Complement factor i and complement factor i cofactor, vectors encoding therefor and therapeutic use

Abstract

To provide a combined preparation for treating or preventing complement-mediated disorders, specifically age-related macular degeneration (AMD) or diabetic retinopathy.SOLUTION: The present invention provides a product comprising (i) a Complement Factor I (CFI) cofactor and (ii) Complement Factor I (CFI), or nucleotide sequences encoding therefor, an isolated polynucleotide comprising nucleotide sequences encoding (i) a Complement Factor I (CFI) cofactor, and (ii) Complement Factor I (CFI), a vector containing the polynucleotide, or a cell containing the polynucleotide.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to agents for use in gene therapy. In particular, the present invention relates to combinations of CFI cofactors, such as complement factor I (CFI) and complement factor H-like protein 1 (FHL1), polynucleotides encoding them, and their use in treating or preventing complement-mediated and complement-related disorders, including complement-mediated eye diseases, such as age-related macular degeneration (AMD). [Background technology]

[0002] The macula is a small area in the retina of the eye, about 3-5 millimeters in size, adjacent to the optic nerve. It is the most sensitive area of ​​the retina and contains the fovea, a concave area that allows for high visual acuity, and a dense concentration of cones, the photoreceptors responsible for color vision.

[0003] Age-related macular degeneration (AMD) is the most common cause of functional blindness in people over 50 years of age in developed countries (Seddon, JM, Epidemiology of age-related macular degeneration. In: Ogden, TE et al., eds., Ryan SJ, ed-in-chief. Retina Vol II. 3rd ed., St. Louis, Mo.: Mosby; 2001: 1039-1050). AMD is associated with neovascularization that arises from the choroidal vasculature and extends into the subretinal space. In addition, AMD is characterized by progressive degeneration of the retina, retinal pigment epithelium (RPE), and underlying choroid (the highly vascular tissue between the retina and sclera and beneath the RPE).

[0004] A variety of factors may be involved in the pathogenesis of AMD, including oxidative stress, inflammation with a possible autoimmune component, genetic background (such as mutations), environmental factors such as smoking and diet, or behavioral factors.

[0005] The clinical progression of AMD is characterized by stages according to changes in the macula. A hallmark of early AMD is the appearance of drusen, which are accumulations of extracellular debris beneath the retina that appear as yellowish spots on the retina during clinical examination and on fundus photography. Drusen are classified by size as small (<63 μm), medium (63–124 μm), and large (>124 μm). They are also considered hard or soft, depending on the appearance of their margins on ophthalmic examination. Hard drusen have a well-defined margin, whereas soft drusen have a less defined and fluid margin. The Age-Related Eye Disease Study (AREDS) Fundus Photography Severity Scale is one of the major classification systems used for this condition.

[0006] Moderate AMD is diagnosed by large drusen and / or any retinal pigment abnormalities. Moderate AMD may cause some vision loss, but like early AMD, it is usually asymptomatic.

[0007] Late AMD has been classified as "dry" and "wet" (exudative or neovascular) forms. Dry AMD is more common than wet AMD, but the dry form can progress to the wet form, and the two occur simultaneously in a significant number of cases. Dry AMD is typically characterized by progressive apoptosis of cells in the RPE layer and the overlying photoreceptor cells, and often cells underlying the choriocapillaris. Confluent areas of RPE cell death with overlying photoreceptor atrophy are called geographic atrophy (GA). Patients with this form of AMD (advanced dry form) experience a slow and progressive deterioration of central vision.

[0008] Wet AMD is characterized by bleeding and / or fluid leakage from abnormal blood vessels growing from the choroidal vasculature (choriocapillaris) under the RPE and macula, which may cause sudden vision loss. It is presumed that much of the vision loss experienced by patients is due to such choroidal neovascularization (CNV) and its secondary complications. A subtype of neovascular AMD is called retinal angiomatous proliferation (RAP). Here, angiomatous proliferation originates in the retina and spreads posteriorly into the subretinal space, eventually communicating with new blood vessels in the choroid in some cases.

[0009] The complement system (CS) has been implicated in early AMD pathogenesis based on the identification of CS components in drusen from eyes of AMD patients. At least 129 drusen-deposited proteins have been identified in AMD, including various apolipoprotein types (E, B, or A-I), several amyloid peptides (P, Aβ, or SA-1), TIMP-3, serum albumin, and specific proteins related to cellular functions (e.g., ATP synthase β subunit, scavenger receptor B2, and retinol dehydrogenase). AMD-derived drusen also contain nearly all complement proteins, including regulatory proteins (CFH, complement receptor 1 (CR1), vitronectin, and clusterin), products of CS activation and degradation (C1q, C3, C3a, C3b, and C5a), and members of the terminal CS pathway, including isolated complex forms of MAC components (i.e., 5, 6, 8 (α, β, and γ), and 9). Accumulating drusen may activate CS, trigger local production of inflammatory mediators and attract leukocytes, which in turn potentiate the local inflammatory state present in AMD.

[0010] Current treatment options for AMD include photodynamic therapy with benzoporphyrins (Arch Ophthalmol (1999) 117:1329-1345) and multiple therapies targeting the vascular endothelial growth factor (VEGF) pathway. Examples of such VEGF-targeted therapies include antibodies such as ranibizumab (sold as Lucentis™, Genentech, Inc.), and bevacizumab (Avastin™, Genentech, Inc., and aflibercept (Eylea™, Bayer). However, although these anti-VEGF antibody therapies are highly effective, they are only approved for the treatment of the wet or neovascular forms of AMD, which account for approximately 10-15% of all AMD patients. Antibody therapies are administered by intravitreal injection once a month in an operating room or clean room, which places a burden on patients, who are typically elderly.

[0011] There are currently no approved treatments for the early stages or advanced (dry) forms of AMD.

[0012] The complement system is a well-documented target for the treatment of many inflammatory diseases. An overactive or inappropriately functioning complement system is implicated in the pathology of many chronic inflammatory conditions, including AMD (Nature Reviews (2015) 14:857-877). As a result, several complement-targeted therapeutics have been proposed or are currently in development, which target the alternative pathway amplification loop / C3b feedback cycle as a means to decrease C3b feedback or increase C3b degradation (Figure 1).

[0013] Lampalizumab (Genentech / Roche) is a humanized monoclonal antibody that inhibits complement factor D and is administered by monthly intravitreal injection to halt the rate of progression of geographic atrophy. Lampalizumab showed a slight reduction in the rate of expansion of geographic atrophy in a phase II clinical trial. However, in a phase III randomized clinical trial involving 906 participants, lampalizumab failed to reduce the expansion of GA when compared to sham over a 48-week period. The results showed an average of approximately 2 mm per year. 2 The results showed a substantial and consistent expansion of GA in

[0014] However, there remains a need for alternative therapies for the treatment of complement-mediated or complement-related diseases, particularly ocular diseases such as AMD, that are effective in the broad AMD population and are not limited to specific genotypes that may predispose individuals to AMD and other complement-related disorders. Summary of the Invention [Problem to be solved by the invention]

[0015] Applicants have identified therapeutic combinations of complement factor I (CFI), CFI cofactors (proteins with cofactor activity in CFI-mediated cleavage of C3b, such as complement factor H-like protein 1 (FHL1), complement factor H (CFH), complement receptor type 1 (CR1) and membrane cofactor protein (MCP)) for delivery to patients to restore or rebalance the overactive complement C3b feedback cycle. These combinations provide CFI and CFI cofactors in a molar ratio that ensures that the cofactors are provided in stoichiometric excess to CFI, ensuring maximum activity of CFI in C3b degradation (and downregulating the overactive complement system). This is important for patients who may have genetic deficiencies in certain complement system proteins and / or for therapies administered to tissues or organs where cofactor levels may be reduced compared to systemic levels, e.g., in the eye or kidney.

[0016] Furthermore, the applicant has provided a bicistronic vector that can be used to deliver and co-express both CFI and CFI cofactor (e.g., FHL1) to patients.In particular, the applicant has successfully designed a functional AAV vector that is produced with good titer and contains nucleotide sequences that code for both CFI and cofactor, thus overcoming the challenges posed by the limited capacity of AAV.The bicistronic vector of the present invention also advantageously allows good expression of both CFI and cofactor, and co-expression of CFI and cofactor in the ratios identified herein as beneficial.

[0017] In one aspect, the present invention provides a product comprising (i) a Complement Factor I (CFI) cofactor, and (ii) Complement Factor I (CFI) or a nucleotide sequence encoding same, as a combined preparation for simultaneous, separate or sequential use in therapy.

[0018] In another aspect, the present invention provides a product comprising (i) Complement Factor H-like protein 1 (FHL1) or Complement Factor H (CFH), and (ii) Complement Factor I (CFI) or a nucleotide sequence encoding same, as a combined preparation for simultaneous, separate or sequential use in therapy.

[0019] In a particular embodiment, the products are used in the treatment of complement mediated disorders, in particular chronic inflammatory conditions, and even more particularly those associated with an overactive complement C3b feedback cycle.

[0020] In another aspect, the present invention provides a product comprising (i) a complement factor I (CFI) cofactor, and (ii) complement factor I (CFI) or a nucleotide sequence encoding same, as a combined preparation for simultaneous, separate or sequential use in treating or preventing complement-mediated disorders, preferably complement-mediated disorders of the eye.

[0021] In another aspect, the present invention provides a product comprising (i) complement factor H-like protein 1 (FHL1) or complement factor H (CFH), and (ii) complement factor I (CFI) or a nucleotide sequence encoding same, as a combined preparation for simultaneous, separate or sequential use in treating or preventing complement-mediated disorders of the eye.

[0022] In some embodiments, the complement factor I (CFI) cofactor is selected from the group consisting of complement factor H-like protein 1 (FHL1); complement factor H (CFH); complement receptor 1 (CR1) or a fragment thereof; and membrane cofactor protein (MCP) or a fragment thereof.

[0023] In some embodiments, the complement factor I (CFI) cofactor is complement factor H (CFH). In some embodiments, the complement factor I (CFI) cofactor is complement receptor 1 (CR1) or a fragment thereof. In some embodiments, the complement factor I (CFI) cofactor is membrane cofactor protein (MCP) or a fragment thereof.

[0024] In a preferred embodiment, the complement factor I (CFI) cofactor is complement factor H-like protein 1 (FHL1).

[0025] In a preferred embodiment, the product comprises (i) complement factor H-like protein 1, and (ii) complement factor I (CFI), or a nucleotide sequence encoding same. In another embodiment, the product comprises (i) complement factor H (CFH); and (ii) complement factor I (CFI), or a nucleotide sequence encoding same. In another embodiment, the product comprises (i) complement receptor 1 (CR1), or a fragment thereof; and (ii) complement factor I (CFI), or a nucleotide sequence encoding same. In another embodiment, the product comprises (i) membrane cofactor protein (MCP), or a fragment thereof; and (ii) complement factor I (CFI), or a nucleotide sequence encoding same.

[0026] In some embodiments, the disorder is associated with overactivity of the complement C3b feedback cycle and / or underactivity of the C3b degradation cycle (see FIG. 1).

[0027] In some embodiments, the disorder is a complement-mediated chronic inflammatory condition of the eye.

[0028] In some embodiments, the disorder is age-related macular degeneration (AMD) or diabetic retinopathy. In other embodiments, the disorder is glaucoma, Stargardt's disease, central serous chorioretinopathy, or retinitis pigmentosa.

[0029] In preferred embodiments, the disease is AMD. In some embodiments, the AMD is dry AMD.

[0030] In some embodiments, the product provides (i) and (ii) to a subject in a molar ratio of (i) to (ii) of at least 2:1, at least 3:1, at least 4:1, at least 5:1, at least 6:1, at least 7:1, at least 8:1, at least 9:1, at least 10:1, at least 15:1, at least 20:1, at least 25:1, at least 30:1, at least 40:1, at least 50:1, or at least 60:1.

[0031] In some embodiments, the product provides (i) and (ii) to a subject in a molar ratio of (i):(ii) of at least 2: 1. In some embodiments, the product provides (i) and (ii) to a subject in a molar ratio of (i):(ii) of at least 3: 1. In preferred embodiments, the product provides (i) and (ii) to a subject in a molar ratio of (i):(ii) of at least 8: 1.

[0032] In some embodiments, (i) and (ii) are provided to a subject in a molar ratio of (i):(ii) of between 2:1 and 34:1, between 2:1 and 25:1, between 2:1 and 15:1, between 2:1 and 12:1, or between 3:1 and 10:1.

[0033] In a preferred embodiment, the product provides (i) and (ii) to a subject in a molar ratio of (i):(ii) of from 3:1 to 10:1.

[0034] The molar ratio provided can be achieved, for example, by delivering protein, polynucleotide or vector to a subject.Protein level can be easily measured by those skilled in the art using techniques known in the art, such as ELISA, for example, as described herein.Similarly, the amount of protein expressed from polynucleotide or its encoding vector can be measured using similar approaches.

[0035] In another aspect, the present invention provides (i) a Complement Factor I (CFI) cofactor, and (ii) an isolated polynucleotide comprising a nucleotide sequence encoding Complement Factor I (CFI).

[0036] In another aspect, the present invention provides (i) an isolated polynucleotide comprising a nucleotide sequence encoding complement factor H-like protein 1 (FHL1) or complement factor H (CFH) and complement factor I (CFI).

[0037] In some embodiments, the complement factor I (CFI) cofactor is selected from the group consisting of complement factor H-like protein 1 (FHL1); complement factor H (CFH); complement receptor 1 (CR1) or a fragment thereof; and membrane cofactor protein (MCP) or a fragment thereof.

[0038] In some embodiments, the complement factor I (CFI) cofactor is complement factor H (CFH). In some embodiments, the complement factor I (CFI) cofactor is complement receptor 1 (CR1) or a fragment thereof. In some embodiments, the complement factor I (CFI) cofactor is membrane cofactor protein (MCP) or a fragment thereof.

[0039] In a preferred embodiment, the complement factor I (CFI) cofactor is complement factor H-like protein 1 (FHL1).

[0040] In a preferred embodiment, the polynucleotide comprises a nucleotide sequence encoding (i) complement factor H-like protein 1 (FHL1) and (ii) complement factor I (CFI). In another embodiment, the polynucleotide comprises a nucleotide sequence encoding (i) complement factor H (CFH); and (ii) complement factor I (CFI). In another embodiment, the polynucleotide comprises a nucleotide sequence encoding (i) complement receptor 1 (CR1) or a fragment thereof and (ii) complement factor I (CFI). In another embodiment, the polynucleotide comprises a nucleotide sequence encoding (i) membrane cofactor protein (MCP) or a fragment thereof and (ii) complement factor I (CFI).

