a regulatory sequence is operably linked to a selected nucleic acid sequence if the regulatory sequence is capable of effecting transcription of the nucleic acid sequence.

[0225] Suitable vectors include plasmids, binary vectors, DNA vectors, mRNA vectors, viral vectors (e.g., gammaretroviral vectors (e.g., murine leukemia virus (MLV)-derived vectors), lentiviral vectors, adenoviral vectors, adeno-associated viral (AAV) vectors, vaccinia viral vectors, and herpes viral vectors), transposon-based vectors, and artificial chromosomes (e.g., yeast artificial chromosomes), such as those described in Maus et al., Annu Rev Immunol (2014) 32:189-225 or Morgan and Boyerinas, Biomedicines 2016 4, 9, both of which are incorporated herein by reference in their entirety. In some embodiments, the lentiviral vector may be pELNS or may be derived from pELNS. In some embodiments, the vector may be a CRISPR / Cas9-encoding vector. In some embodiments, the adeno-associated virus (AAV) vector is selected from AAV serotype 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11, or hybrids and / or mutants thereof. In some embodiments, the AAV vector is an AAV serotype 2 (AAV-2) vector, or hybrids and / or mutants thereof.

[0226] Viral and non-viral delivery systems for introducing genetic material into cells are reviewed, for example, in Nayerossadat et al., Adv Biomed Res. 2012; 1: 27; MacLaren et al., Ophthalmology. 2016, 123(10 Suppl): S98-S106; Petit and Punzo, Discov Med. 2016, 22(121): 221-229; Aguirre, Invest Ophthalmol Vis Sci. 2017, 58(12): 5399-5411; Lundstrom, Diseases. 2018, 6(2): 42, the entire contents of which are incorporated herein by reference. Any suitable nucleotide or vector delivery method may be used in the context of the present invention.

[0227] In some embodiments, the vector may be a eukaryotic vector, e.g., a vector that contains the necessary elements to express a nucleic acid from the vector in a eukaryotic cell, hi some embodiments, the vector may be a mammalian vector, e.g., a mammalian vector that contains a cytomegalovirus (CMV) or SV40 promoter to drive expression.

[0228] The present disclosure also provides a plurality of agents described in this disclosure, such as inhibitory nucleic acids or components of gene editing systems / complexes. The present disclosure also provides nucleic acids and vectors that include or encode a plurality of such agents described in this disclosure.

[0229] The individual agents of a plurality of agents described herein may be identical or non-identical. Similarly, in embodiments where a nucleic acid / vector comprising or encoding an agent described herein comprises / encodes more than one agent described herein, the agents comprised / encoded by the nucleic acid / vector may be identical or non-identical.

[0230] In some embodiments, the nucleic acid / vector may encode one of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 agents, e.g., inhibitory nucleic acids, described herein. In some embodiments, the nucleic acid / vector may encode multiple (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, etc.) copies of a given agent described herein.

[0231] In some embodiments, the multiple agents described herein may be multiple non-identical agents. In some embodiments, the multiple agents may include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 non-identical agents, such as one inhibitory nucleic acid. In some embodiments, a nucleic acid / vector may include / encode multiple non-identical agents described herein.

[0232] The following paragraphs further define multiple non-identical agents in accordance with the multiple agent embodiment described in this disclosure, and also in accordance with the nucleic acid / vector embodiment comprising / encoding multiple non-identical agents described in this disclosure.

[0233] In some embodiments, the non-identical agent may decrease gene and / or protein expression of a non-identical Factor H family protein (e.g., one selected from FHR1, FHR2, FHR3, FHR4, FHR5, FH, and / or FHL-1).

[0234] In some embodiments, the non-identical agents, e.g., inhibitory nucleic acids, may comprise or encode non-identical antisense nucleic acids. In such embodiments, the non-identical antisense nucleic acids may each independently follow any of the embodiments of antisense nucleic acids as described herein above.

[0235] In some embodiments, the non-identical agents, e.g., inhibitory nucleic acids, may comprise or encode antisense nucleic acids that target non-identical target nucleotide sequences. In such embodiments, the non-identical target nucleotide sequences may each independently conform to any of the aspects of the target nucleotide sequence of the antisense nucleic acid, as described above.

[0236] In some embodiments, the target nucleotide sequence of the antisense nucleic acid contained / encoded by the non-identical inhibitory nucleic acid may be that of an RNA encoded by a gene encoding a non-identical Factor H family protein (e.g., selected from FHR1, FHR2, FHR3, FHR4, FHR5, FH, and / or FHL-1). In some embodiments, the target nucleotide sequence may be that of an RNA encoding a non-identical Factor H family protein (e.g., selected from FHR1, FHR2, FHR3, FHR4, FHR5, FH, and / or FHL-1). In some embodiments, the target nucleotide sequence comprises one or more nucleotides of an exon of an RNA encoding a non-identical Factor H family protein (e.g., selected from FHR1, FHR2, FHR3, FHR4, FHR5, FH, and / or FHL-1). In some embodiments, the target nucleotide sequence is the nucleotide sequence of an exon of an RNA encoding a non-identical Factor H family protein (e.g., one selected from FHR1, FHR2, FHR3, FHR4, FHR5, FH, and / or FHL-1).

[0237] It will be appreciated that in some embodiments, it may be desirable to select individual inhibitory nucleic acids of a plurality of non-identical inhibitory nucleic acids to provide for reduced gene and / or protein expression of more than one Factor H family protein (e.g., selected from FHR1, FHR2, FHR3, FHR4, FHR5, FH, and / or FHL-1).

[0238] In some embodiments, the multiple non-identical inhibitory nucleic acids for reducing gene and / or protein expression of more than one Factor H family protein comprise inhibitory nucleic acids for reducing gene and / or protein expression of one of the following combinations of Factor H family proteins: FH and / or FHL-1, FHR1, FHR2, FHR3, FHR4, and FHR5; FH and / or FHL-1, FHR1, FHR2, FHR3, and FHR4; FH and / or FHL-1, FHR1, FHR2, FHR3, and FHR5. HR5;FH and / or FHL-1, FHR1, FHR2, FHR4 and FHR5;FH and / or FHL-1, FHR1, FHR3, FHR4 and FHR5;FH and / or FHL-1, FHR2, FHR3, FHR4 and FHR5;FHR1, FHR2, FHR3, FHR4 and FHR5;FH and / or FHL-1, FHR1, FHR2 and FHR3;FH and / or FHL-1, FHR1, FHR2 and FHR4;FH and / or FHL-1, FHR1, FHR2 and FHR5;FH and / or or FHL-1, FHR1, FHR3 and FHR4; FH and / or FHL-1, FHR1, FHR3 and FHR5; FH and / or FHL-1, FHR1, FHR4 and FHR5; FH and / or FHL-1, FHR2, FHR3 and FHR4; FH and / or FHL-1, FHR2, FHR3 and FHR5; FH and / or FHL-1, FHR2, FHR4 and FHR5; FH and / or FHL-1, FHR3, FHR4 and FHR5; FHR1, FHR2, FHR3 and FHR4; FHR1, FHR2, FHR3 and FHR5;FHR1, FHR2, FHR4 and FHR5;FHR1, FHR3, FHR4 and FHR5;FHR2, FHR3, FHR4 and FHR5;FH and / or FHL-1, FHR1 and FHR2;FH and / or FHL-1, FHR1 and FHR3;FH and / or FHL-1, FHR1 and FHR4;FH and / or FHL-1, FHR1 and FHR5;FH and / or FHL-1, FHR2 and FHR3;FH and / or FHL-1, FHR2 and FHR4;FH and / or FHL-1, FHR2 and FHR5;FH and / or FHL-1, FHR3 and FHR4;FH and / or FHL-1, FHR3 and FHR5;FH and / or FHL-1, FHR4 and FHR5;FHR1, FHR2 and FHR3;FHR1, FHR2 and FHR4;FHR1, FHR2 and FHR5;FHR1, FHR3 and FHR4;FHR1, FHR3 and FHR5;FH and / or FHL-1 and FHR 1;FH and / or FHL-1 and FHR2;FH and / or FHL-1 and FHR3;FH and / or FHL-1 and FHR4;FH and / or FHL-1 and FHR5;FHR1 and FHR2;FHR1 and FHR3;FHR1 and FHR4;FHR1 and FHR5;FHR2 and FHR3;FHR2 and FHR4;FHR2 and FHR5;FHR3 and FHR4;FHR3 and FHR;or FHR4 and FHR5.;

[0239] Suitable combinations of agents, e.g., inhibitory nucleic acids, to be used to reduce gene and / or protein expression of the combinations of Factor H family proteins described in the preceding paragraphs will be readily apparent to one of skill in the art in view of the embodiments of the agents, e.g., inhibitory nucleic acids, disclosed herein.

[0240] In some embodiments, the inhibitory nucleic acids described herein are non-identical to the nucleic acids disclosed in WO 2019 / 215330 A1, the entire contents of which are incorporated herein by reference. In some embodiments, the nucleotide sequence of the inhibitory nucleic acids described herein is non-identical to the nucleotide sequence of the nucleic acids disclosed in WO 2019 / 215330 A1.

[0241] The present disclosure also provides a cell comprising or expressing (i) an agent described in this disclosure, (ii) a nucleic acid comprising or encoding an agent described in this disclosure, and / or (iii) a vector comprising a nucleic acid comprising or encoding an agent described in this disclosure.

[0242] The cell may be a eukaryotic cell, for example, a mammalian cell. The mammal may be a primate (rhesus monkey, cynomolgus monkey, non-human primate, or human) or a non-human mammal (e.g., rabbit, guinea pig, rat, mouse, or other rodent (including any animal in the order Rodentia), cat, dog, pig, sheep, goat, cow (cow, e.g., dairy cow, or any animal in the order Bos), horse (including any animal in the order Equidae), donkey, and non-human primate). In a preferred embodiment, the cell may be a human cell.

[0243] The present disclosure also provides a method for producing a cell containing a nucleic acid or vector described herein, the method comprising introducing a nucleic acid or vector described herein into a cell. In some embodiments, introducing a nucleic acid or vector described herein into a cell comprises transformation, transfection, electroporation, or transduction (e.g., retroviral transduction).

[0244] The present disclosure also provides methods for producing an agent described herein, e.g., an inhibitory nucleic acid, or a nucleic acid comprising or encoding an agent described herein, e.g., an inhibitory nucleic acid, comprising culturing a cell comprising a nucleic acid comprising or encoding an agent described herein, or a vector described herein, under conditions suitable for expression of the nucleic acid or vector by the cell. In some embodiments, the method is performed in vitro.

[0245] The present disclosure also provides agents and articles (inhibitory nucleic acids, gene editing tools / systems and nucleic acids for use therein, nucleic acids comprising / encoding such agents, expression vectors comprising / encoding such agents) described herein or cells described in this disclosure.

[0246] In therapeutic and prophylactic applications, the compositions of the present disclosure are preferably formulated as pharmaceuticals or pharmaceutical compositions (suitable for clinical use). Such compositions may contain agents or cells together with one or more other pharmaceutically acceptable components well known to those skilled in the art. Such components include, but are not limited to, pharmaceutically acceptable carriers, adjuvants, excipients, diluents, fillers, buffers, preservatives, antioxidants, lubricants, stabilizers, solubilizers, surfactants (e.g., wetting agents), masking agents, colorants, flavoring agents, and sweeteners.

[0247] The term "pharmaceutically acceptable," as used herein, refers to compounds, ingredients, substances, compositions, dosage forms, etc. that are within the scope of sound medical judgment and suitable for use in contact with the tissues of the subject (e.g., human) in question without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio. Each carrier, adjuvant, excipient, etc. must also be "acceptable" in the sense of being compatible with the other ingredients of the formulation. Suitable carriers, adjuvants, excipients, etc. can be found in standard pharmaceutical textbooks, e.g., Remington's Pharmaceutical Sciences, 20th ed., 2000, published by Lippincott, Williams & Wilkins; and Handbook of Pharmaceutical Excipients, 2nd ed., 1994.

[0248] The compositions of the present disclosure may be prepared by any method well known in the pharmaceutical industry. Such methods include the step of bringing into association the active compound with the carrier, which constitutes one or more accessory ingredients. Generally, the formulations are prepared by uniformly and intimately bringing into association the active compound with the carrier (e.g., liquid carrier, finely divided solid carrier, etc.), and then, if necessary, shaping the product.

[0249] The compositions may be prepared for topical, parenteral, systemic, intracavitary, intravenous, intraarterial, intramuscular, intrathecal, intraocular, intravitreal, intraconjunctival, subretinal, suprachoroidal, subcutaneous, transdermal, intrathecal, oral, nasal, or transdermal routes of administration, which may include injection or infusion, or eye drops (i.e., ophthalmic administration). Suitable formulations may include the selected agent in a sterile or isotonic vehicle. The formulation and mode of administration may be selected depending on the agent to be administered and the disease to be treated / prevented.