[0041] In some embodiments, the polynucleotide further comprises a nucleotide sequence encoding a CMV promoter. Preferably, the CMV promoter is upstream of the nucleotide sequence encoding (i) and (ii).

[0042] In some embodiments, the polynucleotide further comprises a nucleotide sequence encoding a WPRE regulatory element. Preferably, the WPRE regulatory element is downstream of the nucleotide sequence encoding (i) and (ii).

[0043] In some embodiments, the polynucleotide further comprises a nucleotide sequence encoding a polyA signal. Preferably, the polyA signal is downstream of the nucleotide sequence encoding (i) and (ii).

[0044] In some embodiments, the polynucleotide further comprises a nucleotide sequence encoding a bovine growth hormone poly A signal. Preferably, the bovine growth hormone poly A signal is downstream of the nucleotide sequence encoding (i) and (ii).

[0045] In some embodiments, the polynucleotide further comprises a nucleotide sequence encoding: (a) a CMV promoter, the CMV promoter being upstream of a nucleotide sequence encoding (i) and (ii); and (b) a bovine growth hormone polyA signal, downstream of the nucleotide sequence encoding (i) and (ii).

[0046] In other embodiments, the polynucleotide further comprises a nucleotide sequence encoding: (a) a CMV promoter, the CMV promoter being upstream of a nucleotide sequence encoding (i) and (ii); and (b) a WPRE regulatory element downstream of the nucleotide sequence encoding (i) and (ii); and (c) a bovine growth hormone polyA signal, downstream of the nucleotide sequence encoding (i) and (ii).

[0047] In a preferred embodiment, the WPRE regulatory element is a WPRE3 regulatory element.

[0048] In a preferred embodiment, the nucleotide sequence encoding (i) is upstream of the nucleotide sequence encoding (ii).

[0049] In other embodiments, the nucleotide sequence encoding (ii) is upstream of the nucleotide sequence encoding (i).

[0050] In some embodiments, the nucleotide sequences encoding (i) and (ii) are operably linked by a linker. In some embodiments, the linker is a furin, GSG, 1aa1D or F2A linker. In preferred embodiments, the linker comprises a self-cleaving 2A peptide sequence, such as P2A, or a sequence that includes or is defined by a furin cleavage site, GSG, 11a1D and F2A sequence.

[0051] In some embodiments, the polynucleotide further comprises one or more adeno-associated virus (AAV) inverted terminal repeats (ITRs). In a preferred embodiment, the polynucleotide further comprises two AAV ITRs.

[0052] In some embodiments, the polynucleotide comprises an AAV ITR at its 5' end and an AAV ITR at its 3' end.

[0053] In some embodiments, the polynucleotide comprises: (a) 5′ AAV ITR; (b) CMV promoter; (c) a nucleotide sequence encoding a complement factor I (CFI) cofactor, preferably FHL1; (d) a linker, optionally comprising a furin cleavage site, a GSG, a 11a1D and a F2A sequence; (e) a nucleotide sequence encoding CFI; (f) a poly-A signal, preferably the bovine growth hormone poly-A signal; and (g) 3' AAV ITR Includes.

[0054] In some embodiments, the polynucleotide comprises: (a) 5′ AAV ITR; (b) CMV promoter; (c) a nucleotide sequence encoding a complement factor I (CFI) cofactor, preferably FHL1; (d) a linker, optionally comprising a furin cleavage site, a GSG, a 11a1D and a F2A sequence; (e) a nucleotide sequence encoding CFI; (f) a WPRE regulatory element, preferably a WPRE3 regulatory element; (g) a poly-A signal, preferably the bovine growth hormone poly-A signal; and (h) 3' AAV ITR Includes.

[0055] In a preferred embodiment, the polynucleotide comprises (a) 5′ AAV ITR; (b) CMV promoter; (c) the nucleotide sequence encoding FHL1; (d) a linker containing a furin cleavage site, GSG, 11a1D and F2A sequences; (e) a nucleotide sequence encoding CFI; (f) WPRE3 regulatory element; (g) a poly-A signal, preferably the bovine growth hormone poly-A signal; and (h) 3' AAV ITR Includes.

[0056] In some embodiments, the complement factor I (CFI) cofactor is selected from the group consisting of complement factor H-like protein 1 (FHL1); complement factor H (CFH); complement receptor 1 (CR1) or a fragment thereof; and membrane cofactor protein (MCP) or a fragment thereof.

[0057] In some embodiments, the complement factor I (CFI) cofactor is complement factor H (CFH). In some embodiments, the complement factor I (CFI) cofactor is complement receptor 1 (CR1) or a fragment thereof. In some embodiments, the complement factor I (CFI) cofactor is membrane cofactor protein (MCP) or a fragment thereof.

[0058] In a preferred embodiment, the complement factor I (CFI) cofactor is complement factor H-like protein 1 (FHL1).

[0059] In some embodiments, the AAV ITRs are AAV2 or AAV8 ITRs. In preferred embodiments, the AAV ITRs are AAV2 ITRs.

[0060] In some embodiments, the nucleotide sequence encoding a Complement Factor I (CFI) cofactor (such as FHL1 or CFH) is codon optimized. In some embodiments, the nucleotide sequence encoding a CFI is codon optimized. In preferred embodiments, the nucleotide sequence encoding a Complement Factor I (CFI) cofactor (such as FHL1 or CFH) is codon optimized.

[0061] In some embodiments, the nucleotide sequence encoding FHL1 has at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:12.

[0062] In a preferred embodiment, the nucleotide sequence encoding FHL1 is SEQ ID NO:12.

[0063] In some embodiments, the nucleotide sequence encoding CFI has at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:10.

[0064] In a preferred embodiment, the nucleotide sequence encoding CFI is SEQ ID NO:10.

[0065] In a preferred embodiment, the nucleotide sequence encoding FHL1 is SEQ ID NO:12 and the nucleotide sequence encoding CFI is SEQ ID NO:10.

[0066] In some embodiments, the polynucleotide comprises the nucleotide sequence of SEQ ID NO:22, or a nucleotide sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO:22.

[0067] In some embodiments, the polynucleotide comprises the nucleotide sequence of SEQ ID NO:23, or a nucleotide sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO:23.

[0068] In some embodiments, the polynucleotide is no larger than 5.2, 5.1, 5.0, 4.9, 4.8, or 4.7 kb. In preferred embodiments, the polynucleotide is no larger than 4.7 kb.

[0069] In another aspect, the present invention provides a vector comprising a polynucleotide of the present invention.

[0070] In some embodiments, the vector is an adeno-associated viral (AAV), retroviral, lentiviral, or adenoviral vector.

[0071] In a preferred embodiment, the vector is an AAV vector.

[0072] In some embodiments, the vector is in the form of a viral vector particle.

[0073] In some embodiments, the AAV vector comprises an AAV2 or AAV8 genome.

[0074] In some embodiments, the AAV vector particle comprises AAV2 or AAV8 capsid proteins.

[0075] In some embodiments, the AAV vector particle comprises an AAV2 genome and an AAV2 capsid protein (AAV2 / 2). In other embodiments, the AAV vector particle comprises an AAV2 genome and an AAV8 capsid protein (AAV2 / 8). In other embodiments, the AAV vector particle comprises an AAV8 genome and an AAV8 capsid protein (AAV8 / 8).

[0076] In another aspect, the present invention provides a cell comprising a polynucleotide of the present invention.

[0077] In another aspect, the invention provides a cell transduced with a vector of the invention.

[0078] In another aspect, the invention provides a pharmaceutical composition comprising a polynucleotide, vector or cell of the invention in combination with a pharma- ceutically acceptable carrier, diluent or excipient.

[0079] In a preferred embodiment, the pharmaceutical composition is for intraocular administration.

[0080] In another aspect, the present invention provides a polynucleotide, vector or cell of the invention for use in therapy.

[0081] In another aspect, the invention provides a polynucleotide, vector or cell of the invention for use in treating or preventing an eye disorder.

[0082] In another aspect, the invention provides a polynucleotide, vector or cell of the invention for use in treating or preventing a complement-mediated or complement-associated disorder.

[0083] In another aspect, the invention provides a polynucleotide, vector, or cell of the invention for use in treating or preventing a complement-mediated eye disorder.

[0084] In another aspect, the invention provides a polynucleotide, vector or cell of the invention for use in treating or preventing complement-mediated or complement-associated kidney disorders or complement-mediated or complement-associated disorders of the central nervous system (CNS).

[0085] In another aspect, the present invention provides a method for treating or preventing a complement-mediated or complement-associated eye disorder, comprising administering to a subject in need thereof a polynucleotide, vector or cell of the present invention.

[0086] In another aspect, the present invention provides a method of providing (i) a complement factor I (CFI) cofactor and (ii) complement factor I (CFI) to a subject, comprising delivering a polynucleotide, vector or cell of the present invention to the subject, preferably to the eye of the subject.

[0087] In another aspect, the invention provides a method of providing a subject with (i) complement factor H-like protein 1 (FHL1) or complement factor H (CFH), and (ii) complement factor I (CFI), comprising delivering a polynucleotide, vector, or cell of the invention to an eye of the subject.

[0088] In some embodiments, the disorder is associated with overactivity of the complement C3b feedback cycle and / or underactivity of the C3b degradation cycle (see FIG. 1).

[0089] In some embodiments, the disorder is a chronic complement-mediated or chronic complement-associated inflammatory condition.

[0090] In some embodiments, the disorder is a complement-mediated chronic inflammatory condition of the eye.

[0091] In some embodiments, the disorder is age-related macular degeneration (AMD) or diabetic retinopathy. In other embodiments, the disorder is glaucoma, Stargardt's disease, central serous chorioretinopathy, retinitis pigmentosa or uveitis. Preferably, the uveitis is posterior uveitis.

[0092] In preferred embodiments, the disease is AMD. In some embodiments, the AMD is dry AMD.

[0093] In some embodiments, the subject has been diagnosed with or is at risk for acquiring AMD.

[0094] In some embodiments, the use is for treating or preventing a disorder in a subject: (a) having a lower than normal complement factor I activity or concentration in the eye and / or serum, preferably a concentration of 0-30, 0-20, or 0-10 μg / mL in serum or an equivalent activity; and / or (b) being heterozygous or homozygous for an age-related macular degeneration (AMD)-associated SNP, preferably a rare complement factor I variant;

[0095] In some embodiments, the use is for treating or preventing a disorder in a subject: (a) has a normal level of complement factor I activity or concentration in the eye and / or serum, preferably at least 30 μg / mL, e.g., 30 to 40 μg / mL in serum; and / or (b) not carrying a rare complement factor I mutant allele;

[0096] In another aspect, the present invention provides a polynucleotide, vector or cell of the present invention for use in treating or preventing age-related macular degeneration (AMD). In a preferred embodiment, the AMD is dry AMD.

[0097] In another aspect, the invention provides a polynucleotide, vector or cell of the invention for use in treating or preventing diabetic retinopathy.

[0098] In some embodiments, the formation of geographic atrophy is prevented or reduced and / or the amount of geographic atrophy is reduced.

[0099] In some embodiments, the progression of geographic atrophy is slowed.

[0100] In some embodiments, the increase in the area of ​​geographic atrophy is reduced by at least 10% over a 12 month period following administration to the treated eye of the subject as compared to the untreated eye over the same period. In other embodiments, the increase in the area of ​​geographic atrophy is reduced by at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% over a 12 month period following administration to the treated eye of the subject as compared to the untreated eye over the same period.

[0101] In some embodiments, administration of the polynucleotide, vector or cell increases the level of C3b inactivating and iC3b degrading activity in a subject or in the eye, such as in the subject's retinal pigment epithelium (RPE), optionally to a level that exceeds normal levels in the subject, or in the eye or RPE.

[0102] In another aspect, the invention provides a polynucleotide, vector or cell of the invention for use in improving or restoring vision or visual acuity in a subject suffering from an ocular disorder, such as, for example, an ocular disorder disclosed herein. In another aspect, the invention provides a polynucleotide, vector or cell of the invention for use in alleviating loss of vision or visual acuity, such as, for example, loss of vision or visual acuity associated with an ocular disorder, such as, for example, an ocular disorder disclosed herein.

[0103] In another aspect, the invention provides a polynucleotide, vector or cell of the invention for use in improving or restoring reading speed in a subject suffering from an eye disorder, such as, for example, an eye disorder disclosed herein. In another aspect, the invention provides a polynucleotide, vector or cell of the invention for use in reducing a decrease in reading speed in a subject, such as, for example, a decrease in reading speed associated with an eye disorder, such as, for example, an eye disorder disclosed herein.

[0104] In another aspect, the invention provides a polynucleotide, vector, or cell of the invention for use in reducing or preventing photoreceptor loss and / or retinal pigment epithelium (RPE), e.g., photoreceptor loss and / or RPE associated with an ocular disorder, such as the ocular disorders disclosed herein.

[0105] In some embodiments, the polynucleotide, vector or cell is administered intraocularly.

[0106] In some embodiments, the polynucleotide, vector or cells are administered to the subject's eye by subretinal, direct retinal, suprachoroidal or intravitreal injection.

[0107] In some embodiments, the polynucleotide, vector or cells are administered to the subject's eye by subretinal injection.

[0108] In some embodiments, the polynucleotide or vector of the present invention does not include a hAAT promoter.In some embodiments, the polynucleotide or vector of the present invention does not include an ApoR enhancer.In other embodiments, the polynucleotide or vector of the present invention does not include two ApoR enhancers.

[0109] In some embodiments, a vector of the invention does not comprise an AAV2 genome and an AAV8 capsid protein, i.e., a vector of the invention is not an AAV2 / 8 vector.

[0110] In some embodiments, the polynucleotides, vectors, or cells of the invention are not administered systemically, hi other embodiments, the polynucleotides, vectors, or cells of the invention are not administered intravenously.

[0111] Those skilled in the art will appreciate that the above description of the treatment of AMD is based on the theory of regulating an overactivated complement system (as a result of an overactive C3b feedback cycle and / or an underactive C3b degradation cycle), and therefore (apart from the text specifically relating to intraocular administration) the description applies equally to other chronic inflammatory conditions in which the complement system is involved. Such disorders may be treated by administration of the products, proteins, vectors, cells and compositions described herein, either systemically (e.g., via peripheral venous infusion), locally (e.g., intrathecally), or by direct delivery to target tissues or organs (e.g., liver, kidney). [Brief description of the drawings]

[0112] [Figure 1] C3b feedback (amplification) and degradation (downregulation) cycle of the alternative pathway of vertebrate complement ("I" = complement factor I; "H" = complement factor H; "B" = complement factor B; and "D" = complement factor D). [Diagram 2] (A) Cofactor assay based on ELISA-based measurement of cleavage of C3b to iC3b. Concentrations of CFI and C3b were fixed and CFH or FHL1 was titrated at the indicated ratios. (B) LPS deposition assay based on supplementation of normal serum with complement factor I (F1), complement factor H (FH), or complement factor H-like protein 1 (FHL1). [Diagram 3] Western blot analysis of supernatants from vector transduction of HEK293 cells (RC001, control vector containing wild-type FHL1; GT005, control vector containing wild-type CFI). [Figure 4] ELISA analysis of supernatants from vector transduction of HEK293 cells (RC001, control vector containing wild-type FHL1; GT005, control vector containing wild-type CFI). [Diagram 5]Alkaline gel analysis of vector genome packaging (top panel). Comparison of the ratio of full / empty viral particles determined by qPCR and capsid ELISA (bottom panel) (RC001, control vector containing wild-type FHL1; GT005, control vector containing wild-type CFI). [Figure 6] Western blot analysis (top panel) and C3b cleavage assay using ELISA (bottom panel) (RC001, control vector containing wild-type FHL1; GT005, control vector containing wild-type CFI). [Figure 7] Comparison of complement factor I (FI):complement factor H (FH) ratios in plasma (n=80) and vitreous humor (n=29). [Figure 8] LPS deposition assay to measure C3 deposition. Fl = complement factor I, sCR1 = soluble complement receptor 1, FH = complement factor H, FHL1 = factor H-like protein 1. Numbers above each bar indicate the percent reduction in C3 deposition compared to serum alone. [Figure 9] Western blot analysis of supernatants of in vitro transduced HEK-293 cells. UTC = non-transduced cells, GT005 = CFI-expressing AAV, GT007 = CFI- and FHL1-expressing AAV, RC001 = FHL1-expressing AAV. [Figure 10] C3b Western blot and iC3b ELISA of cofactor assays to test the functional activity of the constructs. (A) Western blot of C3b cofactor assay. (B) iC3b ELISA of C3b cofactor assay. C3b = complement C3b, CFI = complement factor I, FHL1 = complement factor H-like protein 1, UTD = non-transduced cells. [Figure 11] Isolectin stained area of ​​choroidal flatmounts. Isolectin stained area data were non-normally distributed as assessed by the Kolmogorov-Smirnov test, and statistical significance of observed differences was determined using generalized linear model analysis. *=P<0.05; ***=p<0.0001, ****=P<0.00001. Points represent individual laser burn results and lines represent mean values. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0113] As used herein, the terms "comprising," "comprises," and "comprised of" are synonymous with "including" or "includes," or "containing" or "contains," and are inclusive or open-ended and do not exclude additional, unrecited members, elements, or steps. The terms "comprising," "comprises," and "comprised of" also include the term "consisting of." complement system

[0114] The complement system is an integral part of the humoral immune system and is involved in tissue inflammation, cell opsonization, and cell lysis. It provides protection against microorganisms and mediates the clearance of exogenous and endogenous cellular debris from host tissues.

[0115] The complement system cascade is composed of three activation pathways. All pathways ultimately terminate with the central cleavage of the C3 factor and the generation of its active fragments C3a and C3b. C3a is an anaphylatoxin that triggers various chemotactic and proinflammatory responses, such as recruitment of inflammatory cells and increased permeability of the microvasculature, whereas C3b is responsible for opsonization of foreign surfaces covalently bound to C3b. Opsonization by activated C3 fragments (C3b and iC3b) serves three main functions: (i) clearance of cellular debris by phagocytes (e.g., macrophages or microglia) and stimulation of the adaptive immune system (B and T cells), (ii) amplification of complement activation via the formation of surface-bound C3 convertase, and (iii) assembly of C5 convertase.

[0116] The assembly of C5 convertases is responsible for C5 cleavage, which results in the formation of the cytolytic membrane attack complex (MAC) that can generate perforations in the cell membrane, thereby facilitating cell lysis and the elimination of unwanted cells. Through all of these activities, the innate complement cascade supports and promotes the function of downstream mechanisms of the immune system that protect the integrity of host tissues. Overall, activation of the complement pathway results in a proinflammatory response, including the generation of MAC to mediate cell lysis, the release of chemokines to attract inflammatory cells to the site of injury, and enhanced capillary permeability to promote the extravasation of infiltrating leukocytes. Under physiological conditions, complement activation is effectively controlled by the concerted action of soluble and membrane-bound complement regulatory molecules (CRMs). Soluble complement regulators, such as C1 inhibitor, anaphylatoxin inhibitor, C4b-binding protein (C4BP), complement factor H (CFH), complement factor I (CFI), clusterin, and vitronectin, limit the action of complement in human tissues at multiple sites in the cascade reaction. Furthermore, each individual cell is protected from homologous complement attack by surface proteins such as complement receptor 1 (CR1, CD35), membrane cofactor protein (CD46), and glycosylphosphatidylinositol-anchored proteins such as decay accelerating factor (CD55) or CD59 molecules. Notably, host cells and tissues that are insufficiently protected from complement attack may undergo bystander cell lysis.

[0117] The present invention relates to the treatment or prevention of complement-mediated disorders, for example of the eye. For example, the complement-mediated disorder may be a disorder associated with defects in the regulation of the alternative pathway, in particular with hyperactivity of the complement C3b feedback cycle and / or hypoactivity of the C3b degradation cycle.

[0118] In some embodiments, before administration of the product, polynucleotide, vector, cell or pharmaceutical composition of the present invention, the subject has a low level (e.g., lower than normal level) of complement factor I activity, for example, a low level of complement factor I activity in the eye and / or a low serum level of complement factor I activity. The lower than normal level of complement factor I activity may be due to lower than normal expression of normally functioning complement factor I, or at least partial (e.g., heterozygous) expression (normal or lower than normal level) of a non-functional or subfunctional variant of complement factor I (such a subject may carry one or more copies of an AMD-associated SNP, for example, the subject may be homozygous or heterozygous for one of the rare complement factor I variants further described below). Thus, the subject may have a low concentration (e.g., lower than normal concentration) of complement factor I in the eye and / or serum. For a human subject, a normal level of complement factor I activity (activity to inactivate C3b and to degrade iC3b) may be equivalent to that provided by 30-40 μg / mL of complement factor I in the subject's serum. Thus, in subjects with low activity of complement factor I, complement factor I activity in serum may correspond to less than 30 μg / mL and greater than 0 μg / mL of complement factor I (such as 0-20 or 0-10 μg / mL) (these are ranges of serum concentrations of complement factor I, which may include subjects with low concentrations of complement factor I).

[0119] Thus, the subject treated by the present invention may suffer from or be at risk of developing a complement-mediated eye disorder, such as AMD, more particularly dry AMD (e.g., characterized by geographic atrophy). For example, the subject may be homozygous or heterozygous for susceptibility to one or more SNPs associated with a complement-mediated disorder.

[0120] In some embodiments, the subject is at risk of developing AMD.For example, the subject may be homozygous or heterozygous for susceptibility to one or more SNPs associated with AMD, for example, rare mutations in complement factor I that are generally associated with advanced AMD and lead to reduced serum complement factor I levels (Kavanagh et al., (2015) Hum Mol Genet 24:3861-3870). In particular, the subject may carry one or two copies of one or more of the following rare complement factor I variants: rs144082872 (encodes P50A); 4:110687847 (encodes P64L); rs141853578 (encodes G119R); 4:110685721 (encodes V152M); 4:110682846 (encodes G162D); 4:110682801 (encodes N177I); rs146444258 (encodes A240G); rs182078921 (encodes G287R); rs41278047 (encodes K441R); and rs121964913 (encodes R474).

[0121] The invention may further include determining whether a subject is at risk for developing a complement-mediated disorder (e.g., AMD), e.g., by determining whether the subject is homozygous or heterozygous susceptible to one or more SNPs associated with a complement-mediated disorder (e.g., by determining whether the subject is homozygous or heterozygous susceptible to one or more of the rare complement factor I variants associated with AMD described above).

[0122] Alternatively, a subject may have normal levels of endogenous complement factor I activity or concentration, for example in the eye and / or serum, and / or may not carry a rare variant complement factor I allele.

[0123] In some embodiments, administering the product, polynucleotide, vector, cell or pharmaceutical composition of the present invention thereby increases the level of C3b inactivating and iC3b degrading activity in the eye of the subject.In other embodiments, administering the product, polynucleotide, vector, cell or pharmaceutical composition of the present invention thereby increases the level of C3b inactivating and iC3b degrading activity in the eye of the subject to a level that exceeds the normal level in the eye.More specifically, the level of C3b inactivating and iC3b degrading activity is increased in the RPE of the eye. It will be appreciated that C3b inactivating and iC3b degrading activity in a subject following provision of a product of the invention and / or expression of complement factor I and a CFI cofactor, such as complement factor H-like protein 1, from a polynucleotide or vector of the invention may include C3b inactivating and iC3b degrading activity from the subject's endogenous complement factor I (i.e., complement factor I in the subject that is not provided by the product or produced by expression from a polynucleotide or vector), and C3b inactivating and iC3b degrading activity supplied by a product of the invention or produced by expression from a polynucleotide or vector of the invention, such that the total level of C3b inactivating and iC3b degrading activity in the subject is greater than normal levels.

[0124] In some embodiments, the level of C3b inactivating and iC3b degrading activity in a subject, eg, in the eye, is increased to a level at least 5%, 10%, 15%, 20%, or 25% higher than normal levels.

[0125] In other embodiments, the level of C3b inactivating and iC3b degrading activity in a subject, e.g., in the eye, is increased to up to twice normal levels, or to up to 80%, 60%, 40%, or 20% of normal levels.

[0126] For example, the level of C3b inactivating and iC3b degrading activity in a subject, e.g., in the eye, can be increased to a level that is 5-100%, 5-80%, 5-60%, 5-40%, 5-20%, 10-100%, 10-80%, 10-60%, 10-40%, 10-20%, 15-100%, 15-80%, 15-60%, 15-40%, 15-20%, 20-100%, 20-80%, 20-60%, 20-40%, 25-100%, 25-80%, 25-60% or 25-40% of normal levels.

[0127] In some embodiments, administration of a product, polynucleotide, vector, cell or pharmaceutical composition of the invention does not detectably increase the level of C3b inactivating and iC3b degrading activity in the plasma / serum of a subject. In other embodiments, administration of a product, polynucleotide, vector, cell or pharmaceutical composition of the invention does not detectably increase the level of C3b inactivating and iC3b degrading activity in the plasma / serum of a subject to a level higher than normal levels.

[0128] In the previous section, unless clearly not applicable, references to complement factor I and its activity of inactivating C3b and degrading iC3b may be replaced with CFI cofactor, preferably complement factor H or complement factor H-like protein 1, and its ability to act as a cofactor for complement factor I-mediated cleavage of C3b and increase the dissociation rate of C3 convertase and C5 convertase, respectively. In some embodiments, prior to administration of the product, polynucleotide, vector, cell or pharmaceutical composition of the invention, the subject has a low level (e.g., lower than normal level) of complement factor H, e.g., a low level of complement factor H in the eye and / or a low serum level of complement factor H. For a human subject, the normal level of complement factor H may be about 200-500 μg / mL in the subject's serum. Thus, in a subject with a low level of complement factor H, the level in serum may be less than 200 μg / mL, more than 0 pg / mL, e.g., 0-100 μg / mL. Alternatively, the subject may have normal levels of endogenous complement factor H, for example in the eye and / or serum. Complement factor I (CFI)

[0129] Complement factor I (factor I, CFI), also known as C3b / C4b inactivator, is a protein that in humans is encoded by the CFI gene.

[0130] Complement factor I is a serine protease that circulates in a zymogen-like state at a concentration of approximately 35 μg / mL (Roversi et al., (2011) PNAS 108:12839-12844) (Nilsson et al., (2011) Mol Immunol 48:1611-1620). Complement factor I protein is a heavily N-glycosylated heterodimer consisting of two polypeptide chains linked by a single disulfide bond. The heavy chain (50 kDa) contains an N-terminal region; a Fl membrane attack complex (FIMAC) domain; a CD5-like domain or a scavenger receptor cysteine-rich (SRCR) domain; two low density lipoprotein receptor (LDLr) domains; and a C-terminal region of unknown function that is a site of sequence diversity among species (Roversi et al., (2011) PNAS 108:12839-12844). The light chain (38 kDa) contains a serine protease (SP) domain with conserved catalytic residues (Goldberger et al. (1987) J Biol Chem 262:10065-10071).

[0131] Complement factor I inactivates C3b by cleaving it into iC3b, C3d, and C3d,g, and in a similar manner inactivates C4b by cleaving it into C4c and C4d. To properly perform its functions, complement factor I requires the presence of cofactor proteins such as C4b-binding protein (C4BP), complement factor H (CFH), complement receptor 1 (CR1 / CD35), and membrane cofactor protein (MCP / CD46) (Degn et al., (2011) Am J Hum Genet 88:689-705).

[0132] iC3b cannot bind factor B and therefore cannot perpetuate amplification of the complement cascade or activation via the alternative pathway. Thus, when C3b is cleaved to iC3b, neither initiation of the alternative pathway nor activation of the terminal complement cascade occurs.

[0133] iC3b can provide proinflammatory effects by binding to and activating complement receptor 3 (CR3) (CD11b / CD18) on mononuclear phagocytes such as polymorphonuclear leukocytes (mainly neutrophils), NK cells, and macrophages.

[0134] Complement factor I can process iC3b to C3dg through a protease activity that requires the cofactor CR1. C3dg cannot bind to CR3. Because iC3b reacting with the complement receptor CR3 is the primary mechanism by which complement activation causes inflammation, degradation of iC3b to C3dg is essential to reduce complement-induced inflammation (Lachmann (2009) Adv. Immunol. 104:115-149).

[0135] The unique ability of complement factor I to promote the cleavage of C3b to iC3b and accelerate the degradation of iC3b, combined with its relatively low concentration in human serum, which impacts the amount that needs to be delivered for therapeutic effect, makes it a particularly advantageous target.

[0136] In some embodiments, a complement factor I polypeptide can cleave C3b into inactive degradation products, for example, a complement factor I polypeptide can cleave C3b into iC3b.

[0137] In some embodiments, a complement factor I polypeptide can process iC3b into inactive degradation products, for example, a complement factor I polypeptide can process iC3b into C3dg.

[0138] In a preferred embodiment, the complement factor I polypeptide is capable of cleaving C3b into iC3b and processing iC3b into C3dg.

[0139] Suitably, a fragment or derivative of complement factor I may retain at least 50%, 60%, 70%, 80%, 90%, 95% or 100% of the C3b inactivating and iC3b degrading activity of native complement factor I.