[0250] The compositions of the present disclosure may be formulated in liquid form, including gels. Liquid formulations may be formulated for administration by injection or infusion (e.g., via a catheter) into a selected organ or region of the human or animal body. A further aspect of the present disclosure is a method of formulating or producing a medicament or pharmaceutical composition according to the present disclosure, comprising the step of formulating the pharmaceutical composition or medicament by mixing the agent with a pharmaceutically acceptable carrier, adjuvant, excipient, or diluent.

[0251] The agents and articles (e.g., nucleic acids (including inhibitory nucleic acids, expression vectors), cells, and compositions) described herein may be formulated to facilitate delivery to and / or uptake by cells / tissues, such as target cells / tissues of interest. In some embodiments, the inhibitory nucleic acids use a delivery platform described in Hu et al., Sig. Transduc. Tar. Ther. (2020) 5(101) (incorporated herein above).

[0252] The agents and articles described herein may be linked to moieties to facilitate delivery to and / or uptake by cells / tissues. Strategies for facilitating intracellular delivery of molecular cargoes are outlined, for example, in Li et al., Int. J. Mol. Sci. (2015) 16: 19518-19536 and Fu et al., Bioconjug Chem. (2014) 25(9): 1602-1608, which are incorporated herein by reference in their entireties.

[0253] In some embodiments, the agents and articles (e.g., nucleic acids) may be formulated with cationic polymers. In some embodiments, the agents and articles (e.g., nucleic acids) may be encapsulated in nanoparticles or liposomes.

[0254] In some embodiments, the agents and articles described herein may be linked to or otherwise formulated with a moiety to facilitate delivery to and / or uptake by liver cells or tissue.

[0255] In some embodiments, the nucleic acid described herein may be conjugated to N-acetylgalactosamine (GalNAc). In some embodiments, the nucleic acid is conjugated to one or more (e.g., one, two, three, four or more) GalNAc residues. In some embodiments, the nucleic acid is conjugated to one or more (e.g., one, two, three, four or more) GalNAc moieties. In some embodiments, one or more GalNAc moieties may be covalently linked to the 5' or 3' end of the nucleic acid chain.

[0256] In some embodiments, the nucleic acids described herein are conjugated to alpha-tocopherol (i.e., vitamin E). In some embodiments, the nucleic acids are conjugated to one or more (e.g., one, two, three, four, or more) alpha-tocopherol moieties. In some embodiments, the one or more alpha-tocopherol moieties may be covalently linked to the 5' or 3' end of the nucleic acid strand.

[0257] In some embodiments, the nanoparticles are those described in Chen et al., Mol Ther Methods Clin Dev. (2016) 3:16023, which is incorporated herein by reference in its entirety. In some embodiments, the nanoparticles are PLGA, polypeptides, poly(β-amino esters), DOPE, β-cyclodextrin-containing polycations, linear PEI, PAMAM dendrimers, branched PEI, chitosan, or polyphosphoester nanoparticles.

[0258] In some embodiments, the article (e.g., nucleic acid) may be associated (covalently or non-covalently) with a cell-penetrating peptide (e.g., a protein transduction domain, a Trojan horse peptide, an arginine-rich peptide, a vectocell peptide), a cationic polymer, a cationic lipid, or a viral carrier. In some embodiments, the article (e.g., nucleic acid) may be associated with a peptide / polypeptide (e.g., an antibody, a peptide aptamer, a ligand / fragment of a cell surface molecule) or a nucleic acid (e.g., a nucleic acid aptamer) capable of binding to a target cell of interest or an antigen thereof. For example, a liquid-based drug delivery system may be used to administer pharmaceutical agents, such as those described herein, as described in Kalepu et al., Acta Pharmaceutica Sinica B, 2013, 3, 6, 361-372 and Barba et al., Pharmaceutics. 2019 Jul 24;11(8):360, the entire contents of which are incorporated herein by reference.

[0259] Methods for nucleic acid delivery are known in the art and can be found, for example, in Tatiparti K et al., "siRNA Delivery Strategies: A Comprehensive Review of Recent Developments," Thomas Nann. (ed.) Nanomaterials 7.4 (2017): 77, and Lehto T et al., Adv Drug Deliv Rev. 2016, 106(Pt A):172-182, which are incorporated herein by reference in their entireties. For example, nucleic acids can be delivered naked or by using nanoparticles, polymers, peptides, such as cell-penetrating peptides, or by ex vivo transfection. Nanoparticles can be organic, such as micelles, liposomes, proteins, solid lipid particles, solid polymer particles, dendrimers, and polymeric therapeutic agents. Nanoparticles can also be inorganic, such as nanotubes or metal particles, optionally loaded with organic molecules. Viruses offer another nanoparticle delivery option. Nanoparticles may be optimized to improve endocytosis rates, avoid renal clearance and filtration, improve thermal stability, improve pH stability, prevent toxic effects, and improve nucleic acid loading efficiency. Additional encapsulation methods are described, for example, in US2015 / 0157675 A1.

[0260] In some cases, nucleic acids, e.g., nanoparticle-based formulations thereof, may be formulated for pulmonary administration for subsequent delivery to non-pulmonary tissues. See, e.g., US2015 / 0157675 A1, which is incorporated herein by reference in its entirety.

[0261] Therapeutic and prophylactic applications The agents, inhibitory nucleic acids, gene editing tools / systems, nucleic acids, expression vectors and compositions described herein find use in therapeutic and prophylactic methods.

[0262] The present disclosure provides an agent, inhibitory nucleic acid, gene editing tool / system, nucleic acid, expression vector, or composition described herein for use as a pharmaceutical. The present disclosure provides an agent, inhibitory nucleic acid, gene editing tool / system, nucleic acid, expression vector, or composition described herein for use in a method of medical treatment or prevention. Also provided is the use of an agent, inhibitory nucleic acid, gene editing tool / system, nucleic acid, expression vector, or composition described herein in the manufacture of a medicament for treating or preventing a disease or condition. Also provided is a method of treating or preventing a disease or condition, comprising administering to a subject a therapeutically or prophylactically effective amount of an agent, inhibitory nucleic acid, gene editing tool / system, nucleic acid, expression vector, or composition described herein.

[0263] The terms "disorder," "disease," and "condition" may be used interchangeably and refer to a pathological problem with a body part, organ, or system that can be characterized by an identifiable group of signs or symptoms.

[0264] "Treatment" may refer to treating, preventing, or reducing the likelihood of a complement-related disorder, such as those described herein. As used herein, "treatment" may refer, for example, to a reduction in the development or progression of a disease / condition, a reduction in the symptoms of a disease / condition, or a reduction in the pathology of a disease / condition. Treating or alleviating a disease / condition may be effective in preventing the progression of the disease / condition, e.g., preventing the condition from worsening or slowing the rate of development. In some embodiments, treatment or alleviation may lead to an improvement of the disease / condition, e.g., a reduction in the symptoms of the disease / condition, or a reduction in some other correlate of the severity / activity of the disease / condition. Prevention / prophylaxis of a disease / condition may refer to preventing the condition from worsening or preventing the development of a disease / condition, e.g., preventing an early-stage disease / condition from progressing to a later, chronic stage.

[0265] Treating a complement-related disorder as described herein may include modifying at least one cell of a subject to express or contain a nucleic acid provided herein. Treating a complement-related disorder as described herein may include modifying at least one cell of a subject to express or contain a polypeptide provided herein, for example, through a nucleic acid provided herein. Treating a complement-related disorder as described herein may include administering to a subject a vector comprising or consisting of a nucleic acid as described herein.

[0266] The methods may be effective in reducing the development or progression of the disease / condition, alleviating the symptoms of the disease / condition, or reducing the pathology of the disease / condition. The methods may be effective in preventing the progression of the disease / condition, e.g., preventing the disease / condition from worsening or slowing its rate of development. In some embodiments, the methods may lead to an improvement in the disease / condition, e.g., a reduction in the symptoms of the disease / condition, or a reduction in some other correlate of the severity / activity of the disease / condition. In some embodiments, the methods may prevent the development of a later stage of the disease / condition (e.g., a chronic stage or metastasis).

[0267] As used herein, terms such as "develop," "developing," and "development" of a disorder refer to both the onset of the disease and the progression, worsening, or deterioration of the disease state. As used herein, the term "biomarker(s)" refers to one or more measurable indicators of a biological state or abnormality.

[0268] The articles of the present disclosure, e.g., inhibitory nucleic acids, may be used for the treatment / prevention of any disease / condition that would derive therapeutic or prophylactic benefit from reduced gene and / or protein levels of the complement protein(s) targeted by the inhibitory nucleic acid (i.e., the complement protein(s) for which the inhibitory nucleic acid is suitable to reduce / prevent gene and / or protein expression).

[0269] The disease / condition to be treated / prevented according to the present disclosure may be a disease / condition in which the relevant complement protein(s) is / are pathologically implicated, for example, a disease / condition in which increased gene and / or protein expression levels of the relevant complement protein(s) are positively associated with the onset, development or progression of the disease / condition and / or the severity of one or more symptoms of the disease / condition, or a disease / condition in which increased gene and / or protein expression levels of the relevant complement protein(s) are a risk factor for the onset, development or progression of the disease / condition.

[0270] In some embodiments, the disease / condition to be treated / prevented according to the present disclosure is a disease / condition characterized by an increase in the gene and / or protein expression levels of the relevant complement protein(s), e.g., when compared to the gene and / or protein expression levels of the relevant complement protein(s) in the absence of the disease / condition. Treatment according to the methods of the present disclosure may achieve a decrease in the gene and / or protein expression levels of the relevant complement protein(s) in the subject.

[0271] In aspects and embodiments of the present disclosure, the disease / condition to be treated is a complement-related disorder. As used herein, a "complement-related disorder" refers to a disorder, disease, or condition that involves or results from a deficiency or abnormality in the complement system. In some embodiments, a complement-related disorder is a disorder driven by complement activation or complement hyperactivation.

[0272] In any embodiment described herein, the disorder is one in which the complement system or its activation / hyperactivation / dysfunction is pathologically associated. The complement-associated disorder may be any disorder described herein. "Pathologically associated," as used herein, may refer to elevated or decreased protein levels in a disorder compared to a reference value, and / or when a protein contributes to the pathology of the disorder. The selection or combination of complement protein(s) to be detected / determined may depend on the complement-associated disorder of interest and the complement protein(s) useful as biomarkers for said disorder.

[0273] Complement-related disorders may involve disruption of the classical, alternative, and / or lectin complement pathways. In some cases, the disorder may be associated with a deficiency, abnormality, or absence of a regulatory component of the complement system. In some embodiments, the disorder may be associated with the alternative complement pathway, a disruption of the alternative complement pathway, and / or with a deficiency, abnormality, or absence of a regulatory component of the alternative complement pathway. In some cases, the disorder is associated with the complement amplification loop. In some cases, the disorder is associated with inappropriate activation, overactivation, or dysregulation of the complement system as a whole or parts thereof, such as C3 convertase assembly, C3b production, C3b deposition, and / or the amplification loop.

[0274] In some cases, the disorder is associated with any one or more of C3, C3b, iC3b, FI, FH, FHL-1, or FHR1-FHR5. In some cases, the disorder is associated with deficiency or abnormal activity of any one or more of C3, C3b, iC3b, FI, FH, FHL-1, or FHR1-FHR5. In some cases, one or more of these proteins are pathologically associated with the disorder, e.g., have elevated or lower levels compared to a reference value.

[0275] In some cases, the disorder is associated with one or more of CR1, CD46, CD55, C4BP, Factor B (FB), Factor D (FD), SPICE, VCP (or VICE), and / or MOPICE. In some cases, the disorder is associated with deficient or abnormal activity of one or more of CR1, CD46, CD55, C4BP, Factor B, Factor D, SPICE, VCP (or VICE), and / or MOPICE, or where one or more of these proteins is pathologically associated.

[0276] In some embodiments, the disorder may be a disorder associated with C3 or a C3-containing complex, an activity / reaction associated with C3 or a C3-containing complex, or a product of an activity / reaction associated with C3 or a C3-containing complex. That is, in some embodiments, the disorder is a disorder in which C3, a C3-containing complex, an activity / reaction associated with C3 or a C3-containing complex, or a product of said activity / reaction is pathologically implicated. In some embodiments, the disorder may be associated with an increased level of C3 or a C3-containing complex, an increased level of an activity / reaction associated with C3 or a C3-containing complex, or an increased level of a product of an activity / reaction associated with C3 or a C3-containing complex, compared to a control state. In some embodiments, the disorder may be associated with a decreased level of C3 or a C3-containing complex, an decreased level of an activity / reaction associated with C3 or a C3-containing complex, or a decreased level of a product of an activity / reaction associated with C3 or a C3-containing complex, compared to a control state.