[0140] The activity of complement factor I, or its fragments or derivatives, to inactivate C3b and degrade iC3b can be determined using any suitable method known to those skilled in the art. For example, the measurement of the proteolytic activity of complement factor I is described in Hsiung et al. (Biochem.J.(1982)203:293-298). Both hemolytic and agglutination assays of CFI activity are described in Lachmann PJ&Hobart MJ(1978) "Complement Technology" (Handbook of Experimental Immunology 3rd Edition, DM Weir Blackwells Scientific Publications Chapter 5A p17). A more detailed description, including proteolytic assays, is described by Harrison RA(1996) in "Weir's Handbook of Experimental Immunology" (5th Edition, Ed.; Herzenberg Leonore A'Weir DM, Herzenberg Leonard A&Blackwell C Blackwells Scientific Publications Chapter 75 36-37). Agglutination assays are highly sensitive and can be used to detect both the initial (double) clip that converts fixed C3b to iC3b and acquires reactivity with conglutinin, and to detect the final clip to C3dg by starting with fixed iC3b and looking for loss of reactivity with conglutinin. A hemolytic assay is used for conversion of C3b to iC3b, and a proteolytic assay detects all clips.

[0141] In some embodiments, the complement factor I is human complement factor I.

[0142] An example of a human complement factor I protein is the human complement factor I protein having UniProtKB accession number P05156. This exemplary sequence is 583 amino acids in length (disclosed as SEQ ID NO:1), of which amino acids 1-18 form a signal sequence.

[0143] In some embodiments, the amino acid sequence of complement factor I is SEQ ID NO:1.

[0144] In another embodiment, the amino acid sequence of complement factor I is the sequence disclosed as positions 19 to 583 of SEQ ID NO:1. [ka]

[0145] In some embodiments, the amino acid sequence of complement factor I is SEQ ID NO: 9, which corresponds to NCBI Accession No. NP_000195. In other embodiments, the amino acid sequence of complement factor I is the sequence disclosed as positions 19 to 583 of SEQ ID NO: 9. [ka]

[0146] An exemplary wild-type nucleotide sequence encoding complement factor I is the nucleotide sequence having NCBI Accession No. NM_000204, disclosed herein as SEQ ID NO:2. [ka]

[0147] In some embodiments, the nucleotide sequence of complement factor I used in the present invention is codon-optimized. Different cells differ in their usage of certain codons. This codon bias corresponds to the bias of the relative abundance of certain tRNAs in cell types. Expression can be increased by changing the codons in the sequence so that they are adjusted to match the relative abundance of the corresponding tRNA. Similarly, expression can be decreased by deliberately selecting codons whose corresponding tRNAs are known to be rare in certain cell types. Thus, additional translational control is available.

[0148] A preferred nucleotide sequence encoding complement factor I is the nucleotide sequence disclosed as SEQ ID NO:10. [ka]

[0149] A further exemplary codon-optimized nucleotide sequence encoding complement factor I is SEQ ID NO:8. [ka]

[0150] In some embodiments, the nucleotide sequence encoding complement factor I has at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 10, 8 or 2, preferably SEQ ID NO: 10. Preferably, the protein encoded by the nucleotide sequence substantially retains the functional activity of the protein represented by SEQ ID NO: 1 or 9.

[0151] In other embodiments, the nucleotide sequence encoding complement factor I is SEQ ID NO:10, 8 or 2, preferably SEQ ID NO:10.

[0152] In other embodiments, the nucleotide sequence encoding complement factor I has at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 10, 8 or 2, preferably positions 55 to 1752 of SEQ ID NO: 10. Preferably, the protein encoded by the nucleotide sequence substantially retains the functional activity of the protein represented by SEQ ID NO: 1 or 9.

[0153] In another embodiment, the nucleotide sequence encoding complement factor I is SEQ ID NO:10, 8 or 2, preferably positions 55 to 1752 of SEQ ID NO:10.

[0154] In other embodiments, the nucleotide sequence encoding complement factor I encodes an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:1 or 9. Preferably, the amino acid sequence substantially retains the functional activity of the protein represented by SEQ ID NO:1 or 9.

[0155] In other embodiments, the nucleotide sequence encoding complement factor I encodes the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:9.

[0156] In other embodiments, the nucleotide sequence encoding complement factor I encodes an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity to positions 19-583 of SEQ ID NO: 1 or 9. Preferably, the amino acid sequence substantially retains the functional activity of the protein represented by SEQ ID NO: 1 or 9.

[0157] In another embodiment, the nucleotide sequence encoding complement factor I encodes the amino acid sequence from positions 19 to 583 of SEQ ID NO: 1 or 9.

[0158] The advantage of the present invention is that complement factor I is particularly difficult to prepare in the form of purified protein. Therefore, the inventors have devised a method of regulating the complement system, for example to allow the treatment of age-related macular degeneration (AMD), for example by administering complement factor I in the form of an AAV vector comprising a nucleotide sequence encoding complement factor 1. The AAV vector can be administered to the site of interest, for example the eye, to allow in situ translation of complement factor I polypeptide. Complement factor I (CFI) cofactor

[0159] As used herein, the term "Complement factor I (CFI) cofactor" may refer to a protein that can act as a cofactor for the CFI-mediated cleavage of C3b.

[0160] In some embodiments, the complement factor I (CFI) cofactor is selected from the group consisting of complement factor H-like protein 1 (FHL1), complement factor H (CFH); complement receptor 1 (CR1) or a fragment thereof; and membrane cofactor protein (MCP) or a fragment thereof. In some embodiments, the complement factor I (CFI) cofactor is complement factor H In some embodiments, the complement factor I (CFI) cofactor is complement receptor 1 (CR1) or a fragment thereof. In some embodiments, the complement factor I (CFI) cofactors are membrane cofactor proteins (MCPs) or fragments thereof.

[0161] In a preferred embodiment, the complement factor I (CFI) cofactor is complement factor H-like protein 1 (FHL1). Complement factor H (CFH)

[0162] Complement factor H (Factor H, CFH) is a complement regulatory protein.

[0163] Complement factor H is a large (155 kDa) soluble glycoprotein present in human plasma at typical concentrations of 200-300 μg / mL (Hakobyan et al., (2008) 49(5):1983-90). The primary function of complement factor H is to regulate the alternative pathway of the complement system.

[0164] Complement factor H provides cofactor activity for the cleavage of C3b mediated by complement factor 1. Complement factor H also increases the rate of dissociation of the C3bBb complex (C3 convertase) and the (C3b)NBb complex (C5 convertase), thereby reducing the activity of the alternative complement pathway.

[0165] Complement factor H is composed of 20 complement control protein (CCP) modules (also called short consensus repeats or sushi domains), interconnected by short linkers (3–8 amino acid residues) and arranged in an extended head-to-tail fashion. Each of the CCP modules consists of approximately 60 amino acids with four cysteine ​​residue disulfides linked in a 1–3 2–4 configuration and a hydrophobic core built around a nearly invariant tryptophan residue. The CCP modules are numbered 1–20 (from the N-terminus of the protein). CCP1–4 and CCP19–20 bind C3b, while CCP6–8 and CCP19–20 bind GAGs and sialic acids (Schmidt et al., (2008) Journal of Immunology 181:2610-2619).

[0166] Gene therapy using complement factor H has been shown to be able to ameliorate induced AMD-like pathology in mice (Cashman et al. (2015) J. Gene Med. 17:229-243). Mice were subretinally co-injected with: (i) an adenoviral vector expressing complement component C3, previously shown to recapitulate many pathological features of human AMD; and (ii) an adenoviral vector expressing complement factor H. Compared to control animals receiving GFP instead of complement factor H, mice transduced with complement factor H showed a 91% reduction in endothelial cell proliferation, and a 69% attenuation of RPE atrophy. Electroretinography showed improved retinal function in mice administered complement factor H, and immunocytochemistry for rhodopsin and RPE65 was consistent with rescue of photoreceptors and RPE in such animals.

[0167] In some embodiments, the complement factor H polypeptide, or a fragment or derivative thereof, can act as a cofactor for complement factor 1-mediated cleavage of C3b. In some embodiments, the complement factor H polypeptide, or a fragment or derivative thereof, can increase the rate of dissociation of C3 and C5 convertases.

[0168] In a preferred embodiment, the complement factor H polypeptide or a fragment or derivative thereof is capable of acting as a cofactor for complement factor 1-mediated cleavage of C3b and increasing the rate of dissociation of C3 and C5 convertases.

[0169] In some embodiments, the complement factor H is human complement factor H.

[0170] An example of a human complement factor H protein is the human complement factor H protein having UniProtKB Accession No. P08603. This exemplary sequence is 1231 amino acids in length (disclosed as SEQ ID NO:3), of which amino acids 1-18 form a signal sequence.

[0171] In some embodiments, the amino acid sequence of complement factor H is SEQ ID NO: 3. In other embodiments, the amino acid sequence of complement factor H is positions 19-1231 of SEQ ID NO: 3. [ka]

[0172] An example of a nucleotide sequence encoding complement factor H is the nucleotide sequence having NCBI accession number NM_000186.

[0173] In some embodiments, the nucleotide sequence encoding complement factor H is SEQ ID NO:4. [ka]

[0174] In some embodiments, the nucleotide sequence encoding complement factor H has at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 4. Preferably, the protein encoded by the nucleotide sequence substantially retains the functional activity of the protein represented by SEQ ID NO:3.

[0175] In another embodiment, the nucleotide sequence encoding complement factor H is SEQ ID NO:4.

[0176] In other embodiments, the nucleotide sequence encoding complement factor H has at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to positions 55 to 3696 of SEQ ID NO: 4. Preferably, the protein encoded by the nucleotide sequence substantially retains the functional activity of the protein represented by SEQ ID NO: 3.

[0177] In another embodiment, the nucleotide sequence encoding complement factor H is from positions 55 to 3696 of SEQ ID NO:4.

[0178] In other embodiments, the nucleotide sequence encoding complement factor H encodes an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 3. Preferably, the amino acid sequence substantially retains the functional activity of the protein represented by SEQ ID NO:3.

[0179] In another embodiment, the nucleotide sequence encoding complement factor H encodes the amino acid sequence of SEQ ID NO:3.

[0180] In other embodiments, the nucleotide sequence encoding complement factor H encodes an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity to positions 19-1231 of SEQ ID NO: 3. Preferably, the amino acid sequence substantially retains the functional activity of the protein represented by SEQ ID NO: 3.

[0181] In another embodiment, the nucleotide sequence encoding complement factor H encodes the amino acid sequence from positions 19 to 1231 of SEQ ID NO:3. Complement factor H-like protein 1 (FHL1)

[0182] Complement factor H-like protein 1 (FHL1) is a splice variant of complement factor H that contains the first seven CCPs of complement factor H followed by a carboxy-terminal tail of four amino acids (Clark, SJ et al. (2015) J Clin Med 4:18-31).

[0183] In some embodiments, the FHL1 is human FHL1.

[0184] In some embodiments, the amino acid sequence of FHL1 is SEQ ID NO:11. [ka]

[0185] An example of a nucleotide sequence encoding FHL1 is as follows: [ka]

[0186] The nucleotide sequence of FHL1 used in the present invention is preferably codon optimized.

[0187] A preferred nucleotide sequence encoding FHL1 is SEQ ID NO:12. [ka]

[0188] In some embodiments, the nucleotide sequence encoding FHL1 has at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 12 or 16, preferably SEQ ID NO: 12. Preferably, the protein encoded by the nucleotide sequence substantially retains the functional activity of the protein represented by SEQ ID NO:11.

[0189] In another embodiment, the nucleotide sequence encoding FHL1 is SEQ ID NO:12 or 16, preferably SEQ ID NO:12. Complement receptor 1 (CR1)

[0190] Complement receptor 1 (CR1), also known as CD35, is a type I membrane-bound glycoprotein that belongs to the regulator of complement activity (RCA) family. CR1 can be found on the plasma membrane of erythrocytes, eosinophils, monocytes, macrophages, B-lymphocytes, a subpopulation of CD4+ T cells, dendritic cells, Langerhans cells of the skin, and glomerular podocytes.

[0191] CR1 is a single-chain glycoprotein of -200 kDa, the extracellular portion of which is It contains 30 complement control protein repeats (CCPs) or short consensus repeats. Non-membrane-bound soluble CR1 (sCR1) is found in plasma. It can be generated by release from leukocytes by cleavage of the surface form of CR1. The structure of CR1 and sCR1 is described, for example, in Liu, D. et al. (2009) Immunopharmacology and Immunotoxicology 31:524-535.

[0192] In some embodiments, the CR1 or fragment thereof is human CR1 or a fragment thereof.

[0193] An example of a CR1 sequence is: [ka]

[0194] An example of a nucleotide sequence encoding CR1 is as follows: [ka] JPEG2025032238000013.jpg196159

[0195] In some embodiments, CR1 or a fragment thereof is soluble CR1 (sCR1).

[0196] The CR1 fragment is preferably capable of acting as a cofactor for CFI-mediated cleavage of C3b. A person skilled in the art could readily determine such CFI activity using any suitable method known in the art, for example as disclosed herein.

[0197] CR1 contains two known binding sites for C3b (see Liu, D. et al., (2009) Immunopharmacology and Immunotoxicology 31:524-535). In a preferred embodiment, the CR1 fragment contains one or two C3b binding sites.

[0198] Preferably, the fragment of CR1 is a soluble fragment of CR1, generated, for example, by removing the transmembrane and cytoplasmic domains from full-length CR1 and / or by selecting a truncated form of CR1 that contains or consists of a particular CCP.

[0199] Examples of CR1 fragments are known in the art (see, e.g., International Publication No. WO2019138137). For example, a CR1 fragment can include or consist of CCP8-10 (e.g., corresponding to amino acids 491-684 of SEQ ID NO:24) and / or CCP15-17 (e.g., corresponding to amino acids 941-1134 of SEQ ID NO:24).

[0200] In some embodiments, the CR1 fragment comprises CCP8 to 10. In some embodiments, the CR1 fragment comprises CCP15 to 17. In some embodiments, the CR1 fragment comprises CCP8 to 10 and CCP15 to 17.

[0201] In other embodiments, the nucleotide sequence encoding CR1 encodes an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 24. Preferably, the amino acid sequence substantially retains the functional activity of the protein represented by SEQ ID NO:24.

[0202] In other embodiments, the nucleotide sequence encoding CR1 encodes the amino acid sequence of SEQ ID NO:24.

[0203] In other embodiments, the nucleotide sequence encoding CR1 encodes an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity to positions 491-684 of SEQ ID NO: 24. Preferably, the amino acid sequence substantially retains the functional activity of the protein represented by SEQ ID NO:24.

[0204] In another embodiment, the nucleotide sequence encoding CR1 encodes the amino acid sequence from positions 491 to 684 of SEQ ID NO:24.

[0205] In other embodiments, the nucleotide sequence encoding CR1 encodes an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity to positions 941-1134 of SEQ ID NO: 24. Preferably, the amino acid sequence substantially retains the functional activity of the protein represented by SEQ ID NO:24.