[0277] In some embodiments, the disorder may be a disorder associated with C3b or C3b-containing complexes, an activity / reaction associated with C3b or C3b-containing complexes, or a product of an activity / reaction associated with C3b or C3b-containing complexes. That is, in some embodiments, the disorder is a disorder in which C3b, C3b-containing complexes, an activity / reaction associated with C3b or C3b-containing complexes, or a product of said activity / reaction is pathologically implicated. In some embodiments, the disorder may be associated with an increased level of C3b or C3b-containing complexes, an increased level of an activity / reaction associated with C3b or C3b-containing complexes, or an increased level of a product of an activity / reaction associated with C3b or C3b-containing complexes, compared to a control state. In some embodiments, the disorder may be associated with a decreased level of C3b or C3b-containing complexes, an decreased level of an activity / reaction associated with C3b or C3b-containing complexes, or a decreased level of a product of an activity / reaction associated with C3b or C3b-containing complexes, compared to a control state.

[0278] In some embodiments, the disorder may be a disorder associated with any one or more of FH, FHL-1, FI, FHR1-FHR5, FB, FD, CR1 and / or CD46, an activity / response associated with any one or more of FH, FHL-1, FI, FHR1-FHR5, FB, FD, CR1 and / or CD46, or a product of an activity / response associated with any one or more of FH, FHL-1, FI, FHR1-FHR5, FB, FD, CR1 and / or CD46. In some embodiments, the disorder is a disorder in which any one or more of FH, FHL-1, FI, FHR1-FHR5, FB, FD, CR1 and / or CD46, an activity / response associated with any one or more of FH, FHL-1, FI, FHR1-FHR5, FB, FD, CR1 and / or CD46, or a product of said activity / response is pathologically implicated. In some embodiments, the disorder may be associated with decreased levels of any one or more of FH, FHL-1, FI, FHR1-FHR5, FB, FD, CR1 and / or CD46, decreased levels of an activity / response associated with any one or more of FH, FHL-1, FI, FHR1-FHR5, FB, FD, CR1 and / or CD46, or decreased levels of a product of an activity / response associated with any one or more of FH, FHL-1, FI, FHR1-FHR5, FB, FD, CR1 and / or CD46, as compared to a control condition.

[0279] In some embodiments, the disorder may be associated with increased levels of any one or more of FHR1, FHR2, FHR3, FHR4, and / or FHR5, increased levels of an activity / response associated with any one or more of FHR1, FHR2, FHR3, FHR4, and / or FHR5, or increased levels of a product of an activity / response associated with any one or more of FHR1, FHR2, FHR3, FHR4, and / or FHR5, relative to a control condition. See, e.g., Zhu et al., Kidney Int. 2018 Jul;94(1):150-158; Pouw et al., Front Immunol. 2018 Apr 24;9:848, both of which are incorporated herein by reference in their entireties. The method may include determining systemic levels of any combination of FHR1-FHR5, e.g., using the techniques described herein.

[0280] Subjects with elevated levels of FHR1, FHR2, FHR3, FHR4 and / or FHR5, and optionally FH / FHL-1, and / or increased expression of the gene(s) encoding one or more of these proteins, may also benefit from therapeutic or preventive measures by reducing these levels.The expression level of Factor H family proteins may be measured using techniques described herein and / or in the art, for example, as outlined in Roth CM, Curr. Issues Mol. Biol. 2002 4:93-100 and Kukurba KR and Montgomery SB, Cold Spring Harb Protoc. 2015, (11):951-969, which are incorporated herein by reference in their entirety.For example, quantitative PCR, real-time PCR, sequencing techniques such as RNA-seq, next-generation sequencing, microarray, Northern blot, and ribonuclease protection assay (RPA) may be used to measure gene expression. Those skilled in the art will be aware of suitable techniques or techniques for measuring the expression of CFHR1-5, as needed. In some cases, total RNA or cDNA may first be extracted and isolated from a cell sample.

[0281] In certain embodiments, the disorder is characterized by elevated levels of any one or more FH family proteins, such as FHR1, FHR2, FHR3, FHR4, and / or FHR5. The elevated levels may be in a subject. That is, the subject to be evaluated or treated may have (or be determined / already determined to have) elevated levels of one or more of FHR1, FHR2, FHR3, FHR4, and / or FHR5, as assessed, for example, by the methods provided herein. The disorder may be characterized by elevated circulating levels of one or more of FHR1, FHR2, FHR3, FHR4, and / or FHR5 in a blood- or plasma-derived sample, as described herein. The disorder may be characterized by elevated expression of one or more of FHR1, FHR2, FHR3, FHR4, and / or FHR5 by hepatocytes. The disorder may be characterized by elevated levels of one or more of FHR1, FHR2, FHR3, FHR4, and / or FHR5 detected in a tissue of interest, such as the eye, kidney, brain, lung, tumor, vascular tissue, etc. Elevated levels may be determined by comparison to a control value(s) / subject(s) as described herein.

[0282] Not all subjects with a complement-related disorder may have elevated levels of one or more FHR proteins. Thus, some subjects with a complement-related disorder may have elevated levels of one or more FHR proteins, and some subjects with the same complement-related disorder may not have elevated levels. In some cases, for example, as described herein, the presence of elevated levels of one or more FHR proteins may indicate a worse prognosis. Thus, determining the levels of one or more FHR proteins may provide distinct populations of patients who would particularly benefit from treatment with the nucleic acids and proteins described herein, for example, as compared to patients with normal levels of FHR proteins.

[0283] A complement-associated disorder may be characterized, for example, by altered levels of FH and / or FHL-1, either elevated or decreased, in addition to elevated levels of one or more FHR proteins.

[0284] In some embodiments, the disorder may be associated with increased levels of any one or more of FHR1, FHR2, FHR3, and / or FHR5, increased levels of activities / responses associated with any one or more of FHR1, FHR2, FHR3, and / or FHR5, or increased levels of products of activities / responses associated with any one or more of FHR1, FHR2, FHR3, and / or FHR5. In some embodiments, the disorder may be associated with increased levels of FHR4, increased levels of activities / responses associated with FHR4, or increased levels of products of activities / responses associated with FHR4, compared to a control condition. See, e.g., WO 2019 / 215330 and Cipriani et al., Nat Commun 11, 778 (2020), both of which are incorporated herein by reference in their entireties. In some embodiments, the disorder may be associated with increased levels of FHL-1. In some embodiments, the disorder may not be associated with increased levels of FHR4, increased levels of an activity / reaction associated with FHR4, or increased levels of a product of an activity / reaction associated with FHR4, compared to a control condition.

[0285] In some embodiments, the disorder is associated with increased levels of any one or more of C3, C3b, C3 convertase, and / or C3bBb compared to a control state. In some embodiments, the disorder is associated with decreased levels of any one or more of C3, C3b, C3 convertase, and / or C3bBb compared to a control state. In some embodiments, the disorder is associated with increased levels of iC3b compared to a control state. In some embodiments, the disorder is associated with decreased levels of iC3b compared to a control state. In some embodiments, the disorder is associated with increased levels of any one or more of C3a, C3f, C3c, C3dg, C3d, and / or C3g compared to a control state. In some embodiments, the disorder is associated with decreased levels of any one or more of C3a, C3f, C3c, C3dg, C3d, and / or C3g compared to a control state.

[0286] In some cases, the methods described herein find use in treating or preventing a disorder that would benefit from one or more of: a decrease in the level or activity of one or more of C3bBb-type C3 convertase, C3bBb3b-type C5 convertase, and / or C4b2a3b-type C5 convertase, as compared to a reference value(s); a decrease in the level of one or more of C3, C3b, C3a, iC3b, FHR1, FHR2, FHR3, FHR4, FHR5, C5b, and / or C5a; or an increase in the level of one or more of iC3b, C3f, C3c, C3dg, C3d, C3g, FH, FHL-1, FI, FH, FHL-1, FHR1, FHR2, FHR3, FHR4, and / or FHR5.

[0287] In some cases, the methods described herein find use in the treatment or prevention of disorders that would benefit from a decrease in the level or activity of one or more of FHR1, FHR2, FHR3, FHR4 and / or FHR5, and optionally FHL-1.

[0288] The disorder may be an ocular disorder. In some embodiments, the disease or condition to be treated or prevented as described herein is a complement-related ocular disease. In some embodiments, the disorder is macular degeneration. In some embodiments, the disorder may be selected from age-related macular degeneration (AMD), choroidal neovascularization (CNV), macular dystrophy, and diabetic maculopathy, i.e., one or more of these. As used herein, the term "AMD" includes early AMD, intermediate AMD, late / advanced AMD, geographic atrophy ("dry" (i.e., non-exudative) AMD), and "wet" (i.e., exudative or neovascular) AMD, each of which may be detected, treated, and / or prevented as described herein. In some embodiments, the disease or condition to be treated or prevented is a combination of the above diseases / conditions, such as "dry" and "wet" AMD. In some embodiments, the disease or condition to be treated or prevented is not "wet" AMD or choroidal neovascularization. AMD is generally defined as causing vision loss in subjects aged 50 and older. In some embodiments, the subject to be treated is 50 years old or older, i.e., at least 50 years old.

[0289] As used herein, "early AMD" refers to a stage of AMD characterized by the presence of medium-sized drusen, generally up to 200 μm in diameter, in Bruch's membrane adjacent to the RPE layer. Subjects with early AMD typically do not experience significant vision loss. As used herein, "intermediate AMD" refers to a stage of AMD characterized by large drusen and / or pigmentary changes in the retina. Intermediate AMD may be accompanied by some degree of vision loss. As used herein, "late AMD" refers to a stage of AMD characterized by the presence of drusen and vision loss, e.g., severe central vision loss, due to damage to the macula. In all stages of AMD, "retinal pseudodrusen" (RPD) or "retinal drusen" (which may also be referred to as subretinal drusen-like deposits (SSD)) may also be present, which refers to the accumulation of extracellular material in the subretinal space between the neurosensory retina and the RPE. "Late AMD" includes "dry" and "wet" AMD. In "dry" AMD (also known as geographic atrophy), there is a gradual destruction of the light-sensitive cells in the macula that carry visual information to the brain and the supporting tissues below the macula. In "wet" AMD (also known as choroidal neovascularization, neovascularization, and exudative AMD), abnormal blood vessels grow under and into the retina. These vessels can leak fluid and blood, which can lead to swelling and damage to the macula and subsequent scarring. Damage can be rapid and severe.

[0290] In some embodiments, the disorder is early-onset macular degeneration (EOMD). As used herein, "EOMD" refers to a phenotypically severe subtype of macular degeneration, which shows onset at a much younger age than classic AMD and causes longer-term substantial vision loss. Affected individuals may exhibit an early-onset drusen phenotype, which includes uniform, small, slightly elevated, yellow subretinal nodules randomly distributed in the macula, also known as "basal drusen" or "corneal drusen." EOMD may also be referred to as "intermediate-onset macular degeneration." EOMD subsets are described, for example, in Boon CJ et al. Am J Hum Genet 2008; 82(2):516-23; van de Ven JP et al. Arch Ophthalmol 2012; 130(8):1038-47; and Taylor, RL et al., Ophthalmol. 2019, 126, 1410-1421, all of which are incorporated herein by reference in their entirety. Like other types of macular degeneration, EOMD is associated with impaired complement regulation and disrupted factor H activity. In some embodiments, the subject to be treated is 49 years old or younger. In some embodiments, the subject to be treated is between 15 and 49 years old, i.e., 15 to 49 years old. In some embodiments, the disease or condition to be treated is macular dystrophy. Macular dystrophy is a genetic condition, usually caused by a mutation in a single gene, resulting in degeneration of the macula.

[0291] In some embodiments, the disorder is kidney-related, such as nephropathy / nephropathic disorder. In some cases, the disorder is neurological and / or neurodegenerative disorder. In some cases, the disorder is autoimmune-related, such as autoimmune disease. In some cases, the disorder is inflammation-related, such as inflammatory disease. In some cases, the disorder is characterized by C3 deposition, such as glomerular disease (e.g., Skerka et al., 2013, supra).

[0292] Many FHR proteins have been implicated in complement-related kidney disorders. FH, FHL-1, FHR1, FHR2, FHR3, and FHR5 have been implicated in IgA nephropathy (see, e.g., Poppelaars et al., J Clin Med. 2021, 10(20):4715; Zhu et al., Kidney Int. 2018 Jul, 94(1):150-158). Poppelaars et al. suggest that FHR1 and FHR5 compete with the regulatory function of factor H, such that FHR proteins amplify alternative pathway activation and thereby stimulate the development and progression of IgA nephropathy.

[0293] FHR5 has been implicated in C3 glomerulopathy and renal injury (see, e.g., Medjeral-Thomas et al., Kidney Int Rep. 2019, 4(10):1387-1400), as well as glomerular injury and renal damage (see, e.g., Malik et al., PNAS, 2021, 118(13) e2022722118). Abnormal FHR hybrid proteins have also been reported in C3 glomerulopathy and are thought to compete with FH for C3b binding and regulation (see, e.g., Wong & Kavanagh, Semin Immunopathol. 2018; 40(1):49-64). Wong & Kavanagh also discuss the involvement of FH, FHR1, and FHR3 in atypical hemolytic uremic syndrome (aHUS) and paroxysmal nocturnal hemoglobinuria (PNH). FHR1 and FHR5 have also been detected in glomeruli from patients with dense deposit disease (DDD) / membranoproliferative glomerulonephritis type II. See, e.g., Sethi et al., Kidney Int. 2009, 75(9):952-60, and Abrera-Abeleda et al., J Med Genet. 2006, 43(7):582-589.

[0294] Schafer et al., Front Immunol. 2016, 7:542, describes the role of elevated FHR3 in a selection of autoimmune diseases, including renal disease and rheumatoid arthritis patients. Goicoechea de Jorge et al., PNAS 2013, 110(12):4685-90, discloses that CFH mutations increase susceptibility to aHUS, DDD, and meningococcal sepsis, and that FHR3 is involved in the pathogenesis of systemic lupus erythematosus (SLE). They also report that dimerization of FHR proteins, such as FHR1, FHR2, and FHR5, competes with FH for C3b binding and enhances its ability to deregulate complement activation. Legatowicz-Koprowska et al., Reumatologia. 2020, 58(6): 357-366, report the absence of complement cascade proteins in patients with primary Sjögren's syndrome. Increased levels of FHR proteins have been associated with antineutrophil cytoplasmic autoantibody (ANCA) vasculitis (Skerka et al., Br J Pharmacol. 2021 Jul;178(14):2823-2831).

[0295] Activation of the alternative complement pathway and upregulation of FHR proteins have been suggested to play a role in the development of atherosclerosis (see, e.g., Speidel et al., J Thromb Haemost. 2011, 9(3):428-40; Malik et al., Circulation. 2010, 122(19):1948-56; Machalinska et al., Acta Ophthalmol. 2012, 90(8):695-703; and Irmscher et al., Nature Scientific Reports, volume 11, Article number: 22511 (2021)).

[0296] In some embodiments, the disorder is hemolytic uremic syndrome (HUS), atypical hemolytic uremic syndrome (aHUS), DEAP HUS (FHR plasma protein deficiency and autoantibody positive form of hemolytic uremic syndrome), autoimmune uveitis, membranoproliferative glomerulonephritis type II (MPGN II), sepsis, Henoch-Schönlein purpura (HSP), IgA nephropathy, chronic kidney disease, paroxysmal nocturnal hemoglobinuria (PNH), autoimmune hemolytic anemia (AIHA), systemic lupus erythematosus (SLE), Sjogren's syndrome (SS), rheumatoid arthritis (RA), C3 glomerulopathy (C3G), dense deposit disease (DDD), C3 nephritic factor glomerulonephritis (C3NF), GN), FHR5 nephropathy, hereditary angioedema (HAE), acquired angioedema (AAE), encephalomyelitis, atherosclerosis, multiple sclerosis (MS), stroke, Parkinson's disease, and Alzheimer's disease.

[0297] In some cases, the disorder is cancer. Complement activation plays a role in the development and progression of cancer. DeCordova et al., Immunobiology. 2019, 224(5):625-631, reported that FHR5 is secreted by primary tumor cells from glioblastoma multiforme (GBM) patients and can be used by the cells to resist complement-mediated lysis. Afshar-Kharghan, J Clin Invest. 2017, 127(3):780-789, reported that expression of complement factors, including FHR proteins, increases in malignant tumors, potentially outcompeting FH and leading to complement dysfunction. Alternatively, when tumors become hypoxic, this may then lead to downregulation of FH expression and thus increased complement inflammatory activity; therefore, inhibiting complement activation is a therapeutic option (e.g., Pio et al., Semin Immunol. 2013, 25(1):54-64). FH has been reported as a biomarker for lung cancer, squamous cell lung cancer, bladder cancer, ovarian cancer, liver cancer, and SCC (e.g., Revel et al., Antibodies (Basel). 2020, 9(4): 57).

[0298] The cancer may be a liquid or blood cancer, such as leukemia, lymphoma, or myeloma. In other cases, the cancer is a solid cancer, such as breast cancer, lung cancer, liver cancer, colorectal cancer, nasopharyngeal cancer, renal cancer, or glioma. In some cases, the cancer is located in the liver, bone marrow, lung, spleen, brain, pancreas, stomach, or intestine. In some cases, the cancer is lung cancer. In some cases, the cancer is glioblastoma, such as glioblastoma multiforme (GBM). In some embodiments, the complement-related disorder is indoleamine 2,3-dioxygenase 1 (IDO)-expressing cancer, such as those described in WO2022 / 058447.

[0299] In some cases, the disorder is neurodegeneration or a neurodegenerative disease. The disorder may involve progressive atrophy and loss of neuronal function. The disorder may be selected from Parkinson's disease, Alzheimer's disease, dementia, stroke, Lewy body disease, amyotrophic lateral sclerosis (ALS), multiple sclerosis (MS), Huntington's disease, and prion disease.

[0300] The role of complement in various diseases is discussed in, for example, Morgan, B.P., Complement in the pathogenesis of Alzheimer's disease. Semin Immunopathol, 2018. 40(1): pp. 113-124; Halbgebauer, R. et al., Janus face of complement-driven neutrophil activation during sepsis. Semin Immunol, 2018. 37: pp. 12-20; Ma, Y. et al., Significance of the Complement System in Ischemic Stroke: A Comprehensive Review. Aging Dis, 2019. 10(2): pp. 429-462; Bonifati and Kishore, Role of complement in neurodegeneration and neuroinflammation. Mol Immunol. 2007 Feb;44(5):999-1010; Kleczko, E.K. et al., Targeting the Complement Pathway as a Therapeutic Strategy in Lung Cancer. Front Immunol, 2019. 10: p. 954; and Schafer N. et al., Complement Regulator FHR-3 Is Elevated Either Locally or Systemically in a Selection of Autoimmune Diseases, Front Immunol. 2016; 7: 542. For example, FHL-1 is more highly expressed than FH in certain tumor cell lines (Junnikkala et al. (2000) J. Immunol. 164: 6075-81), and glioblastoma tumors have been shown to express FHR protein (DeCordova et al. (2019) Immunobiology 224: 625-631), both of which are incorporated herein by reference in their entireties.It would be advantageous if it were possible to measure and distinguish between FH family proteins.

[0301] Elevated levels of FHR1 and FHR3 have been found in plasma from Alzheimer's disease patients. See, e.g., Chen & Xia, J Alzheimers Dis. 2020, 76(1): 349-368; and Ashton et al., Alzheimers Dement (Amst). 2015, 1(1): 48-60 (also see Clark and Bishop J Clin Med. 2015 Jan; 4(1): 18-31). Thus, elevated levels of FHR proteins are associated with dementia-related disorders. Increased levels of FHR proteins (FHR1, 2, and 5) are associated with multiple sclerosis. See, e.g., Loveless et al., Brain Pathol. 2018 Jul; 28(4): 507-520. Pouw and Ricklin, Semin Immunopathol. 2021, 43(6):757-771, discuss the role of FHR proteins as FH competitors and review the adverse effects of complement activation in the central nervous system, such as in the context of Alzheimer's disease, Parkinson's disease, schizophrenia, myasthenia gravis (MG), amyotrophic lateral sclerosis (ALS), and Guillain-Barré syndrome (GBS).

[0302] Increased circulating FH levels in subjects with altered glucose tolerance have been associated with insulin resistance and metabolic disorders (Moreno-Navarrete et al., Diabetes. 2010, 59(1):200-9).

[0303] In some embodiments, the complement-related disorder is an infectious disease. Complement is a key component of the innate immune system, which is involved in defense against foreign pathogens, including bacteria, viruses, fungi, and parasites. Complement activation leads to a robust and efficient proteolytic cascade, which leads to the opsonization and lysis of pathogens and the generation of classical immune responses through the production of potent pro-inflammatory molecules. The role of complement in innate and adaptive immune responses is reviewed, for example, in Dunkelberger, J., Song, WC. Cell Res 2010; 20, 34-50, and Rus H et al., Immunol Res. 2005; 33(2):103-12, which are incorporated herein by reference in their entirety.

[0304] In some embodiments, the complement-related disorder is infection with severe acute respiratory syndrome-associated coronavirus (SARSr-CoV). In some embodiments, the complement-related disorder is infection with SARS-CoV-2. In some embodiments, the complement-related disorder is a disease / condition caused or exacerbated by SARS-CoV-2 infection, such as COVID-19 or another disease / condition in which SARS-CoV-2 infection is a contributing factor. WO2022 / 058447 describes significantly elevated levels of FHR1, FHR2, FHR3, FHR4, FHR5, and FHL-1 in the blood of COVID-19 patients with severe disease compared to healthy control subjects.

[0305] All references in the above paragraphs are incorporated herein in their entirety. Thus, in some embodiments, a complement-associated disorder (e.g., characterized by elevated levels of one or more of FHR1, FHR2, FHR3, FHR4, and / or FHR5) is a condition characterized by macular degeneration, age-related macular degeneration (AMD), geographic atrophy ("dry" (i.e., non-exudative) AMD), early AMD, early-onset macular degeneration (EOMD), intermediate AMD, late / advanced AMD, "wet" (neovascular or exudative) AMD, choroidal neovascularization (CNV), retinal dystrophies, hemolytic uremic syndrome (HUS), atypical hemolytic uremic syndrome (aHUS), DEAP HUS (FHR plasma protein deficiency and autoantibody positive form of hemolytic uremic syndrome), autoimmune uveitis, kidney injury / damage / dysfunction, glomerular disease, membranoproliferative glomerulonephritis type II (MPGN), or other conditions characterized by elevated levels of FHR1, FHR2, FHR3, FHR4, and / or FHR5. II), sepsis, Henoch-Schönlein purpura (HSP), IgA nephropathy, chronic kidney disease, paroxysmal nocturnal hemoglobinuria (PNH), autoimmune hemolytic anemia (AIHA), systemic lupus erythematosus (SLE), Sjögren's syndrome (SS), rheumatoid arthritis (RA), C3 glomerulopathy (C3G), dense deposit disease (DDD), C3 nephritic factor glomerulonephritis (C3 NF) GN), FHR5 nephropathy, hereditary angioedema (HAE), acquired angioedema (AAE), encephalomyelitis, atherosclerosis, antineutrophil cytoplasmic autoantibody (ANCA) vasculitis, neurodegenerative / neurodegenerative disease, dementia, multiple sclerosis (MS), Lewy body disease, amyotrophic lateral sclerosis (ALS), Huntington's disease, prion disease, cancer, lung cancer, glioblastoma, e.g., glioblastoma multiforme (GBM), stroke, insulin resistance, diabetes, infectious disease, Parkinson's disease, and / or Alzheimer's disease.

[0306] Administration of the articles of the present disclosure is preferably in a "therapeutically effective" or "prophylactically effective" amount, that is, an amount sufficient to provide a therapeutic or prophylactic benefit to a subject. The actual amount administered, as well as the rate and time course of administration, will depend on the nature and severity of the disease / condition and the particular article being administered. Prescribing treatment, e.g., determining dosage, etc., is within the responsibility of general practitioners and other physicians and typically takes into account the disease / disorder to be treated, the condition of the individual subject, the site of delivery, the method of administration, and other factors known to physicians. Examples of the techniques and protocols mentioned above can be found in Remington's Pharmaceutical Sciences, 20th Edition, 2000, published by Lippincott, Williams & Wilkins.

[0307] Administration of the articles of the present disclosure may be topical, parenteral, systemic, intracavitary, intravenous, intraarterial, intramuscular, intrathecal, intraocular, intravitreal, intraconjunctival, subretinal, suprachoroidal, subcutaneous, intradermal, intrathecal, oral, nasal, or transdermal, or as eye drops (i.e., ophthalmic administration).

[0308] In some aspects and embodiments according to the present disclosure, the agent, inhibitory nucleic acid, gene editing tool / system, nucleic acid, expression vector, or composition described herein is administered to the liver, for example, to one or more hepatocytes.In some cases, the agent, inhibitory nucleic acid, gene editing tool / system, nucleic acid, expression vector, or composition described herein is administered to the blood (i.e., intravenous / intra-arterial administration).In some cases, the agent, inhibitory nucleic acid, gene editing tool / system, nucleic acid, expression vector, or composition described herein is administered subcutaneously.

[0309] Some aspects and embodiments according to the present disclosure may include targeted delivery of the articles of the present disclosure, i.e., the concentration of the related agent in a subject is increased in one part of the body compared to other parts of the body, and / or the related agent is delivered via a modified release technology. Exemplary methods for nucleic acid delivery are described herein above. In some embodiments, the method includes intravenous, intraarterial, intramuscular, or subcutaneous administration, and the related agent is formulated in a targeted agent delivery system. Suitable targeted delivery systems include, for example, nanoparticles, liposomes, micelles, beads, polymers, metal particles, dendrimers, antibodies, aptamers, nanotubes, or micro-sized silica rods. Such systems may also include magnetic elements to direct the agent to a desired organ or tissue. Suitable nanocarriers and delivery systems will be apparent to those skilled in the art. In some cases, the related agent is formulated for targeted delivery to a specific organ(s) or tissue(s). In some cases, the related agent is delivered to the liver. In some cases, the method involves intravenous, intraarterial, intramuscular, or subcutaneous administration, and the associated agent is formulated for targeted delivery to the liver.