[0206] In another embodiment, the nucleotide sequence encoding CR1 encodes the amino acid sequence from positions 941 to 1134 of SEQ ID NO:24. Membrane Cofactor Proteins (MCPs)

[0207] Membrane cofactor protein (MCP), also known as CD46, is a type I membrane protein that functions as a regulatory part of the complement system and acts as a cofactor for CFI.

[0208] The extracellular domain of MCP contains four short consensus repeats (SCRs).

[0209] In some embodiments, the MCP or fragment thereof is human MCP or a fragment thereof.

[0210] An example of an MCP sequence is: [ka]

[0211] An example of a nucleotide sequence encoding MCP is as follows: [ka]

[0212] In some embodiments, the MCP or fragment thereof is a soluble MCP.

[0213] The MCP fragment is preferably capable of acting as a cofactor for CFI-mediated cleavage of C3b. One of skill in the art could readily determine such CFI activity using any suitable method known in the art, e.g., as disclosed herein.

[0214] Preferably, the fragment of MCP is a soluble fragment of MCP, generated, for example, by removing the transmembrane domain from a full-length MCP fragment and / or by selecting a truncated form of MCP that contains or consists of specific SCRs.

[0215] Preferably, the fragment of MCP is a soluble fragment of MCP, e.g., generated by removing the transmembrane domain from full-length MCP and / or by selecting MCP truncations that include or consist of specific SCRs. In some embodiments, the MCP fragment includes SCRs 2 and 3. In some embodiments, the MCP fragment includes SCRs 2, 3 and 4.

[0216] In other embodiments, the nucleotide sequence encoding MCP encodes an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 26. Preferably, the amino acid sequence substantially retains the functional activity of the protein represented by SEQ ID NO:26.

[0217] In other embodiments, the nucleotide sequence encoding MCP encodes the amino acid sequence of SEQ ID NO:26. Linker

[0218] In preferred embodiments, the nucleotide sequence encoding (i) is upstream of the nucleotide sequence encoding (ii). In other embodiments, the nucleotide sequence encoding (ii) is upstream of the nucleotide sequence encoding (i).

[0219] In some embodiments, the nucleotide sequences encoding (i) and (ii) are operably linked by a linker. In some embodiments, the linker comprises a self-cleaving 2A peptide sequence, such as a sequence that includes or is defined by a furin cleavage site, GSG, 11a1D, and F2A sequence.

[0220] In some embodiments, the linker is SEQ ID NO:17. [ka]

[0221] In other embodiments, the linker has at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 17. Preferably, the linker substantially retains the functional activity of SEQ ID NO: 17.

[0222] By "operably linked" it is understood that the individual components are linked together in a manner permitting them to carry out their functions substantially unimpeded. product

[0223] A product of the invention can be a composition (e.g., a pharmaceutical composition) comprising, for example, (i) a complement factor I (CFI) cofactor, (ii) complement factor I (CFI), or a nucleotide sequence encoding same, in a mixture. Alternatively, the product can be, for example, a kit comprising a preparation of (i) a complement factor I (CFI) cofactor and (ii) complement factor I (CFI) or a nucleotide sequence encoding same, and optionally instructions for administering the preparations simultaneously, sequentially or separately to a subject in need thereof.

[0224] A product of the invention can be a composition (e.g., a pharmaceutical composition) comprising, for example, (i) Complement Factor H-like protein 1 (FHL1) or Complement Factor H (CFH), and (ii) Complement Factor I (CFI) or a nucleotide sequence encoding same, in a mixture. Alternatively, the product can be a kit comprising, for example, a preparation of (i) Complement Factor H-like protein 1 (FHL1) or Complement Factor H (CFH), and (ii) Complement Factor I (CFI) or a nucleotide sequence encoding same, and optionally instructions for administering the preparations simultaneously, sequentially or separately to a subject in need thereof. Protein transduction

[0225] As an alternative to delivery of polynucleotides to cells, the products and agents of the invention can be delivered to cells by protein transduction.

[0226] Protein transduction may be via vector delivery (Cai, Y. et al., (2014) Elife 3:e01911; Maetzig, T. et al., (2012) Curr. Gene Ther. 12:389-409). Vector delivery involves engineering a viral particle (e.g., a lentiviral particle) to contain the protein to be delivered to a cell. Thus, when the engineered viral particles enter a cell as part of their natural life cycle, the protein contained in the particle is carried into the cell.

[0227] Protein transduction may be via protein delivery (Gaj, T. et al., (2012) Nat. Methods 9:805-7). Protein delivery may also be achieved, for example, by utilizing a vehicle (e.g., liposomes) or by administering the protein itself directly to the cells. Polynucleotides

[0228] The polynucleotides of the present invention may comprise DNA or RNA, preferably DNA. They may be single-stranded or double-stranded. Those skilled in the art will understand that, as a result of the degeneracy of the genetic code, many different polynucleotides can code for the same polypeptide. Furthermore, those skilled in the art will understand that, using conventional techniques, nucleotide substitutions that do not affect the polypeptide sequence encoded by the polynucleotide of the present invention can be made to reflect the codon usage of any particular host organism in which the polypeptide of the present invention is expressed.

[0229] The nucleotide sequences of the invention disclosed herein may contain or lack a stop codon at their 3' end, depending, for example, on their location in a bicistronic vector. Thus, the disclosure encompasses the SEQ ID NOs disclosed herein with or without a stop codon.

[0230] Polynucleotides may be modified by any method available in the art. Such modifications may be carried out in order to enhance the in vivo activity or life span of polynucleotides of the invention.

[0231] Polynucleotides, such as DNA polynucleotides, may be produced recombinantly, synthetically, or by any means available to those of skill in the art. They may also be cloned by standard techniques.

[0232] Longer polynucleotides will generally be generated using recombinant means, for example, using polymerase chain reaction (PCR) cloning techniques. This involves creating a pair of primers (e.g., about 15-30 nucleotides) that flank the target sequence desired to be cloned, contacting the primers with mRNA or cDNA obtained from an animal or human cell, performing the polymerase chain reaction under conditions that result in amplification of the desired region, isolating the amplified fragment (e.g., by purifying the reaction mixture on an agarose gel), and recovering the amplified DNA. The primers may be designed to contain suitable restriction enzyme recognition sites so that the amplified DNA can be cloned into a suitable vector.

[0233] In some embodiments, the polynucleotide comprises or consists of the nucleotide sequence of SEQ ID NO:22, or a nucleotide sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO:22. [ka] JPEG2025032238000018.jpg7159

[0234] In some embodiments, the polynucleotide comprises or consists of the nucleotide sequence of SEQ ID NO:23, or a nucleotide sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO:23. [ka] JPEG2025032238000020.jpg241159 Eye structure

[0235] The agents disclosed herein can be delivered to the eye of a mammal, preferably a human, in connection with the treatment or prevention of ocular diseases such as age-related macular degeneration (AMD).

[0236] Those skilled in the art of treating eye disorders will have a detailed and thorough understanding of the structure of the eye, however, the following structures are described which are particularly relevant to the present invention. retina

[0237] The retina is a multi-layered membrane that lines the posterior chamber of the eye and senses images of the visual world that are transmitted to the brain via the optic nerve. From the inside to the outside of the eye, the retina includes layers of the neurosensory retina and the retinal pigment epithelium, and the choroid lies outside the retinal pigment epithelium. Neurosensory retina and photoreceptor cells

[0238] The neurosensory retina holds the photoreceptor cells that directly sense light. It contains the following layers: the inner limiting membrane (ILM); the nerve fiber layer; the ganglion cell layer; the inner plexiform layer; the inner nuclear layer; the outer plexiform layer; the outer nuclear layer (the nuclei of the photoreceptors); the outer limiting membrane (ELM); and the photoreceptors (the inner and outer segments of the rods and cones).

[0239] Those skilled in the art will have a detailed understanding of photoreceptor cells. Briefly, photoreceptor cells are specialized neurons located in the retina that convert light into biological signals.

[0240] Photoreceptor cells include rods and cones and are distributed differently throughout the retina.

[0241] Rod cells are found primarily in the outer retina. They are highly sensitive and provide vision at low light levels. On average, there are about 125 million rod cells in a normal human retina.

[0242] Cone cells are found throughout the retina, but are particularly concentrated in the fovea, a depression in the neurosensory retina responsible for central, high-resolution vision. Cone cells are less sensitive than rod cells. On average, there are 6-7 million cone cells in a normal human retina. retinal pigment epithelium The retinal pigment epithelium (RPE) is a pigmented layer of cells located just outside the neurosensory retina. The RPE performs multiple functions, including transporting nutrients and other substances to photoreceptor cells and absorbing scattered light to improve vision. choroid

[0243] The choroid is a vascular layer that lies between the RPE and the outer sclera of the eye. The choroid's vasculature allows the supply of oxygen and nutrients to the retina. Age-related Macular Degeneration (AMD)

[0244] The clinical progression of age-related macular degeneration (AMD) is characterized by stages according to changes in the macula. A hallmark of early AMD is the appearance of drusen, which are accumulations of extracellular debris beneath the retina that appear as yellow spots in the retina during clinical examination and on fundus photography. Drusen are classified by size as small (<63 μm), medium (63–124 μm), and large (>124 μm). They are also considered hard or soft, depending on the appearance of their margins on ophthalmic examination. Hard drusen have well-defined margins, whereas soft drusen have less-defined and fluid margins. The Age-Related Eye Disease Study (AREDS) Fundus Photography Severity Scale is one of the major classification systems used for this condition.

[0245] AMD is classified as "dry" and "wet" (exudative or neovascular) forms. Dry AMD is more common than wet AMD, but the dry form can progress to the wet form, and the two occur simultaneously in a significant number of cases. Dry AMD is typically characterized by progressive apoptosis of cells in the RPE layer, the overlying photoreceptor cells, and often the cells underlying the choriocapillaris layer. Confluent areas of RPE cell death with overlying photoreceptor atrophy are referred to as geographic atrophy (GA). Patients with this form of AMD experience a slow, progressive deterioration in central vision.

[0246] Wet AMD is characterized by bleeding and / or fluid leakage from abnormal blood vessels growing from the RPE and choroidal vasculature (choriocapillaris) beneath the macula, which can cause sudden vision loss. It is presumed that much of the vision loss experienced by patients is due to such choroidal neovascularization (CNV) and its secondary complications.

[0247] The treatment or prevention of AMD described herein may reduce or prevent the appearance of the above AMD phenotypes. Preferably, the treatment of AMD allows for the maintenance or improvement of visual function.

[0248] In some embodiments, treating or preventing AMD results in the prevention or reduction of the formation of geographic atrophy.In other embodiments, treating or preventing AMD slows the progression of geographic atrophy.For example, it results in at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90% reduction in the increase in GA area over 12 months after administration to the treated eye of the subject, compared with the untreated eye over the same period.In other embodiments, treating or preventing AMD results in the treatment of geographic atrophy, for example, the reduction of the amount of geographic atrophy.

[0249] In some embodiments, treating or preventing AMD results in the prevention or reduction of the formation of drusen. In other embodiments, treating or preventing AMD results in the reduction of existing drusen, e.g., a reduction in the size and / or number of existing drusen.

[0250] In some embodiments, treating or preventing AMD results in the prevention or reduction of complement deposition, hi other embodiments, treating or preventing AMD results in the reduction of existing complement deposition.

[0251] In some embodiments, treating or preventing AMD results in improved or restored vision or visual acuity, hi other embodiments, treating or preventing AMD reduces vision or visual acuity loss.

[0252] In some embodiments, treating or preventing AMD results in an improvement or restoration of reading speed in a subject. In other embodiments, treating or preventing AMD results in a reduction in the decline in reading speed in a subject.

[0253] In some embodiments, treating or preventing AMD results in the reduction or prevention of photoreceptor and / or retinal pigment epithelium (RPE) loss. diabetic retinopathy

[0254] Diabetic retinopathy is a condition characterized by damage to the blood vessels of the retina caused by high blood sugar levels associated with diabetes. Left untreated, diabetic retinopathy can lead to blindness.

[0255] Although subjects with mild diabetic retinopathy may have good vision, certain types of diabetic retinopathy, namely diabetic macular edema (DMO) and proliferative diabetic retinopathy (PDR), can threaten a subject's vision.

[0256] Diabetic macular edema is characterized by fluid leaking from damaged blood vessels at the back of the eye. The leaked fluid accumulates in the macula, which leads to swelling and blurred vision. This eventually leads to a decline in central vision that may make reading or driving impossible. Side vision usually remains normal.

[0257] Proliferative diabetic retinopathy is characterized by the closure of retinal blood vessels, which leads to the growth of abnormal and fragile blood vessels on the surface of the retina. This can lead to permanent loss of vision due to bleeding into the eye, scarring, and retinal detachment. Non-proliferative retinopathy is the early stage of diabetic retinopathy, and if left untreated, can lead to proliferative retinopathy. Therefore, treatment is intended for all stages and types of diabetic retinopathy. vector

[0258] A vector is a tool that enables or facilitates the transfer of entities from one environment to another. Adeno-associated virus (AAV) vectors

[0259] In one aspect, the invention provides an AAV vector comprising a polynucleotide of the invention.

[0260] Preferably, the AAV vector is in the form of an AAV vector particle.

[0261] Methods for preparing and modifying viral vectors and viral vector particles, such as those derived from AAV, are well known in the art.

[0262] An AAV vector may comprise an AAV genome, or a fragment or derivative thereof.

[0263] AAV is known to be capable of packaging genomes up to 5.2 kb in size (Dong, J.-Y. et al. (1996) Human Gene Therapy 7:2101-2112).

[0264] AAV genome is a polynucleotide sequence, which can code for the functions required for the production of AAV particles.These functions include the functions that are operated in the replication and packaging cycle of AAV in host cells, including the encapsidation of AAV genome into AAV particles.Naturally occurring AAV is replication-defective and depends on the provision of helper functions in trans to complete the replication and packaging cycle.Therefore, the AAV genome of the AAV vector of the present invention is typically replication-defective.

[0265] The AAV genome can be in a single-stranded form, either positive or negative sense, or in a double-stranded form. The use of the double-stranded form can bypass the DNA replication step in the target cell, thus facilitating expression of the transgene.

[0266] AAV genome can be derived from any naturally occurring serotype, isolate or clade of AAV.Therefore, AAV genome can be the whole genome of naturally occurring AAV.As known to those skilled in the art, naturally occurring AAV can be classified according to different biological systems.

[0267] Generally, AAV is referred to in terms of their serotype.Serotype corresponds to the variant subspecies of AAV, which has a unique reactivity that can be used to distinguish it from other variant subspecies by the expression profile of capsid surface antigen.Typically, the virus with a certain AAV serotype does not cross-react effectively with the neutralizing antibody specific for any other AAV serotype.