[0310] The particular mode and / or site of administration may be selected according to the location where gene and / or protein expression of a Factor H family protein (e.g., FHR protein) needs to be reduced. In some cases, the method includes intravenous and / or intra-arterial administration. In some cases, the method includes administration to the eye. If reduced expression of a gene encoding a Factor H family protein (e.g., FHR protein) is needed, administration may be to the liver. In some cases, the related agent is delivered to one or more hepatocytes.

[0311] The administration of the agent or article of the present disclosure may be alone or in combination with other treatments, either simultaneously or sequentially, depending on the condition to be treated. Simultaneous administration refers to administration together with another therapeutic agent, for example, as a pharmaceutical composition (combined preparation) containing both agents, or immediately after each other, and optionally via the same administration route, for example, into the same tissue, artery, vein, or other blood vessel. Sequential administration refers to the separate administration of one agent followed by another agent after a predetermined time interval. It is not necessary for the two agents to be administered by the same route, although in some embodiments they are administered by the same route. The time interval may be any time interval.

[0312] In some embodiments, the therapeutic or prophylactic intervention described herein may further comprise the administration of another agent for the treatment / prevention of a complement-associated disorder. Exemplary agents for the treatment / prevention of complement-associated disorders for use in such embodiments include C1 inhibitors, C5 inhibitors, C5a inhibitors, C5aR antagonists, C3 inhibitors, C3a inhibitors, C3b inhibitors, C3aR antagonists, classical pathway inhibitors, alternative pathway inhibitors, FH replacement therapy, and / or MBL pathway inhibitors. Specific complement-targeting therapies include, without limitation, human C1 elastase inhibitor (C1-INH), eculizumab (Soliris®, Alexion; a humanized monoclonal IgG2 / 4 antibody targeting C5), APL-2 (Apellis), Mubodina (Adienne Pharma and Biotech), Elzidina (Adienne Pharma and Biotech), POT-4 (a cyclic peptide inhibitor of C3;Alcon), rituximab (Biogen Idec, Genentech / Roche), ofatumumab (Genmab, GSK), compastatin analogues, soluble and targeted forms of CD59, PMX53 and PMX205 (Cephalon / Teva), JPE-1375 (Jerini), CCX168 (ChemoCentryx), NGD-2000-1 (formerly Neurogen), Cinryze (Shire), Berinert (CSL Behring), Cetor (Sanquin), Ruconest / Conestat alfa (Pharming), TNT009 (True North), OMS721 (Omeros), CLG561 (Novartis), AMY-101 (Amyndas), APL-1 (Apellis), APL-2 (Apellis), Mirococept (MRC), Lampalizumab (FCD4514) S, Genentech / Roche), ACH-4471 (Achillion), ALXN1210 (Alexion), Tesidolumab / LFG316 (Novartis / Morphosys), Coversin (Akari), RA101495 (Ra Pharma), Zimura (ARC1905, Ophthotech), ALN-CC5 (Alnylam), IFX-1 (InflaRx), ALXN1007 (Alexion), Avacopan / CCX168 (Chemocentryx), and / or, for example, Ricklin and Lambris, Adv Exp Med Biol. 2013, 734: 1-22; Ricklin and Lambris, Semin Immunol. 2016, 28(3):208-22; Melis JPM et al., Mol Immunol. 2015 67(2):117-130; Thurman JM, Nephrol Dial Transplant, 2017 32: i57-i64; Cashman SM et al., PLoS One. 2011, 6(4):e19078; all of which are incorporated herein by reference in their entirety. Bora NS et al., J Biol Chem. 2010, 285(44):33826-33;and one or more of the therapeutic agents described in Clark et al., J Clin Med 2015, 4(1):18-31, WO 2018 / 224663 and WO 2019 / 138137.

[0313] Other therapeutic agents or techniques suitable for use in connection with the present disclosure may include nutritional therapy, photodynamic therapy (PDT), laser photocoagulation, anti-VEGF (vascular endothelial growth factor) therapy, and / or additional therapies known in the art. See, e.g., Al-Zamil WM and Yassin SA, Clin Interv Aging. 2017 Aug 22;12:1313-1330. Anti-VEGF therapy may include agents such as ranibizumab (Lucentis, manufactured by Genentech / Novartis), Avastin (Genentech), bevacizumab (off-label Avastin), and aflibercept (Eylea® / VEGF Trap-Eye from Regeneron / Bayer). Additional agents or techniques suitable for use in the present disclosure include APL-2 (Apellis), AdPEDF (GenVec), encapsulated cell technology (ECT; Neurotech), squalamine lactate (EVIZON TM , Genaera), OT-551 (antioxidant eye drops, Othera), anecortave acetate (Retaane®, Alcon), bevasiranib (siRNA, Acuity Pharmaceuticals), pegaptanib sodium (Macugen®), and AAVCAGsCD59 (clinical trial identifier: NCT03144999).

[0314] In some embodiments, the method includes further therapy or prophylactic intervention, for example, for the treatment / prevention of cancer. In some embodiments, the therapy or prophylactic intervention is selected from chemotherapy, immunotherapy, radiation therapy, surgery, vaccination, and / or hormonal therapy. In some embodiments, the therapy or prophylactic intervention includes leukapheresis. In some embodiments, the therapy or prophylactic intervention includes stem cell transplantation.

[0315] Chemotherapy and radiotherapy refer to the treatment of cancer with drugs or ionizing radiation (e.g., radiotherapy using X-rays or gamma rays), respectively. Drugs can be chemical entities, such as small molecule drugs, antibiotics, DNA intercalators, protein inhibitors (e.g., kinase inhibitors), or biological agents, such as antibodies, antibody fragments, aptamers, nucleic acids (e.g., DNA, RNA), peptides, polypeptides, or proteins. Drugs can be formulated as pharmaceutical compositions or medicaments. Formulations can include one or more drugs (e.g., one or more active agents) together with one or more pharmaceutically acceptable diluents, excipients, or carriers.

[0316] Multiple doses of the articles of the present disclosure may be provided, and one or more, or each, of the doses may be accompanied by the simultaneous or sequential administration of another therapeutic agent. The multiple doses may be separated by a predetermined time interval, which may be selected to be one of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or 31 days, or 1, 2, 3, 4, 5, or 6 months. For example, doses may be administered once every 7, 14, 21, or 28 days (plus or minus 3, 2, or 1 day).

[0317] The articles of the present disclosure may be formulated in sustained release delivery systems to release the agent(s), inhibitory nucleic acid(s), nucleic acid(s), expression vector(s), and composition(s) at a predetermined rate. The sustained release delivery systems may maintain a constant drug / therapeutic agent concentration for a specified period of time. In some embodiments, the nucleic acid(s), nucleic acid(s), expression vector(s), and composition(s) described herein are formulated in liposomes, gels, implants, devices, or drug-polymer conjugates, such as hydrogels.

[0318] Choices regarding therapeutic or preventive interventions In some aspects, the present disclosure provides methods that include selecting a treatment for and / or treating a subject / patient having or identified as having a complement-associated disorder by determining levels of complement proteins, e.g., as described herein.

[0319] The methods described herein may be diagnostic, prognostic, and / or predictive of the risk of onset or progression of a complement-related disorder. Diagnostic methods may be used to determine disease diagnosis or severity, prognostic methods aid in predicting the likely course of disease in a defined clinical population under standard treatment conditions, and predictive methods predict the likely response to treatment in terms of efficacy and / or safety, thus aiding clinical decision-making. Such methods may be used prior to administration of an agent described herein, such as an inhibitory nucleic acid or gene-editing tool / system. Subjects with elevated levels of FHR1, FHR2, FHR3, FHR4, and / or FHR5 may also derive therapeutic or prophylactic benefit from reducing the activity levels of any one or more of the aforementioned proteins, for example, using any of the agents described herein.

[0320] In some aspects, the subject: (a) determining in a blood sample obtained from the subject the level of complement proteins selected from one or more of FHR1, FHR2, FHR3, FHR4 and / or FHR5, and optionally FH and / or FHL-1; (b) If the level of one or more of FHR1, FHR2, FHR3, FHR4 and / or FHR5, and optionally FHL-1, as determined in (a), is elevated when compared to the level of that complement protein(s) in the blood in a control subject who does not have a complement-related disorder, then the subject is selected for treatment with an agent described herein, e.g., an inhibitory nucleic acid, gene editing tool / system, nucleic acid, expression vector, or composition. The method may select for therapeutic or prophylactic intervention as described in this disclosure by methods comprising the steps.

[0321] The subject may then be treated with an agent as described herein. It also offers: (a) determining in a blood sample obtained from the subject the level of complement proteins selected from one or more of FHR1, FHR2, FHR3, FHR4 and / or FHR5, and optionally FH and / or FHL-1; (b) determining whether the levels of FHR1, FHR2, FHR3, FHR4 and / or FHR5, and optionally FHL-1, are elevated when compared to the levels of that complement protein(s) in the blood in control subjects who do not have a complement-related disorder; and (c) administering to the subject an agent described herein, e.g., an inhibitory nucleic acid, a gene editing tool / system, a nucleic acid, an expression vector, or a composition. The present invention relates to a method for treating / preventing a complement-associated disorder, comprising the steps of:

[0322] Also provided is an agent, gene editing tool / system, inhibitory nucleic acid, nucleic acid, expression vector or composition described herein for use as a medicament, for example, the agent is a compound used in a method of treating / preventing a complement-associated disorder, comprising: (a) determining in a blood sample obtained from the subject the level of complement proteins selected from one or more of FHR1, FHR2, FHR3, FHR4 and / or FHR5, and optionally FH and / or FHL-1; (b) determining whether the levels of FHR1, FHR2, FHR3, FHR4 and / or FHR5, and optionally FHL-1, are elevated when compared to the levels of those complement proteins in the blood in control subjects who do not have a complement-related disorder; and (c) administering to the subject an agent, gene editing tool / system, inhibitory nucleic acid, nucleic acid, expression vector, or composition described herein. The method may be provided for use in the method, comprising the steps of:

[0323] Also provided is the use of an agent, gene editing tool / system, inhibitory nucleic acid, nucleic acid, expression vector or composition described herein in the manufacture of a medicament for treating / preventing a complement-associated disorder, comprising: (a) determining in a blood sample obtained from the subject the level of complement proteins selected from one or more of FHR1, FHR2, FHR3, FHR4 and / or FHR5, and optionally FH and / or FHL-1; (b) determining whether the level of one or more of FHR1, FHR2, FHR3, FHR4 and / or FHR5, and optionally FHL-1, is elevated when compared to the level of that complement protein in the blood in a control subject who does not have a complement-related disorder; and (c) administering to the subject an agent, gene editing tool / system, inhibitory nucleic acid, nucleic acid, expression vector, or composition described herein. The use includes the steps of:

[0324] Any of the methods described herein may comprise: (a) determining in a blood sample obtained from the subject the level of complement proteins selected from one or more of FHR1, FHR2, FHR3, FHR4 and / or FHR5, and optionally FH and / or FHL-1; (b) determining whether the level of a complement protein (e.g., one or more of FHR1, FHR2, FHR3, FHR4, and / or FHR5, and optionally FHL-1) is elevated when compared to the level of the complement protein(s) in the blood in a control subject without a complement-related disorder; and / or (c) administering to the subject an agent, gene editing tool / system, inhibitory nucleic acid, nucleic acid, expression vector, or composition described herein to reduce gene and / or protein expression of one or more complement proteins found to be elevated in step (b). The method may include one or more of the following steps:

[0325] The methods provided herein may be useful for determining a subject's risk of developing a serious complement-related disorder, e.g., the methods are useful for distinguishing between subjects who may develop a mild complement-related disorder and subjects who are at risk for serious disease, and / or for identifying subjects who are likely to develop serious disease. The methods described herein may also be useful for assessing whether a treatment for a complement-related disorder is effective or successful, or whether it remains effective or successful.

[0326] As described herein, the methods may include determining whether the level of a complement protein is altered, e.g., increased or decreased, when compared to the level of that complement protein in the blood in a control subject without a complement-related disorder or in a subject with a complement-related disorder that is not associated with the altered level of that complement protein. In some cases, the methods may include determining the relative concentrations of the complement proteins compared to each other, e.g., the level of a complement protein may be elevated when compared to the level of a different complement protein, which may be unaltered or decreased, in the same subject or when compared to a control subject.

[0327] In some embodiments, the levels of complement proteins are determined using any suitable technique known in the art and available to one of skill in the art, hi some embodiments, the levels of complement proteins are determined by mass spectrometry and / or by digesting the proteins with endoproteinase GluC, such as those described herein.

[0328] In some embodiments, the methods described herein are carried out in vitro or ex vivo. For example, a sample may be obtained from a subject of interest or a control subject, and the level of complement proteins may be determined to determine whether the subject has a complement-related disorder or is at risk of developing such a disorder, and the steps involving digesting at least one complement protein may be carried out in vitro or ex vivo. Method steps involving treating a subject may also be carried out in vivo.

[0329] The methods described herein can be useful in monitoring the success of therapeutic or preventive interventions, including past or ongoing treatments, for complement-related disorders. In some embodiments, the methods described herein can include administering an agent, inhibitory nucleic acid, nucleic acid, expression vector, or composition described herein to a subject who has / has been determined to have a complement-related disorder, and then re-determining the levels of one or more of FHR1, FHR2, FHR3, FHR4, and / or FHR5, and optionally FH and / or FHL-1, after such therapeutic / prophylactic intervention. Such methods are useful for determining the effectiveness of therapeutic / prophylactic interventions and the progression of the disorder.

[0330] In some aspects, the present disclosure provides methods for predicting whether a subject is at risk for developing a complement-related disorder, has a complement-related disorder, is in need of a therapy / prophylactic intervention for a complement-related disorder, will respond to a therapy / prophylactic intervention for a complement-related disorder, and / or is / has responded to a therapy / prophylactic intervention for a complement-related disorder, based on the analysis of a sample from the subject described herein. The methods can also be used to determine whether a subject is at risk for developing a disorder and / or is at risk for progression, progression, or worsening of a disorder.

[0331] The methods described herein can be used to determine whether a subject is at risk for the onset or progression of a complement-related disorder, e.g., as described herein. The complement-related disorder may be macular degeneration, e.g., EOMD and / or AMD. In some cases, the disorder is selected from EOMD, AMD, geographic atrophy ("dry" (i.e., non-exudative) AMD), early AMD, intermediate AMD, late / advanced AMD, "wet" (neovascular or exudative) AMD, choroidal neovascularization (CNV), and retinal dystrophy. In some cases, the subject has or is suspected of having a complement-related disorder. In some cases, the disorder is AMD. In some cases, the disorder is EOMD.

[0332] Accordingly, the present disclosure provides a method for determining whether a subject is at risk for developing macular degeneration, e.g., EOMD and / or AMD, comprising: (a) digesting one or more complement proteins in a sample obtained from the subject with endoproteinase GluC to obtain one or more peptides, for example, the one or more complement proteins selected from one or more of FHR1, FHR2, FHR3, FHR4 and / or FHR5, and optionally FH and / or FHL-1; (b) determining the level of one or more peptides by mass spectrometry; (c) using the results of (b) to determine the level of one or more complement proteins; and (d) determining that the subject has or is at risk of developing macular degeneration if the levels of FHR1, FHR2, FHR3, FHR4 and / or FHR5, and optionally FHL-1, are elevated when compared to the levels of that complement protein(s) in the blood in control subjects who do not have a complement-related disorder. The method includes the steps of:

[0333] Also provided herein is a method for assessing a subject's tendency or predisposition to develop a complement-associated disorder, comprising steps (a) through (d) above. The methods described herein may also be useful for assessing whether a therapeutic / prophylactic intervention for a complement-associated disorder is / was effective or successful.

[0334] In some aspects, the methods described herein can be useful for determining whether a subject is likely to respond, likely not to respond, or whether a subject is responding to a therapeutic intervention, which should allow the patient to receive the most effective therapy for their particular pathological needs.

[0335] In some cases, the subject has or is suspected to have a complement-related disorder. In some cases, the method provided herein comprises determining whether the subject has or is suspected to have a complement-related disorder. In some cases, the disorder is AMD. In some cases, the disorder is EOMD.

[0336] In some aspects, the present disclosure provides methods for treating or preventing a complement-associated disorder in a subject, the method comprising administering a therapeutically or prophylactically effective amount of an agent, inhibitory nucleic acid, gene editing tool / system, nucleic acid, expression vector, or composition described herein, wherein the treated subject has been determined to have an atypical presence or level of one or more complement proteins when compared to control subjects and / or reference value(s), e.g., detected / determined as described herein. In some aspects, the subject has been determined to be at risk for developing a complement-associated disorder and / or has been identified as having a complement-associated disorder.

[0337] In other aspects, the present disclosure provides an agent, inhibitory nucleic acid, gene editing tool / system, nucleic acid, expression vector, or composition described herein for use in a method of treating or preventing a complement-associated disorder in a subject, the method comprising administering a therapeutically or prophylactically effective amount of the agent, inhibitory nucleic acid, gene editing tool / system, nucleic acid, expression vector, or composition, wherein the subject has been determined to have an atypical presence or level of one or more complement proteins when compared to a reference value(s), e.g., determined as described herein. In some aspects, the subject has been determined to be at risk of developing a complement-associated disorder and / or has been identified as having a complement-associated disorder.

[0338] In some aspects, there is provided use of an agent, inhibitory nucleic acid, gene editing tool / system, nucleic acid, expression vector, or composition described herein in the manufacture of a medicament for treating or preventing a complement-associated disorder in a subject, wherein the subject has / has been determined to have an atypical presence or level of one or more complement proteins when compared to a reference value(s), e.g., determined as described herein. In some aspects, the subject has been determined to be at risk of developing a complement-associated disorder and / or has been identified as having a complement-associated disorder.

[0339] Also provided is a method of treating or preventing a complement-related disorder in a subject, or an agent, inhibitory nucleic acid, gene editing tool / system, nucleic acid, expression vector, or composition described herein for use in a method of treating or preventing a complement-related disorder in a subject, the method comprising administering a therapeutically or prophylactically effective amount of the agent, inhibitory nucleic acid, gene editing tool / system, nucleic acid, expression vector, or composition described herein, wherein the subject has / has been determined to have an atypical presence or level of one or more complement proteins when compared to a reference value(s), e.g., determined as described herein. In some aspects, the subject has been determined to be at risk of developing a complement-related disorder and / or has been identified as having a complement-related disorder.

[0340] In various aspects provided herein, a subject to be administered a therapy or preventative intervention has an atypical presence or level of at least one complement protein, preferably one or more of FH, FHL-1, FHR1, FHR2, FHR3, FHR4, FHR5, FI, C3, C3b, C3a, iC3b, C3f, C3c, C3dg, and / or C3d. In some embodiments, the subject has an atypical presence or level of one or more of FHR1, FHR2, and / or FHR3, and optionally FHR4 and / or FHR5, and / or FHL-1. A subject may benefit from a therapy or preventative intervention that reduces the level of any complement protein that is increased compared to a reference value(s) and / or that increases the level of any complement protein that is decreased compared to a reference value(s).

[0341] The methods provided herein for assessing the risk of developing a complement-related disorder, i.e., the risk of its onset or progression, or for identifying subjects having / at risk for a complement-related disorder, may be performed in combination with additional diagnostic methods and / or tests for such disorders that would be known to those skilled in the art. In some cases, the method for assessing the risk of developing a complement-related disorder includes additional techniques selected from: CH50 or AH50 measurement via hemolytic assay, measurement of neoantigen formation during MAC complex (C5b, C6, C7, C8, C9) generation, C3 deficiency screening, mannose-binding lectin assay, immunochemical assays for quantifying individual complement components, flow cytometry for evaluating cell-associated regulatory proteins such as CD55, CD59, and CD35, and / or renal function tests. See, e.g., Shih AR and Murali MR, Am. J. Hematol. 2015, 90(12):1180-1186; Ogedegbe HO, Laboratory Medicine, 2007, 38(5):295-304; and Gowda S et al., N Am J Med Sci. 2010, 2(4): 170-73, which are incorporated herein by reference in their entireties.

[0342] In some cases, the methods provided herein for assessing the risk of developing AMD and / or EOMD include: dark adaptation testing, contrast sensitivity testing, e.g., Pelli Robson, vision testing using, e.g., a Snellen chart and / or an Amsler grid, the Farnsworth-Munsell 100 Hue Test and Maximum Color Contrast Sensitivity Test (MCCS) to assess color vision and color contrast sensitivity, preferential hyperacusis perimetry (PHP), fundus photography of the back of the eye, fundus examination, fundus autofluorescence, optical coherence tomography, angiography, e.g., fluorescein angiography, fundus fluorescein angiography, iodocyanine green angiography, optical coherence tomography angiography, adaptive optics retinal imaging, deep learning analysis of fundus images, electroretinography, and / or further evaluation techniques selected from ocular atrophy, retinal pigment changes, exudative changes, e.g., hemorrhage, hard exudates, subretinal / sub-RPE / intraretinal fluid, and / or the presence of drusen.

[0343] The methods described herein may take into account lifestyle factors known to contribute to the risk of developing a complement-related disorder. For example, lifestyle factors that may cause or contribute to AMD include smoking, being overweight, high blood pressure, and having a family history of AMD.

[0344] The methods provided herein may include determining the presence or absence of a genetic profile in a subject characterized by polymorphisms in the subject's genome associated with complement dysregulation. The polymorphisms may be found in or near genes such as CCL28, FBN2, ADAM12, PTPRC, IGLC1, HS3ST4, PRELP, PPID, SPOCK, APOB, SLC2A2, COL4A1, MYOC, ADAM19, FGFR2, C8A, FCN1, IFNAR2, C1NH, C7, and ITGA4. The genetic profile associated with complement dysregulation may include one or more, often multiple, single nucleotide polymorphisms, such as those listed in Tables I and II of US 2010 / 0303832, the entire contents of which are incorporated herein by reference.

[0345] Genetic factors are believed to play a role in the development of AMD and EOMD.Therefore, any of the assessment or therapeutic / preventive methods described herein can be combined with the method of assessing AMD-related and / or EOMD-related and / or macular dystrophy-related gene variants.In some cases, the complement-related disorders described herein can include genetic factors and / or genetic risk factors.

[0346] In some cases, the method provided herein may comprise determining the presence or absence of one or more genetic factors associated with AMD and / or EOMD in a subject, such as one or more AMD or EOMD-related gene variants.In some cases, the method comprises screening (directly or indirectly) for the presence or absence of one or more genetic factors.In some embodiments, the genetic factor(s) are genetic risk factor(s).In some embodiments, the subject has been determined to have one or more of these risk factors.In some embodiments, the method of the present disclosure comprises determining whether the subject has one or more of these risk factors.

[0347] In some embodiments, one or more genetic factors may be located at or near the RCA locus, for example, in the CFH / CFHR gene / CFH locus, on chromosome 1. In some embodiments, the presence of one or more AMD risk variants in the CFH locus increases disease risk through increased FHR protein levels.

[0348] One or more genetic factors: Y402H (i.e., rs1061170 C ), rs1410996 C, I62V(rs800292), A473A(rs2274700), R53C, D90G, D936E(rs1065489), R1210C, IVS1(rs529825), IVS2insTT, IVS6 (rs3766404), A307A (rs1061147), IVS10 (rs203674), rs3753396, R1210C, rs148553336, rs191281603, rs In some embodiments, the genetic factor may be located in one or more of: CFH selected from rs35292876 and rs800292; CFHR4 selected from rs6685931 and rs1409153; CFI selected from G119R and rs141853578; CFB, e.g., rs4151667; C2, e.g., rs9332739; C9, e.g., P167S; and / or C3, e.g., K155Q. In some embodiments, the genetic factor may be located in one or more of: Y402H (i.e., rs1061170 C ). In some embodiments, the genetic factor is rs3753396. In some embodiments, the genetic factor is rs6685931 and / or rs1409153. In some embodiments, the genetic factor is intronic KCNT2 rs61820755. In some embodiments, the genetic factor is not rs6685931.

[0349] In some embodiments, the genetic factor is rs61820755 and may be associated with FHL-1. In any embodiment herein, genetic risk factors may be present in combination with elevated levels of one or more FHR proteins. One or more genetic factors in the CFH locus may be selected from intergenic CFHR1 / CFHR4 rs149369377 and / or rs61820755 for FHR-1, CFHR2 rs4085749 for FHR-2, intronic CFH rs70620 for FHR-3, rs12047098 for FHR-4, and intronic KCNT2 rs72732232 for FHR-5. The presence of any one or more of these genetic factors indicates that the subject has or may develop a complement-related disorder.

[0350] The one or more genetic risk factors are selected from rs10922109, rs570618, rs121913059 (R1210C), rs148553336, rs187328863, rs61818925, rs35292876, and rs191281603.

[0351] The one or more genetic factors may be selected from rs113721756 on chromosome 10, rs111260777 on chromosome 11, rs117468955 on chromosome 12, rs200404865 on chromosome 13, rs4790395 on chromosome 17, and rs117115124 on chromosome 19. These factors may be present separately from or in addition to genetic factors at the CFH locus. These factors may be present in combination with elevated FHR-3 levels.