[0268] AAV serotypes include AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10 and AAV11, as well as recombinant serotypes recently identified from primate brain, such as Rec2 and Rec3. Any of these AAV serotypes can be used in the present invention.

[0269] In some embodiments, the AAV vector particle is an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, Rec2 or Rec3 AAV vector particle.

[0270] In some embodiments, the AAV may be an AAV1, AAV2, AAV5, AAV7, or AAV8 serotype.

[0271] In some embodiments, the AAV may be of the AAV2 or AAV8 serotype.

[0272] In some embodiments, the AAV may be of the AAV2 serotype. In other embodiments, the AAV may be of the AAV8 serotype.

[0273] The capsid protein may be a mutated capsid protein as disclosed in WO 2008 / 124724, which is incorporated herein by reference.

[0274] In some embodiments, the AAV vector comprises an AAV8 capsid with a Y733F mutation.

[0275] Reviews of AAV serotypes can be found in Choi et al. (2005) Curr. Gene Ther. 5:299-310 and Wu et al. (2006) Molecular Therapy 14:316-27. The sequence of the AAV genome, or the sequence of an element of the AAV genome, such as the ITR sequence, the rep gene or the cap gene, for use in the present invention may be derived from the following accession numbers for the AAV whole genome sequence: Adeno-associated virus 1 NC_002077, AF063497; Adeno-associated virus 2 NC_001401; Adeno-associated virus 3 NC_001729; Adeno-associated virus 3B NC_001863; Adeno-associated virus 4 NC_001829; Adeno-associated virus 5 Y18065, AF085716; Adeno-associated virus 6 NC_001862; Avian AAV ATCC VR-865 AY186198, AY629583, NC_004828; Avian AAV strain DA-1 NC_006263, AY629583; Bovine AAV NC_005889, AY388617.

[0276] AAVs can also be referred to in terms of clades or clones. This refers to the phylogenetic relationship of naturally occurring AAVs, typically referring to a phylogenetic group of AAVs that can be traced back to a common ancestor and includes all its descendants. Additionally, AAVs can be referred to in terms of specific isolates, i.e., genetic isolates of specific AAVs found in nature. The term genetic isolates refers to populations of AAVs that have undergone limited genetic mixing with other naturally occurring AAVs, thereby defining distinct populations that can be recognized at the genetic level.

[0277] Those skilled in the art can select the appropriate serotype, clade, clone or isolate of AAV for use in the present invention based on general knowledge. For example, AAV5 capsid has been shown to efficiently transduce primate cone photoreceptors, as evidenced by the successful correction of inherited color blindness (Mancuso et al. (2009) Nature 461:784-7).

[0278] The AAV serotype determines the tissue specificity of infection (or tropism) of the AAV. Thus, preferred AAV serotypes for use in the AAV administered to a patient according to the present invention are those that have a natural tropism or high efficiency for infection of target cells in the eye. In some embodiments, the AAV serotypes for use in the present invention transduce cells of the neurosensory retina, retinal pigment epithelium and / or choroid.

[0279] Typically, the AAV genome of a naturally occurring serotype, isolate, or clade of AAV contains at least one inverted terminal repeat (ITR). The ITR sequence acts in cis to provide a functional origin of replication, allowing integration and excision of the vector from the genome of the cell. In a preferred embodiment, one or more ITR sequences flank a nucleotide sequence encoding complement factor I, and / or complement factor H or FHL1. The AAV genome also typically contains packaging genes, such as the rep gene and / or the cap gene, which encode the packaging function of the AAV particle. The Rep gene encodes one or more of the proteins Rep78, Rep68, Rep52, and Rep40 or variants thereof. The cap gene encodes one or more capsid proteins, such as VP1, VP2, and VP3 or variants thereof. These proteins constitute the capsid of the AAV particle. Capsid variants are described below.

[0280] The promoter is operably linked to each of the packaging genes. Specific examples of such promoters include p5, p19, and p40 promoters (Laughlin et al., (1979) Proc. Natl. Acad. Sci. USA 76:5567-5571). For example, p5 and p19 promoters are commonly used to express the rep gene, and p40 promoter is commonly used to express the cap gene.

[0281] As mentioned above, the AAV genome used in the AAV vector of the present invention can therefore be the complete genome of a naturally occurring AAV. For example, a vector containing a complete AAV genome may be used to prepare an AAV vector or vector particle in vitro. However, although such a vector can in principle be administered to a patient, this will rarely be done in practice. Preferably, the AAV genome will be derivatized for the purpose of administering to a patient. Such derivatization is standard in the art, and the present invention encompasses the use of any known derivative of the AAV genome, and derivatives that can be produced by applying techniques known in the art. Derivatization of the AAV genome and AAV capsid is reviewed in Coura and Nardi (2007) Virology Journal 4:99 and Choi et al., and Wu et al. (see above).

[0282] The derivative of AAV genome includes any shortened or modified form of AAV genome that allows the expression of transgene from the AAV vector of the present invention in vivo.Typically, AAV genome can be significantly shortened to contain minimal viral sequence and still retain the above-mentioned functions.This is preferred for safety reasons, to reduce the risk of vector recombination with wild-type virus and to avoid the induction of cellular immune response due to the presence of viral gene protein in target cell.

[0283] Typically, the derivative will contain at least one inverted terminal repeat (ITR), preferably two or more ITRs, for example two or more ITRs. One or more of the ITRs can be derived from AAV genomes with different serotypes, or can be chimeric or mutant ITRs. A preferred mutant ITR is one that has trs (terminal resolution site) deletion. This deletion allows the genome to continue replicating, generating a single-stranded genome that contains both coding and complementary sequences, i.e., a self-complementary AAV genome. This allows bypass of DNA replication in target cells, thus facilitating the expression of the transgene.

[0284] The one or more ITRs are preferably flanked on either end by nucleotide sequences encoding complement factor I, and / or complement factor H or FHL1 (e.g., complement factor H or FHL1). The inclusion of one or more ITRs is preferred to support the formation of concatemers of the vectors of the invention in the nucleus of the host cell, e.g., after conversion of single-stranded vector DNA to double-stranded DNA by the action of host cell DNA polymerase.

[0285] The one or more ITRs are preferably flanked at either end by nucleotide sequences encoding complement factor I and / or CFI cofactors (e.g., complement factor H or FHL1). The inclusion of one or more ITRs is preferred to aid in the formation of concatemers of the vectors of the invention in the nucleus of the host cell, for example, after conversion of single-stranded vector DNA to double-stranded DNA by the action of host cell DNA polymerase. The formation of such episomal concatemers protects the vector construct during the life of the host cell, thereby allowing long-term expression of the transgene in vivo.

[0286] In a preferred embodiment, ITR elements are the only sequence that is retained from the native AAV genome in the derivative.Therefore, the derivative preferably does not contain the rep gene and / or cap gene of the native genome and any other sequence of the native genome.This is preferred for the reasons mentioned above, and also to reduce the possibility that the vector will be integrated into the host cell genome.In addition, reducing the size of the AAV genome allows for greater flexibility in incorporating other sequence elements (such as regulatory elements) into the vector in addition to the transgene.

[0287] Thus, the following parts may be removed in the derivatives of the present invention: one inverted terminal repeat (ITR) sequence, the replication (rep) gene and the capsid (cap) gene. However, in some embodiments, the derivatives may further include one or more rep and / or cap genes or other viral sequences of the AAV genome. Naturally occurring AAV integrates at a high frequency at a specific site on human chromosome 19, and shows negligible random integration frequency, so retention of integration ability in the vector may be acceptable in a therapeutic environment.

[0288] Where the derivatives include capsid proteins, i.e., VP1, VP2 and / or VP3, the derivatives may be chimeric, shuffled, or capsid-modified derivatives of one or more naturally occurring AAVs. In particular, the present invention encompasses the provision of capsid protein sequences from different serotypes, clades, clones, or isolates of AAV within the same vector (i.e., pseudotyped vectors).

[0289] Chimeric, shuffled, or capsid-modified derivatives are typically selected to provide one or more desired functions to the AAV vector. Thus, these derivatives may show improved efficiency of gene delivery, reduced immunogenicity (humoral or cellular), altered tropism range, and / or improved targeting of specific cell types compared to AAV vectors that contain naturally occurring AAV genomes, such as AAV2. Improved efficiency of gene delivery may be affected by improved receptor or co-receptor binding at the cell surface, improved internalization, improved transport within the cell and to the nucleus, improved uncoating of viral particles, and improved conversion of single-stranded genome to double-stranded form. Improved efficiency may also be related to altered tropism range or targeting of specific cell populations, so that the vector dose is not diluted by administration to tissues where it is not needed.

[0290] Chimeric capsid proteins include those generated by recombination between two or more capsid coding sequences of naturally occurring AAV serotypes. This may be done, for example, by a marker rescue approach in which a non-infectious capsid sequence of one serotype is co-transfected with a capsid sequence of a different serotype, and a directed selection is used to select the capsid sequence with the desired properties. The capsid sequences of different serotypes can be altered by homologous recombination in cells to generate novel chimeric capsid proteins.

[0291] Chimeric capsid proteins also include those generated by manipulating the capsid protein sequence to transfer particular capsid protein domains, surface loops, or particular amino acid residues between two or more capsid proteins, for example between two or more capsid proteins of different serotypes.

[0292] Shuffled or chimeric capsid proteins can also be generated by DNA shuffling or by error-prone PCR. Hybrid AAV capsid genes can be generated by randomly fragmenting sequences of related AAV genes, such as those encoding capsid proteins of multiple different serotypes, and then reassembling the fragments in a self-priming polymerase reaction, which can also generate crossovers at regions of sequence homology. A library of hybrid AAV genes thus created by shuffling the capsid genes of several serotypes can be screened to identify viral clones with desired functions. Similarly, error-prone PCR can be used to randomly mutate AAV capsid genes to generate a diverse library of variants that can be selected for desired properties.

[0293] The sequence of the capsid gene may also be genetically modified to introduce specific deletions, substitutions or insertions with respect to the native wild-type sequence. In particular, the capsid gene may be modified by inserting the sequence of an unrelated protein or peptide within the open reading frame of the capsid coding sequence or at the N-terminus and / or C-terminus of the capsid coding sequence.

[0294] The unrelated protein or peptide may advantageously act as a ligand for a particular cell type, thereby resulting in improved binding to the target cell or improving the specificity of the targeting of the vector to a particular cell population. An example may be the use of RGD peptides to block uptake in the retinal pigment epithelium, thereby facilitating transduction of the surrounding retinal tissue (Cronin et al. (2008) ARVO Abstract:D1048). The unrelated protein may also be an epitope or affinity tag that aids in the purification of the viral particles as part of the production process. The insertion site is typically selected so as not to interfere with other functions of the viral particle (viral particle internalization, transport, etc.). The skilled person can identify suitable sites for insertion based on general knowledge. Particular sites are disclosed in Choi et al., referenced above.

[0295] The present invention further encompasses providing sequences of the AAV genome in an order and organization different from that of the native AAV genome.The present invention also encompasses replacing one or more AAV sequences or genes with sequences from another virus or chimeric genes composed of sequences from two or more viruses.Such chimeric genes can be composed of sequences from two or more related viral proteins of different viral species.

[0296] An AAV vector of the invention may take the form of a nucleotide sequence comprising the AAV genome or a derivative thereof and a sequence encoding complement factor I, and / or complement factor H or FHL1 transgene or a derivative thereof.

[0297] The AAV particles of the present invention include transcapsid forms, in which the AAV genome or derivatives with ITRs of one serotype are packaged into the capsid of a different serotype.The AAV particles of the present invention also include mosaic forms, in which a mixture of unmodified capsid proteins from two or more different serotypes constitutes the viral capsid.The AAV particles also include chemically modified forms, which carry ligands adsorbed on the capsid surface.For example, such ligands can include antibodies for targeting specific cell surface receptors.

[0298] Thus, for example, AAV particles of the present invention include those having an AAV2 genome and AAV2 capsid proteins (AAV2 / 2), those having an AAV2 genome and AAV5 capsid proteins (AAV2 / 5), and those having an AAV2 genome and AAV8 capsid proteins (AAV2 / 8), as well as those having an AAV2 genome and capsid proteins of multiple serotypes.

[0299] AAV vectors may contain multiple copies (e.g., 2, 3, etc.) of the nucleotide sequences referred to herein.

[0300] In some embodiments, the polynucleotide further comprises one or more AAV ITRs. In a preferred embodiment, the polynucleotide further comprises two AAV ITRs. In some embodiments, the polynucleotide comprises an AAV ITR at its 5' end and an AAV ITR at its 3' end. In some embodiments, the AAV ITR is an AAV2 or AAV8 ITR. In a preferred embodiment, the AAV ITR is an AAV2 ITR.

[0301] In some embodiments, the polynucleotide comprises a 5' AAV ITR having a nucleotide sequence of SEQ ID NO:18, or a nucleotide sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO:18. [ka]

[0302] In some embodiments, the polynucleotide further comprises a nucleotide sequence of SEQ ID NO:20, or a nucleotide sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity immediately adjacent to the 3' end of the 5' ITR. [ka]

[0303] In some embodiments, the polynucleotide comprises a 3' AAV ITR having a nucleotide sequence of SEQ ID NO:19, or a nucleotide sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO:19. [ka]

[0304] In some embodiments, the polynucleotide further comprises a nucleotide sequence of SEQ ID NO:21, or a nucleotide sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity immediately adjacent to the 5' end of the 3' ITR. [ka] Promoters and Regulatory Sequences

[0305] The polynucleotide or vector of the present invention may also comprise elements that allow expression of complement factor I and CFI cofactors, such as complement factor H and / or FHL1 transgenes, in vitro or in vivo. These may also be referred to as expression control sequences. Thus, the polynucleotide or vector typically comprises an expression control sequence (e.g., a promoter sequence) operably linked to the nucleotide sequence encoding the transgene.

[0306] In some embodiments, the polynucleotide or vector comprises a nucleotide sequence encoding: (a) a CMV promoter, optionally upstream of a nucleotide sequence encoding a complement factor I (CFI) cofactor, such as complement factor I and / or complement factor H and / or FHL1; (b) a WPRE regulatory element, optionally downstream of a nucleotide sequence encoding a complement factor I (CFI) cofactor, such as complement factor I and / or complement factor H and / or FHL1; and / or (c) a polyA signal, such as a bovine growth hormone polyA signal, optionally downstream of a nucleotide sequence encoding a complement factor I (CFI) cofactor, such as complement factor I and / or complement factor H and / or FHL1.