[0352] Any combination of genetic factors is contemplated, such as those described herein or additional factors, including detection / assessment of: The methods described herein may involve detecting a combination of risk factors to assess a subject's risk for developing a complement-associated disorder, e.g., detecting whether one or both risk factors are present in a subject, for example: rs10922109 and intergenic CFHR1 / CFHR4 rs149369377 rs10922109 and CFHR2 rs4085749 rs10922109 and intronic CFH rs70620 rs10922109 and intergenic CFHR1-CFHR4 rs12047098 rs570618 and intergenic CFHR1 / CFHR4 rs149369377 rs570618 and CFHR2 rs4085749 rs148553336 and intronic KCNT2 rs72732232 rs61818925 and CFHR2 rs4085749; and / or rs61818925 and intergenic CFHR1-CFHR4 rs12047098.

[0353] Assessment of the presence of any of the genetic risk factors provided herein may be combined with detection of any one or more of FHR1, FHR2, FHR3, FHR4, and / or FHR5, and optionally FHL-1, as described herein. For example, the presence of genetic factor rs10922109 may be assessed in combination with detection of any one or more of FHR-1, FHR-2, FHR-3, and / or FHR-4; rs570618 may be assessed in combination with detection of FHR-1 and / or FHR-2; rs61818925 may be assessed in combination with detection of FHR-2 and / or FHR-4; and rs148553336 may be assessed in combination with detection of FHR-5.

[0354] In any embodiment or method herein, the method may include determining that a subject is likely to have or develop a complement-associated disorder if one or more genetic factors, such as those described herein, are present.

[0355] Accordingly, provided herein are methods for identifying a subject having or at risk of developing a complement-related disorder, comprising evaluating the subject for one or more genetic risk factors, such as any of those described herein or others, and determining that if one or more genetic risk factors are present in the subject, the subject is likely to have or develop a complement-related disorder.

[0356] Provided herein are methods for determining whether a subject has or is at risk for developing a complement-related disorder, comprising evaluating the subject for one or more genetic risk factors, such as any of those described herein or others, and determining that the subject has a high likelihood of having or developing a complement-related disorder if one or more genetic risk factors are present in the subject.

[0357] Also provided are methods for selecting and / or administering therapeutic / prophylactic interventions to subjects / patients who have or have been identified as having a complement-associated disorder, e.g., using the above steps.

[0358] Also provided are methods for selecting a subject for therapeutic / prophylactic intervention with an agent, inhibitory nucleic acid, gene editing tool / system, nucleic acid, expression vector, or composition described herein; methods for selecting for a subject an agent, inhibitory nucleic acid, gene editing tool / system, nucleic acid, expression vector, or composition described herein; methods of treatment; an agent, inhibitory nucleic acid, gene editing tool / system, nucleic acid, expression vector, or composition described herein for use in treatment; and use of an agent, inhibitory nucleic acid, gene editing tool / system, nucleic acid, expression vector, or composition described herein in the manufacture of a medicament for the treatment of a complement-related disorder, wherein the method comprises assessing genetic risk factors (alone or in combination with determining the level of a complement protein, e.g., an FHR protein as described herein, and / or determining whether one or more of FHR1, FHR2, FHR3, FHR4 and / or FHR5, and optionally FHL-1, are elevated when compared to the level of complement proteins in the blood in control subjects who do not have a complement-related disorder as described herein).

[0359] Any such methods that include detecting and assessing genetic risk factors may include, for example, administering to a subject who has been determined to have or be likely to develop a complement-associated disorder, an agent, inhibitory nucleic acid, gene editing tool / system, nucleic acid, expression vector, or composition described herein.

[0360] Other suitable genetic risk factors and gene variants are known in the art and include, for example, those described in Edwards AO et al., Science 2005, 308(5720):421-4; Hageman GS et al., Proc Natl Acad Sci US A. 2005, 102(20):7227-7232; Haines JL et al., Science 2005, 308(5720):419-21; Klein RJ et al., Science 2005, 308(5720):385-389; Fritsche et al., Nat Genet. 2016, 48(2):134-43; US 2010 / 0303832; Clark S et al., J Clin Med. 2015, 4(1):18-31; Cipriani, V. et al., Nat Commun. 2020, 11, 778; or as described in Hageman GS et al., Hum Genomics. 2011, 5, 420 (2011).

[0361] In some cases, the methods provided herein further comprise determining the presence or absence of one or more genetic factors associated with EOMD, such as one or more EOMD-associated gene variants, in the subject. In some cases, the method comprises screening (directly or indirectly) for the presence or absence of one or more genetic factors. In some embodiments, the genetic factor(s) are genetic risk factor(s). In some embodiments, the subject has been determined to have one or more such risk factors. In some embodiments, the methods of the present disclosure comprise determining whether the subject has one or more such risk factors. In some embodiments, the subject may have one or more risk factors for early-onset macular degeneration (EOMD).

[0362] EOMD is thought to be caused by monogenic inheritance of rare variants in the CFH gene (see, e.g., Boon CJ et al. Am J Hum Genet 2008; 82(2):516-23; van de Ven JP et al. Arch Ophthalmol 2012; 130(8):1038-47; Yu Y et al. Hum Mol Genet 2014; 23(19):5283-93; Duvvari MR et al. Mol Vis 2015; 21:285-92; Hughes AE et al. Acta Ophthalmol 2016; 94(3):e247-8; Wagner et al. Sci Rep 2016; 6:31531; Taylor RL et al. Ophthalmology. 2019 Mar 21. pii: S0161-6420(18):33171-3). In some embodiments, the subject may have one or more EOMD-associated gene variants. EOMD-associated gene variants are described, for example, in Servais A et al. Kidney Int, 2012; 82(4):454-64 and Dragon-Durey MA et al. J Am Soc Nephrol 2004; 15(3):787-95, the entire contents of which are incorporated herein by reference. In some embodiments, the subject may have one or more of the following EOMD-associated genetic variants: CFH c.1243del, p.(Ala415Profs*39) het; CFH c.350+1G>T het; CFH c.619+1G>A het; CFH c.380G>A, p.(Arg127His); CFH c.694C>T, p.(Arg232Ter); or CFH c.1291T>A, p.(Cys431Ser).

[0363] In some cases, the methods provided herein include screening for deletions within the RCA locus (a region of DNA sequence located on chromosome 1 extending from the CFH gene through the CD46 (MCP) gene) that are associated with AMD and / or EOMD risk or protection.

[0364] Methods for determining the presence or absence of genetic factors include restriction fragment length polymorphism identification (RFLPI) of genomic DNA, random amplification polymorphism detection (RAPD) of genomic DNA, amplified fragment length polymorphism detection (AFLPD), multiple locus variable number tandem repeat (VNTR) analysis (MLVA), SNP genotyping, multilocus sequence typing, PCR, DNA sequencing, such as Sanger sequencing or next-generation sequencing, allele-specific oligonucleotide (ASO) probe, and oligonucleotide microarray or bead.Other suitable methods are described, for example, in Edenberg HJ and Liu Y, Cold Spring Harb Protoc; 2009; doi:10.1101 / pdb.top62, and Tsuchihashi Z and Dracopoli NC, Pharmacogenomics J., 2002, 2:103-110.

[0365] In some embodiments, the subject is determined to have one or more genetic factors related to AMD and / or EOMD, for example, one or more AMD-related and / or EOMD-related gene variants, or is determined to have macular dystrophy, and is therefore selected for therapeutic or preventive treatment with the agent, inhibitory nucleic acid, gene editing tool / system, nucleic acid, expression vector, or composition described herein.In some embodiments, the subject has been determined to have one or more of these genetic factors.In some embodiments, the method provided herein comprises determining whether the subject has one or more of these genetic factors.Examples of such methods and genetic factors are described herein. Accordingly, provided herein are methods of diagnosing, treating, or preventing a complement-associated disorder in a subject, wherein the subject has / is determined to have / possess one or more genetic causes of AMD and / or EOMD, and the subject has / is determined to have / have an atypical presence or level of one or more complement proteins when compared to a reference value(s), e.g., detected / determined as described herein; and optionally, the method comprises administering an agent, inhibitory nucleic acid, gene editing tool / system, nucleic acid, expression vector, or composition described herein.

[0366] Provided are methods of treating or preventing a complement-associated disorder in a subject, wherein the subject is characterized as having an atypical presence or level of one or more complement proteins, e.g., detected / determined as described herein.

[0367] Also provided is a complement-targeting therapeutic agent for use in a method of treating or preventing a complement-associated disease in a subject, wherein the subject is characterized as having an atypical presence or level of one or more complement proteins, e.g., detected / determined as described herein.

[0368] Methods for assessing complement-associated disorders The present disclosure also provides methods for assessing the risk of onset, progression, or development of a complement-related disorder. The complement-related disorder may be any disorder in which the complement system or its activation / overactivation / dysregulation is pathologically associated. The complement-related disorder may be any disorder described herein. The methods described herein may be useful in monitoring the success of treatment, including past or current treatment, for a complement-related disorder. Such treatment may involve one or more agents as described herein.

[0369] In some aspects, provided are methods for identifying a subject having or at risk of developing a complement-associated disorder, comprising: (a) determining in a blood sample obtained from the subject the level of a complement protein selected from one or more of FHR1, FHR2, FHR3, FHR4 and / or FHR5; (b) determining that the subject has or is likely to develop a complement-related disorder if the level of the complement protein determined in (a) is elevated when compared to the level of that complement protein in the blood of a control subject who does not have a complement-related disorder. The method includes the steps of:

[0370] In some aspects, a method for determining whether a subject has or is at risk for developing a complement-associated disorder comprises: (a) determining in a blood sample obtained from the subject a level of a complement protein selected from one or more of FHR1, FHR2, FHR3, FHR4 and / or FHR5; (b) determining that the subject has or is at increased risk for developing a complement-related disorder if the level of the complement protein determined in (a) is elevated when compared to the level of that complement protein in the blood of a control subject who does not have a complement-related disorder. The method includes the steps of:

[0371] In some embodiments, step (a) comprises determining the levels of two of the complement proteins selected from FHR1, FHR2 and / or FHR3. In some embodiments, step (a) comprises determining the levels of three of the complement proteins selected from FHR1, FHR2 and FHR3.

[0372] In some embodiments, step (a) comprises or further comprises determining the level of FHR4 and / or FHR5. The methods described herein may include determining that the subject has or is likely to develop a complement-related disorder if the level of FHR4 and / or FHR5 is elevated when compared to the level of that complement protein in the blood in a control subject who does not have a complement-related disorder.

[0373] In some embodiments, step (a) comprises or further comprises determining the level of FH and / or FHL-1. The method may comprise determining the level of FHL-1, alone or in combination with other complement protein(s), and determining that the subject has or is likely to develop a complement-related disorder if the level of FHL-1 is elevated when compared to the level of FHL-1 in the blood of a control subject who does not have a complement-related disorder. The level of FH and / or FHL-1 may be increased or decreased compared to the control subject.

[0374] The determination of the levels of two or more complement proteins may be performed simultaneously, concurrently, or sequentially. The complement proteins may be detected in the same assay or in one or more separate assays. The determination of the level of a second or subsequent complement protein may be performed simultaneously with, before, or after the determination of the level of the first complement protein. In some embodiments, steps (a) and (b) may be repeated one or more times on the same subject at appropriate time intervals to assess the progression of a complement-related disorder.

[0375] Any aspect or embodiment described herein may include, for example, determining the level (e.g., expression) of any one of the following genes / proteins in a subject: a)FHR1; b) FHR2; c)FHR3; d)FHR4; e)FHR5; f) FHR1 and FHR2; g) FHR1 and FHR3; h) FHR1 and FHR4; i) FHR1 and FHR5; j) FHR2 and FHR3; k) FHR2 and FHR4; l)FHR2 and FHR5; m) FHR3 and FHR4; n)FHR3 and FHR5; o)FHR4 and FHR5; p)FHR1, FHR2 and FHR3; q) FHR1, FHR2 and FHR4; r) FHR1, FHR2 and FHR5; s)FHR1, FHR3 and FHR4; t)FHR1, FHR3 and FHR5; u) FHR1, FHR4 and FHR5; v) FHR2, FHR3 and FHR4; w) FHR2, FHR3 and FHR5; x) FHR2, FHR4 and FHR5; y) FHR3, FHR4 and FHR5; z) FHR1, FHR2, FHR3 and FHR4; aa) FHR1, FHR2, FHR3 and FHR5; bb) FHR1, FHR2, FHR4 and FHR5; cc) FHR2, FHR3, FHR4 and FHR5; dd) FHR1, FHR3, FHR4 and FHR5; or ee) FHR1, FHR2, FHR3, FHR4 and FHR5; Or any of (a) to (ee) combined with determination of levels of FH and / or FHL-1, for example FH and / or FHL-1 in addition to FHR1, FHR2, FHR3; or FH and / or FHL-1 in addition to FHR1, FHR2, FHR3, FHR4, FHR5.