[0307] In a preferred embodiment, the polynucleotide or vector comprises: (a) 5′ AAV ITR; (b) CMV promoter; (c) a nucleotide sequence encoding a complement factor I (CFI) cofactor, preferably FHL1; (d) a linker, optionally defined by a furin cleavage site, GSG, 11a1D and F2A sequences; (e) a nucleotide sequence encoding CFI; (f) a WPRE regulatory element, preferably a WPRE3 regulatory element; (g) bovine growth hormone polyA signal; and (h) 3' AAV ITR.

[0308] Any suitable promoter can be used, and its selection can be easily performed by those skilled in the art. The promoter sequence can be constitutively active (i.e., operable in any host cell background), or can be active only in a specific host cell environment, thus allowing the target expression of the transgene in a specific cell type (e.g., tissue-specific promoter). The promoter can show inducible expression in response to the presence of another factor, for example, a factor present in the host cell. In any event, when the vector is administered for therapy, it is preferred that the promoter is functional in the background of the target cell.

[0309] In some embodiments, the promoter preferably exhibits retinal cell-specific expression, so that the transgene can be expressed only in a retinal cell population. Thus, expression from the promoter can be retinal cell-specific, for example, restricted to only cells of the neurosensory retina and retinal pigment epithelium.

[0310] Preferred promoters that are not retinal cell specific include chicken beta actin (CBA) promoter, optionally combined with cytomegalovirus (CMV) enhancer element. An exemplary promoter for use in the present invention is the CAG promoter, for example, the promoter used in rAVE expression cassette (GeneDetect.com).

[0311] In a preferred embodiment, the polynucleotide or vector comprises a CMV promoter.

[0312] An example of a CMV promoter sequence is as follows: [ka]

[0313] In some embodiments, the polynucleotide or vector comprises a promoter having a nucleotide sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 13. Preferably, the nucleotide sequence substantially retains the functional activity of the promoter represented by SEQ ID NO:13.

[0314] In other embodiments, the polynucleotide or vector comprises a promoter having the nucleotide sequence of SEQ ID NO:13.

[0315] Further examples of promoter sequences are as follows: [ka]

[0316] In some embodiments, the polynucleotide or vector comprises a promoter having a nucleotide sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 5. Preferably, the nucleotide sequence substantially retains the functional activity of the promoter represented by SEQ ID NO:5.

[0317] In other embodiments, the polynucleotide or vector comprises a promoter having the nucleotide sequence of SEQ ID NO:5.

[0318] Examples of promoters based on human sequences that direct retina-specific gene expression include rod and cone rhodopsin kinase (Allocca et al., (2007) J. Virol. 81:11372-80), cone-only PR2.1 (Mancuso et al., (2009) Nature 461:784-7) and / or retinal pigment epithelium RPE65 (Bainbridge et al., (2008) N. Engl. J. Med. 358:2231-9) or VMD2 (Esumi et al., (2004) J. Biol. Chem. 279:19064-73).

[0319] The polynucleotide or vector of the present invention may also comprise one or more additional regulatory sequences that may act pre- or post-transcriptionally.The regulatory sequence may be part of the natural transgene locus or may be a heterologous regulatory sequence.The polynucleotide or vector of the present invention may comprise a portion of the 5'-UTR or 3'-UTR from the natural transgene transcript.

[0320] Regulatory sequences are any sequences that facilitate the expression of transgenes, i.e., increase the expression of transcripts, improve the nuclear export of mRNA, or increase its stability. Such regulatory sequences include, for example, enhancer elements, posttranscriptional regulatory elements, and polyadenylation sites.

[0321] A preferred polyadenylation site is the bovine growth hormone polyA (bGH polyA) signal.

[0322] An example of the bovine growth hormone polyA (bGH polyA) signal is: [ka]

[0323] A further example of the bovine growth hormone polyA (bGH polyA) signal is as follows: [ka]

[0324] In some embodiments, the polynucleotide or vector comprises a polyadenylation signal having a nucleotide sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 14 or 6, preferably SEQ ID NO: 14. Preferably, the nucleotide sequence substantially retains the functional activity of the polyadenylation signal represented by SEQ ID NO: 14 or 6.

[0325] In another embodiment, the polynucleotide or vector comprises a polyadenylation signal having the nucleotide sequence of SEQ ID NO: 14 or 6, preferably SEQ ID NO: 14.

[0326] In the context of the polynucleotides or vectors of the invention, such regulatory sequences are cis-acting, however, the invention also encompasses the use of trans-acting regulatory sequences located on additional genetic constructs.

[0327] A preferred post-transcriptional regulatory element for use in the AAV vectors of the invention is the woodchuck hepatitis post-transcriptional regulatory element (WPRE) or a variant thereof.

[0328] An example of a WPRE is: [ka] The WPRE is a tripartite element that contains gamma, alpha, and beta elements in the given order. A truncated version of the WPRE (also called WPRE3; Choi, J.-H. et al. (2014) Molecular Brain 7:17), which contains only minimal gamma and alpha elements, may be used in the present invention.

[0329] An example of a WPRE3 sequence is as follows: [ka]

[0330] In some embodiments, the polynucleotide or vector comprises a post-transcriptional regulatory element having a nucleotide sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 15 or 7, preferably SEQ ID NO: 15. Preferably, the nucleotide sequence substantially retains the functional activity of the post-transcriptional regulatory element represented by SEQ ID NO: 15 or 7.

[0331] In other embodiments, the polynucleotide or vector comprises a post-transcriptional regulatory element having the nucleotide sequence of SEQ ID NO: 15 or 7, preferably SEQ ID NO: 15.

[0332] Another regulatory sequence that can be used in the polynucleotide or vector of the present invention is a scaffold attachment region (SAR). Additional regulatory sequences can be readily selected by those skilled in the art. Method of administration

[0333] The products, polynucleotides or vectors of the invention can be administered systemically (e.g., by peripheral venous injection), and can be administered locally or regionally (e.g., to the CNS system by intrathecal injection). In a preferred embodiment, the products, polynucleotides or vectors are administered intraocularly.

[0334] The term "intraocular" refers to the inside of the eye, and thus, intraocular administration relates to administration to the inside of the eye of a subject.

[0335] In some embodiments, the product, polynucleotide or vector is administered to the subject's eye by subretinal, direct retinal, suprachoroidal or intravitreal injection, hi some embodiments, said administration is performed robotically.

[0336] The volume of the pharmaceutical composition to be injected may be, for example, about 10 to 500 μL, for example, about 50 to 500, 100 to 500, 200 to 500, 300 to 500, 400 to 500, 50 to 250, 100 to 250, 200 to 250, or 50 to 150 μL. The volume may be, for example, about 10, 50, 100, 150, 200, 250, 300, 350, 400, 450, or 500 μL. Preferably, the volume of the pharmaceutical composition to be injected is 100 μL.

[0337] Those skilled in the art are familiar with and well able to perform individual subretinal, direct retinal, suprachoroidal or intravitreal injections.

[0338] Preferably, the product, polynucleotide or vector is administered by subretinal injection. In some embodiments, the product, polynucleotide, vector or pharmaceutical composition comprising same is administered no more than once, or no more than twice during the subject's lifetime. subretinal injection

[0339] Subretinal injection is an injection into the subretinal space, i.e., beneath the neurosensory retina. During a subretinal injection, the injected material is directed toward the photoreceptor cells and the retinal pigment epithelium (RPE) layer, creating a space between them.

[0340] If the injection is made through a small retinotomy, retinal detachment can occur. The detached raised layer of the retina created by the injected material is called a "bleb."

[0341] The hole created by the subretinal injection must be small enough so that the injected solution does not flow significantly back into the vitreous cavity after administration. Such flow back may be particularly problematic when the drug is injected, since the effect of the drug is directed away from the target area. Preferably, the injection creates a self-sealing entry point in the neurosensory retina. That is, when the injection needle is removed, the hole created by the needle reseals so that the injected material is hardly or substantially released from the hole.

[0342] To facilitate this process, specialized subretinal needles are commercially available (e.g., DORC 41G Teflon subretinal needle, Dutch Ophthalmic Research Center International BV, Zuidland, The Netherlands). These are needles designed to perform subretinal injections.

[0343] As long as no damage to the retina occurs during injection, and as long as a sufficiently small needle is used, virtually all of the injected material remains localized between the detached neurosensory retina and the RPE at the localized site of retinal detachment (i.e., does not flow back into the vitreous cavity). Indeed, the typical persistence of a bleb over a short time frame indicates that there is usually little escape of the injected material into the vitreous. The bleb may dissipate over a longer time frame as the injected material is absorbed.

[0344] Visualization of the eye, and in particular the retina, can be performed pre-operatively using, for example, optical coherence tomography.

[0345] The volume of the pharmaceutical composition injected may be, for example, about 10-500 μL, for example, about 50-500, 100-500, 200-500, 300-500, 400-500, 50-250, 100-250, 200-250 or 50-150 μL. The volume may be, for example, about 10, 50, 100, 150, 200, 250, 300, 350, 400, 450 or 500 μL. Preferably, the volume of the pharmaceutical composition injected is 100 μL. A larger volume may increase the risk of the retina elongating, but a smaller volume will be difficult to see. Two-step subretinal injection

[0346] The product, polynucleotide or vector of the present invention can be delivered with increased precision and safety by using a two-step method in which a first solution is injected subretinally to cause localized retinal detachment.The first solution does not contain the product, polynucleotide or vector.A second subretinal injection is then used to deliver the drug containing the product, polynucleotide or vector to the subretinal fluid of the bleb created by the first subretinal injection.Since the injection that delivers the drug is not used to detach the retina, a certain volume of solution can be injected in this second step.

[0347] In some embodiments, the subretinal injection of the vector comprises: (a) administering to a subject a solution by subretinal injection in an amount effective to at least partially detach the retina to form a subretinal bleb, wherein the solution does not contain a product, polynucleotide, or vector; (b) administering a pharmaceutical composition by subretinal injection into the bleb formed by step (a), the pharmaceutical comprising a product, a polynucleotide, or a vector; Includes.

[0348] The volume of the solution injected in step (a) to at least partially detach the retina may be, for example, about 10-1000 μL, e.g., about 50-1000, 100-1000, 250-1000, 500-1000, 10-500, 50-500, 100-500, 250-500 μL. The volume may be, for example, about 10, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 μL.

[0349] The volume of the pharmaceutical composition injected in step (b) may be, for example, about 10-500 μL, for example, about 50-500, 100-500, 200-500, 300-500, 400-500, 50-250, 100-250, 200-250 or 50-150 μL. The volume may be, for example, about 10, 50, 100, 150, 200, 250, 300, 350, 400, 450 or 500 μL. Preferably, the volume of the pharmaceutical composition injected in step (b) is 100 μL. A larger volume may increase the risk of the retina elongating, but a smaller volume will be difficult to see.

[0350] The drug-free solution (i.e., the "solution" in step (a)) may be formulated similarly to the drug-containing solution, as described below. A preferred drug-free solution is a balanced salt solution (BSS) or similar buffer that is compatible with the pH and osmolality of the subretinal space. Intraoperative visualization of the retina

[0351] Under certain circumstances, such as during end-stage retinal degeneration, it is difficult to identify the retina because it is thin, transparent, and difficult to see against the destroyed thick pigment epithelium on which it rests. The use of blue vitreous dyes (e.g., BrilliantPeel®, Geuder; MembraneBlue-Dual®, Dore) facilitates identification of the retinal hole created for the retinal detachment procedure (i.e., step (a) of the two-stage subretinal injection method of the present invention), so that medication can be administered through the same hole without the risk of reflux back into the vitreous cavity.

[0352] The use of blue vital dye also identifies any areas of the retina where a thick internal limiting membrane or epiretinal membrane is present, because injection through either of these structures would prevent clear access to the subretinal space. Furthermore, contraction of either of these structures in the immediate postoperative period can result in elongation of the retinal entry hole, which can result in backflow of medication into the vitreous cavity. suprachoroidal injection

[0353] The products, polynucleotides or vectors of the invention can be delivered to the suprachoroidal space using an ab externo approach utilizing a microcatheter (e.g., Peden et al. (2011) PLoS One 6(2):e17140). In this method, a limbal periclinal incision is made to expose the bare sclera, followed by a sclerotomy to expose the bare choroid. A microcatheter (such as iTrack250A from iScience Interventional, optionally connected to an illumination system such as the iLumin laser diode-based microillumination system (iScience Interventional)) is introduced into the suprachoroidal space and advanced posteriorly toward the optic nerve head. After manipulation of the tip of the microcatheter to the desired location, injection of the product, polynucleotide or vector forms a bleb in the retina and choroid.

[0354] Thus, in some embodiments, the product, polynucleotide or vector is delivered to the suprachoroid by a method that includes: (i) introducing a microcatheter into the suprachoroidal space; (ii) advancing the microcatheter within the suprachoroidal space until the tip is near the diseased area of ​​the retina; and (iii) injecting the product, polynucleotide, or vector from the tip of the microcatheter to form a bleb.

[0355] In some embodiments, the above administration steps are performed directly by the robot. Pharmaceutical compositions and injection solutions

[0356] The agent of the present invention, for example, product, polynucleotide or vector, can be formulated into pharmaceutical compositions.These compositions can contain, in addition to the agent, pharma- ceutically acceptable carriers, diluents, excipients, buffers, stabilizers, or other materials that are well known in the art.Such materials must be non-toxic and must not interfere with the effectiveness of the active ingredient.The exact nature of the carrier or other materials can be determined by those skilled in the art according to the administration route, for example, subretinal, direct retinal, suprachoroidal or intravitreal injection.

[0357] Pharmaceutical compositions are typically in liquid form. Liquid pharmaceutical compositions generally contain liquid carriers such as water, petroleum, animal or vegetable oils, mineral oil or synthetic oil. May contain saline, magnesium chloride, dextrose or other sugar solution, or glycols such as ethylene glycol, propylene glycol or polyethylene glycol. In some cases, surfactants such as pluronic acid (PF68) 0.001% may be used.

[0358] For injection at affected site, the active ingredient can be in the form of an aqueous solution that is pyrogen-free and has suitable pH, isotonicity and stability.Those skilled in the art can fully prepare suitable solutions using isotonic vehicles such as sodium chloride injection, Ringer's injection, or lactated Ringer's injection.If necessary, preservatives, stabilizers, buffers, antioxidants and / or other additives can be included.

[0359] For delayed release, the drug may be included in a pharmaceutical composition formulated for sustained release, such as in microcapsules formed from biocompatible polymers by methods known in the art or in a liposome carrier system. Treatment method

[0360] All references herein to treatment include curative, palliative and prophylactic treatment, and it will be understood that in the context of the present invention, references to prevention relate more generally to prophylactic treatment. Treatment may also include preventing the progression of the severity of the disease.

[0361] The treatment of mammals, especially humans, is preferred, however, both human and veterinary treatment is within the scope of the present invention.