[0376] The selection or combination of the complement protein(s) to be detected may depend on the complement-related disorder of interest and the complement protein(s) that are useful biomarkers for each disorder. For example, detecting one or more of FHR1, FHR2, FHR3, FHR4, FHR5, FH, and / or FHL-1L-1 predicts AMD risk, while other specific complement proteins and combinations thereof predict other complement-related disorders. For example, see the disorders and references described herein. The present disclosure allows for accurate detection and differentiation of any one or more of the complement proteins described herein, and thus allows the absolute levels of the proteins to provide information on the likelihood of disorder onset and / or progression according to the fluctuations in protein levels in each disorder. Complement protein(s) may be detected in a sample obtained from a subject. For example, a sample may be obtained to provide information on the appropriate treatment and / or progression of the disorder.

[0377] In some cases, any aspect described herein may include determining the level of any one or more complement proteins selected from, for example, FHR1, FHR2, FHR3, FHR4, FHR5, FH, and / or FHL-1 in a blood sample obtained from a subject, and then determining that the subject has or is likely to develop a complement-related disorder if the level of the complement protein(s) is altered when compared to the level of that complement protein(s) in the blood of a control subject without a complement-related disorder. The term "altered," as used herein, refers to increased or decreased levels of a complement protein(s), e.g., the level of one or more complement proteins may be higher or lower when compared to the level of those complement proteins in the blood of a control subject without a complement-related disorder. In some cases, the level of a complement protein may be decreased when compared to the level of that complement protein in the blood of a control subject without a complement-related disorder. In some cases, when the levels of two or more complement proteins are determined, the levels of one or more complement proteins may be elevated, while the levels of one or more different complement proteins may be decreased, when compared to the levels of these complement proteins in the blood in control subjects who do not have a complement-related disorder.

[0378] In some embodiments, the level of complement proteins is determined using any suitable technique known in the art and available to those skilled in the art. In some embodiments, the level of complement proteins is determined by mass spectrometry and / or digestion of proteins with endoproteinase GluC, for example, as described herein. Determining the level of complement protein(s) may involve detecting any combination of peptides produced by digestion with GluC, as described herein. The level of complement proteins may be determined, for example, using enzyme-linked immunosorbent assay (ELISA / EIA), as described, for example, in van Beek et al., Front Immunol. 2017; 8: 1328; van Beek et al., Front Immunol. 2018; 9: 1727; and Pouw et al., PLoS One. 2016 Mar 23; 11(3): e0152164, the entire contents of which are incorporated herein by reference. Levels of complement proteins may be determined, for example, using Western or dot blotting with appropriate antibodies, HPLC, protein immunoprecipitation or immunoelectrophoresis.

[0379] Any aspect described herein may include an initial step of obtaining a sample and / or at least one protein, such as a complement protein, from a subject. Suitable sources of samples are described herein. The methods described herein may include determining levels of circulating FHR1, FHR2, and / or FHR3, circulating FHR4 and / or FHR5, and optionally circulating FH and / or FHL-1. Circulating proteins may be present, for example, in blood or lymph.

[0380] Any of the methods described herein may include determining the levels of one or more of C3, C3b, C3a, iC3b, C3f, C3c, C3dg, and / or C3d, for example, as described herein.

[0381] In some aspects, a method for determining whether a subject has or is at risk for developing a complement-associated disorder comprises: (a) digesting at least one complement protein selected from FHR1, FHR2, FHR3, FHR4 and / or FHR5 in a sample obtained from a subject with endoproteinase GluC to obtain one or more peptides; (b) determining the level of one or more peptides by mass spectrometry; (c) using the results of (b) to determine the level of one or more complement proteins; and (d) determining that the subject has or is at risk for developing a complement-related disorder if the level of one or more complement proteins determined in (c) is elevated when compared to the level of that complement protein(s) in the blood in a control subject who does not have a complement-related disorder. The method includes the steps of:

[0382] In some embodiments, the method further comprises digesting one or both of FH and / or FHL-1 with endoproteinase GluC to obtain one or more peptides, determining the level of the one or more peptides by mass spectrometry, and / or using the results of mass spectrometry to determine the level of FH and / or FHL-1. Exemplary combinations of complement proteins for use in the methods of the present disclosure are described above.

[0383] Also described herein is the use of complement proteins selected from one or more of FHR1, FHR2, FHR3, FHR4 and / or FHR5, and optionally FH and / or FHL-1, to identify a subject having or at risk of developing a complement-related disorder, or to determine whether a subject has or is at risk of developing a complement-related disorder, comprising: (a) determining in a blood sample obtained from the subject the level of complement proteins selected from one or more of FHR1, FHR2, FHR3, FHR4 and / or FHR5, and optionally FH and / or FHL-1; and (b) determining that the subject has or is likely to develop a complement-related disorder if the levels of FHR1, FHR2, FHR3, FHR4, and / or FHR5, and optionally FHL-1, are elevated when compared to the levels of those complement proteins in the blood in control subjects who do not have a complement-related disorder. The use includes the steps of:

[0384] Also provided is the use of complement proteins selected from one or more of FHR1, FHR2, FHR3, FHR4 and / or FHR5, and optionally FH and / or FHL-1, as biomarkers, for example, to identify a subject having or at risk of developing a complement-related disorder, or to determine whether a subject has or is at risk of developing a complement-related disorder, said use comprising the steps described herein above.

[0385] In one aspect, provided is the use of endoproteinase GluC in a method for determining the presence and / or level of complement proteins, e.g., in a sample or a subject, e.g., according to the methods described herein. Also provided is a method for identifying a subject having or at risk of developing a complement-related disorder, comprising: a) digesting at least one complement protein in a sample obtained from a subject with endoproteinase GluC to obtain one or more peptides; b) determining the presence and / or levels of one or more peptides by mass spectrometry; and (c) using the results of (b) to determine that the subject has or is likely to develop a complement-associated disorder. The method further comprises the steps of:

[0386] Also provided is a method of selecting a subject for treatment of a complement-associated disorder with complement-targeting therapy, comprising: a) digesting at least one complement protein in a sample obtained from a subject with endoproteinase GluC to obtain one or more peptides; b) determining the presence and / or levels of one or more peptides by mass spectrometry; and (c) Using the results of (b), determine whether the subject is in need of complement-targeting therapy. The use of GluC in the method comprises the steps of:

[0387] In some embodiments, the methods described herein are performed in vitro or ex vivo. For example, a sample may be obtained from a subject of interest and / or a control subject, and the determining step is performed in vitro or ex vivo.

[0388] In the methods described herein, the level of complement protein(s) is compared to a reference value level or reference level, sometimes referred to as a control. In some cases, the level of complement protein(s) is compared to the level of the same complement protein in a control subject who does not have a complement-related disorder. The reference value may itself be obtained from a control subject, or may be obtained from a control sample. Data or values ​​obtained from, for example, a sample from the individual to be tested may be compared to data or values ​​obtained from a control sample. In some cases, the control is the spouse, partner, or friend of the subject.

[0389] As used herein, the term "reference value" refers to a known measurement value used for comparison during analysis. In some cases, the reference value is one or a set of test values ​​obtained from an individual or group with a defined health condition. The reference value may also be one or a set of test values ​​obtained from a control. In some cases, the reference value is / has been obtained by determining the level of a complement protein in a subject known not to have a complement-related disorder. In some cases, the reference value is / has been obtained by determining the level of a complement protein in a subject with a complement-related disorder not associated with elevated levels of FHR protein(s), e.g., a subset of subjects in whom FHR protein is not considered a pathological factor. In some cases, the reference value is established by previously determining the level or amount of a complement protein from the individual to be tested, for example, at an earlier stage of disease progression or before the onset of disease. The reference value may be taken from a sample obtained from the same subject or a different subject(s). The sample may be derived from the same tissue / cell / body fluid as the sample used according to the present disclosure. The reference value may be a standard value, a standard curve, or a standard dataset. Values / levels that deviate significantly from the reference value may be described as atypical values / levels.

[0390] In some cases, the control may be a reference sample or a reference dataset, or one or more values ​​from the sample or dataset. The reference value may be obtained from a reference sample or a reference dataset. The reference value may be obtained from one or more samples previously obtained from one or more subjects known to not have a complement-related disorder and / or known or expected to not be at risk for developing a complement-related disorder. The reference value may be obtained from one or more samples previously obtained from one or more subjects known to have a complement-related disorder. The reference value may be obtained from one or more samples previously obtained from one or more subjects known to be at risk for developing a complement-related disorder. The reference value may be a consensus level or average, or mean value calculated from a reference dataset, e.g., average protein levels. The reference dataset / value may be obtained from a large-scale study of subjects known to have a complement-related disorder, e.g., AMD, as described herein.

[0391] The reference value may be obtained from one or more samples previously obtained from one or more subjects who are in the same family as the subject of interest, or from one or more subjects who are not in the same family as the subject of interest.

[0392] The reference value may be obtained from one or more samples that have previously been obtained and / or analyzed from the individual / subject / patient to be tested, for example, a sample obtained from an individual in which the individual is at an earlier stage of a complement-associated disorder, or a sample obtained from an individual prior to the onset of a complement-associated disorder.

[0393] Reference values ​​may be obtained by analyzing samples taken from control subjects in parallel with samples from individuals to be tested. Alternatively, control values ​​may be obtained from a database or other previously obtained values. Reference values ​​may be determined contemporaneously with the methods disclosed herein or may be previously determined.

[0394] The control subject from whom the sample is obtained / obtained may be undergoing treatment for a complement-associated disorder and / or may be receiving a complement-associated therapy / therapeutic agent. The control may be a positive control in which the target molecule is known to be present or expressed at high levels, or a negative control in which the target molecule is known to be absent or expressed at low levels.

[0395] A sample from one or more control subjects may contain any one, two, three, four, five, or six of the seven: FHR1, FHR2, FHR3, FHR4, FHR5, FH, and / or FHL-1. In some cases, each complement protein is in a separate control sample. In some cases, a control sample ...

Claims

1. A composition for use in a method of treating or preventing a complement-related disorder, comprising a nucleic acid for reducing the expression of one or more genes and / or proteins of the H factor family, wherein said nucleic acid comprises a nucleic acid sequence consisting of a sequence having at least 70% sequence identity with SEQ ID NO: 178, said composition.

2. The composition according to claim 1, wherein said one or more H factor family proteins are selected from FHR1, FHR2, FHR3, FHR4 and FHR5.

3. The composition according to claim 1, wherein said nucleic acid comprises or targets a nucleotide sequence comprising SEQ ID NO:

158.

4. The composition according to claim 1, wherein said nucleic acid comprises or consists of an antisense nucleic acid having at least 85% sequence identity with SEQ ID NO: 178 or encodes the same.

5. The composition according to claim 1, wherein said nucleic acid is siRNA, shRNA, miRNA or an antisense oligonucleotide.

6. The composition according to claim 1, wherein said nucleic acid does not reduce the expression of the genes and / or proteins of FH and / or FHL-1.

7. The composition according to any one of claims 1 to 6, wherein said nucleic acid is encoded by a nucleic acid contained in an expression vector.

8. The composition according to any one of claims 1 to 6, wherein said nucleic acid is contained in the composition together with a pharmaceutically acceptable carrier, diluent, excipient or adjuvant.

9. The composition according to any one of claims 1 to 6, wherein said nucleic acid is contained in a cell.

10. The subject has or has been determined to have a complement-related disorder, and / or, wherein the complement-related disorder is selected from age-related macular degeneration, age-related macular degeneration (AMD), geographic atrophy ("dry" (i.e., non-exudative) AMD), early AMD, early-onset macular degeneration (EOMD), intermediate AMD, late / progressive AMD, "wet" (neovascular or exudative) AMD, choroidal neovascularization (CNV), retinal dystrophy, hemolytic uremic syndrome (HUS), atypical hemolytic uremic syndrome (aHUS), DEAP HUS (FHr plasma protein deficiency and autoantibody-positive type of hemolytic uremic syndrome), autoimmune uveitis, kidney injury / damage / dysfunction, glomerular disease, type II membranoproliferative glomerulonephritis (MPGN II), sepsis, Henoch-Schönlein purpura (HSP), IgA nephropathy, chronic kidney disease, paroxysmal nocturnal hemoglobinuria (PNH), autoimmune hemolytic anemia (AIHA), systemic lupus erythematosus (SLE), Sjögren's syndrome (SS), rheumatoid arthritis (RA), C3 glomerulopathy (C3G), dense deposit disease (DDD), C3 nephritic factor glomerulonephritis (C3 NF GN), FHR5 nephropathy, hereditary angioedema (HAE), acquired angioedema (AAE), encephalomyelitis, atherosclerosis, antineutrophil cytoplasmic autoantibody (ANCA) vasculitis, neurodegenerative / neurodegenerative diseases, dementia, multiple sclerosis (MS), Lewy body disease, amyotrophic lateral sclerosis (ALS), Huntington's disease, prion disease, cancer, lung cancer, glioblastoma, such as glioblastoma multiforme (GBM), stroke, insulin resistance, diabetes, infectious diseases, Parkinson's disease, and / or Alzheimer's disease. The composition according to any one of claims 1 to 6.

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