[0362] As used herein, the term "combination" or the terms "in combination," "used in combination," or "combined preparation" can refer to the combined administration of two or more agents simultaneously, sequentially, or separately.

[0363] As used herein, the term "concurrently" means that the agents are administered simultaneously, i.e., at the same time.

[0364] As used herein, the term "sequential" means that the agents are administered one after the other.

[0365] The term "individually" as used herein means that the agents are administered independently of each other, but within a time interval that allows the agents to exhibit a combined, preferably synergistic effect. Thus, administering "individually" may allow one agent to be administered, for example, within 1 minute, 5 minutes, or 10 minutes from another agent. Variants, derivatives, analogues, homologues and fragments

[0366] In addition to the specific proteins and nucleotides mentioned herein, the present invention also encompasses the use of variants, derivatives, analogs, homologs and fragments thereof.

[0367] In the context of the present invention, a variant of any given sequence is a sequence in which a particular sequence of residues (whether amino acid or nucleic acid residues) has been modified such that the polypeptide or polynucleotide in question substantially retains its function. Variant sequences can be obtained by addition, deletion, substitution, modification, replacement and / or alteration of at least one residue present in the naturally occurring protein.

[0368] The term "derivative" as used herein in relation to a protein or polypeptide of the invention includes any of the substitutions, alterations, modifications, substitutions, deletions and / or additions of one (or more) amino acid residues from or to the sequence, provided that the resulting protein or polypeptide substantially retains at least one of its endogenous functions.

[0369] The term "analog" as used herein with respect to a polypeptide or polynucleotide includes any mimetic, ie, a compound that retains at least one of the endogenous functions of the polypeptide or polynucleotide it mimics.

[0370] Typically, amino acid substitutions can be made, for example, from 1, 2 or 3 to 10 or 20 substitutions, provided that the modified sequence substantially retains the required activity or ability. Amino acid substitutions can include the use of non-naturally occurring analogs. Proteins used in the present invention can also have deletions, insertions or substitutions of amino acid residues that produce silent changes and result in functionally equivalent proteins. Deliberate amino acid substitutions can be made based on similarity in the polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphipathic nature of the residues, so long as endogenous function is retained. For example, negatively charged amino acids include aspartic acid and glutamic acid, positively charged amino acids include lysine and arginine, and amino acids with uncharged polar head groups with similar hydrophilicity values ​​include asparagine, glutamine, serine, threonine, and tyrosine.

[0371] Conservative substitutions may be made, for example, according to the following table: Amino acids in the same block in the second column and preferably in the same line in the third column may be substituted for each other: [Table 1]

[0372] The term "homolog" as used herein means an entity that has a certain homology with the wild-type amino acid sequence and the wild-type nucleotide sequence. The term "homology" may be equivalent to "identity".

[0373] A homologous sequence may comprise an amino acid sequence that may be at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85% or 90% identical to the subject sequence, preferably at least 95% or 97% or 99% identical. Typically, a homolog will contain the same active site etc. as the subject amino acid sequence. Although homology can also be considered in terms of similarity (i.e. amino acid residues having similar chemical properties / functions), in the context of the present invention it is preferred to express homology in terms of sequence identity.

[0374] A homologous sequence may comprise a nucleotide sequence that may be at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85% or 90% identical to the subject sequence, preferably at least 95% or 97% or 99% identical. Homology may also be considered in terms of similarity, but in the context of the present invention, it is preferred to express homology in terms of sequence identity. Preferably, reference to a sequence having a percent identity to any one of the SEQ ID NOs detailed herein refers to a sequence having the stated percent identity over the entire length of the SEQ ID NO referenced.

[0375] Homology comparisons can be performed by eye, or more usually, with the aid of readily available sequence comparison programs. These commercially available computer programs can calculate the percentage of homology or identity between two or more sequences.

[0376] The percentage of homology can be calculated across adjacent sequences, i.e., one sequence is aligned with the other and each amino acid in one sequence is directly compared to the corresponding amino acid in the other sequence, one residue at a time. This is called a "non-gapped" alignment. Typically, such non-gapped alignments are only performed over a relatively short number of residues.

[0377] Although this is a very simple and consistent method, it does not take into account that, for example, in an otherwise identical sequence pair, a single insertion or deletion in a nucleotide sequence can cause the next codon to be out of alignment. This can lead to a large drop in percent homology when a global alignment is performed. As a result, most sequence comparison methods are designed to produce an optimal alignment that takes into account possible insertions and deletions without excessively penalizing the overall homology score. This is achieved by inserting "gaps" in the sequence alignment to try to maximize local homology.

[0378] However, these more complex methods assign a "gap penalty" for each gap that occurs in the alignment, so that for the same number of identical amino acids, a sequence alignment with as few gaps as possible, reflecting a higher relatedness between the two compared sequences, will achieve a higher score than one with many gaps. "Affine gap costs" are typically used to impose a relatively high cost for the presence of a gap and a smaller penalty for each subsequent residue in the gap. This is the most commonly used gap scoring system. High gap penalties naturally result in optimized alignments with fewer gaps. Most alignment programs allow gap penalties to be modified. However, when using such software for sequence comparisons, it is preferred to use the default values. For example, when using the GCG Wisconsin Bestfit package, the default gap penalty for amino acid sequences is -12 for a gap and -4 for each extension.

[0379] Calculation of maximum percentage homology therefore first requires the creation of an optimal alignment, taking into account gap penalties. A suitable computer program for carrying out such an alignment is the GCG Wisconsin Bestfit package (University of Wisconsin, USA; Devereux et al. (1984) Nucleic Acids Res. 12:387). Examples of other software capable of carrying out sequence comparisons include, but are not limited to, the BLAST package (Ausubel et al. (1999) ibid. - Chapter 18), FASTA (Atschul et al. (1990) J. Mol. Biol. 403-410) and the GENEWORKS suite of comparison tools. Both BLAST and FASTA are available for offline and online searching (see Ausubel et al. (1999) ibid., pages 7-58 to 7-60). However, for some applications it is preferred to use the GCG Bestfit program. Another tool called BLAST2 sequences is also available for comparing protein and nucleotide sequences (see FEMS Microbiol. Lett. (1999) 174:247-50; FEMS Microbiol. Lett. (1999) 177:187-8).

[0380] Although the final percent homology can be measured in terms of identity, the alignment process itself is typically not based on an all-or-nothing pairwise comparison. Instead, a scaled similarity score matrix is ​​commonly used that assigns a score to each pairwise comparison based on chemical similarity or evolutionary distance. An example of such a matrix commonly used is the BLOSUM62 matrix, which is the default matrix for the BLAST suite of programs. GCG Wisconsin programs usually use either the public default values ​​or a custom symbol comparison table if provided (see user manual for details). For some applications, it is preferred to use the public default values ​​of the GCG package, or, in the case of other software, a default matrix such as BLOSUM62.

[0381] Once the software has produced an optimal alignment, it can calculate percent homology, preferably percent sequence identity. The software typically does this as part of the sequence comparison and generates a numerical result.

[0382] A "fragment" of full-length complement factor I or a complement factor I (CFI) cofactor (such as complement factor H or FHL1) is also a variant, and the term typically refers to a selected region of a polypeptide or polynucleotide of interest functionally or, for example, in an assay. A "fragment" thus refers to an amino acid or nucleic acid sequence that is a portion of the full-length polypeptide or polynucleotide.

[0383] Such variants can be prepared using standard recombinant DNA techniques such as site-directed mutagenesis. When an insertion is made, a synthetic DNA can be made that encodes the insertion with 5' and 3' flanking regions that correspond to the naturally occurring sequence on either side of the insertion site. The flanking regions contain convenient restriction sites that correspond to sites in the naturally occurring sequence, so that the sequence can be cleaved with an appropriate enzyme(s) and the synthetic DNA ligated to the cleavage. The DNA is then expressed according to the invention to produce the encoded protein. These methods are merely illustrative of the many standard techniques known in the art for manipulating DNA sequences, and other known techniques may be used.

[0384] Those skilled in the art will understand that they may combine all features of the invention disclosed herein without departing from the scope of the invention disclosed.

[0385] Preferred features and embodiments of the present invention will now be described, by way of non-limiting examples.

[0386] The practice of the present invention will employ, unless otherwise indicated, conventional techniques of chemistry, biochemistry, molecular biology, microbiology and immunology that are within the capabilities of those skilled in the art. Such techniques are explained in the literature. See, e.g., Sambrook, J., Fritsch, E F and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press; Ausubel, F M et al. (1995 and periodic supplements) Current Protocols in Molecular Biology, Chapters 9, 13 and 16, John Wiley & Sons; Roe, B., Crabtree, J. and Kahn, A. (1996) DNA Isolation and Sequencing: Essential Techniques, John Wiley & Sons; Polak, J M and McGee, J. O'D. (1990) In Situ Hybridization: Principles and Practice, Oxford University Press; Gait, M J (1984) Oligonucleotide Synthesis: A Practical Approach, IRL Press; and Lilley, D M and Dahlberg, J E (1992) Methods in See Enzymology: DNA Structures Part A: Synthesis and Physical Analysis of DNA, Academic Press. Each of these general texts is incorporated herein by reference. Working Example Example 1 Cofactor assays

[0387] Recombinant complement factor I (CFI), cofactors (complement factor H (CFH) or complement factor H-like protein 1 (FHL1)) and C3b were incubated together for 20 min at 37°C.

[0388] CFI and C3b concentrations were fixed, and CFH or FHL1 was titrated at the indicated ratios.

[0389] Cleavage of C3b to iC3b was quantified by ELISA.

[0390] The results of these studies are shown in FIG. 2A.

[0391] From these results it can be concluded that the minimum functional CFI:CFH / FHL1 molar ratio is 1:2. Measurement of normal serum CFk cofactor ratios

[0392] Concentrations of complement factor I (CFI), complement factor H (CFH), and complement factor H-like protein 1 (FHL1) were measured in normal human serum using ELISA.

[0393] The molar ratio of CFk cofactors in normal serum was found to be 1:8.3. Comparison of CFI:cofactor ratios between plasma and ocular fluids

[0394] We then compared the levels of CFI and CFH in both plasma and ocular fluid, and the data showed that in contrast to plasma, where the cofactor is present in several-fold molar excess relative to the CFI enzyme, this ratio is reversed in the vitreous and aqueous humor (Figure 7). Complement deposition assay for lipopolysaccharide (LPS)

[0395] Microtiter plates were coated with 1 μg / well LPS and incubated overnight at 4°C.

[0396] Plates were then washed and incubated with normal human serum supplemented with different amounts of recombinant complement factor I (CFI), complement factor H (CFH), or complement factor H-like protein 1 (FHL1) for 1 h at 37° C. Plates were then washed extensively and complement deposition was measured with mouse anti-C3d antibody (Abeam) and donkey anti-mouse (Jackson ImmunoResearch) used as secondary antibody.

[0397] FIG. 2B shows the effect of CFI, CFH, and FHL1 supplementation on complement deposition to LPS.

[0398] The x-axis shows the ratio of complement factor I (CFI) to complement factor H plus complement factor H-like 1 (CFH and FHL1, "cofactors"). Changes in the ratio were only achieved by the addition of recombinant CFI, CFH, or FHL1.

[0399] Although a starting molar ratio of 1:8.3 (the natural ratio in normal serum) already has an excess of cofactor compared to the enzyme, the additional benefit of reduced complement deposition, and therefore activation, is achieved when the ratio is further increased. This additional benefit may be related to an additional function of CFH and FHL1 (decay accelerating activity, DAA). Both CFH and FHL1 have DAA in that they compete with complement factor B (FB) for C3b binding, displacing FB from C3b and thereby "decaying" the alternative pathway convertases.

[0400] From these experiments it can be concluded that below the minimum CFI:CFH / FHL1 molar ratio of 1:2 the cofactor becomes limiting. Example 2 Generation of bicistronic plasmids

[0401] Recombinant AAV transgene plasmids (designated RC204, RC206-210 and RC212-218) were constructed containing the AAV2 5' and 3' inverted terminal repeats (ITRs) flanking the cassettes described in Table 1. [Table 2]

[0402] The 5'ITR flanking sequence used was SEQ ID NO:20.

[0403] The CMV promoter sequence used was SEQ ID NO:13.

[0404] The FHL1 sequence used was SEQ ID NO: 16. The codon-optimized FHL1 (FHL1-CO) sequence used was SEQ ID NO: 12.

[0405] The furin-F2A linker sequence used was SEQ ID NO:17.

[0406] The CFI sequence used was SEQ ID NO: 2. The codon-optimized CFI (CFI-CO) sequence used was SEQ ID NO: 10.

[0407] The WPRE3 sequence used was SEQ ID NO:15.

[0408] The bovine growth hormone polyA (BGHpA) sequence used was SEQ ID NO:14.

[0409] The 3'ITR flanking sequence used was SEQ ID NO:21.

[0410] The overall sequences of RC212 and RC218 are SEQ ID NOs:22 and 23, respectively. Comparison of vectors by transduction Vector production in adherent HEK293

[0411] Individual transfections of HEK293 cells with the 12 plasmids (RC204, RC206–210, and RC212–217) were performed using the following protocol. Day 1: HEK293 cells were dissociated and counted using a ViCell. Cells were plated at 6x10 in 10mL DMEM / Glutamax+10% FBS. 5 cells / cm 2 The cells were seeded onto 10 cm plates. Day 2: Confluency was checked and found to be 70-80%.

[0412] The FBS medium was replaced with DMEM / Glutamax+5% FBS.

[0413] After 4 hours, cells were transfected with 5 μg total plasmid DNA per plate (1.25 μg construct DNA, 1.25 μg RepCap plasmid, 2.5 μg helper plasmid) using PEI at a DNA:PEI ratio of 1:3 in duplicate. 1.2x5μg DNA was diluted in 2x1mL PBS. 2.2x15μL PEI was added and incubated for 20 minutes. 3.1 mL of DNA / PEI was added dropwise to the cells. Day 3: 15 mM sodium butyrate was added. Day 5: Media was harvested by pooling replicates and centrifuged at 1000 rpm for 10 min to remove cell debris.

[0414] The supernatant was transferred to a new tube and AAVanced (AAV110A-1, Cambridge Bioscience) was added at 1 / 5 of the volume.

[0415] The mixture was incubated at 4° C. for 72 hours.

[0416] Day 5: The mixture was centrifuged at 1000 rpm for 30 min at 4° C. The supernatant was then discarded and the pellet was resuspended in 500 μL of PBS. The pellet was centrifuged at 1500 g for 3 min, after which the supernatant was again discarded. The pellet was resuspended in 1 / 100 of the original supernatant volume and stored at −80° C. until titration. Transduction of HEK293 cells with vectors

[0417] Individual transduction of HEK293 cells with 12 viral vectors (RC204, RC206-210, and RC212-217) was carried out using the following protocol. Day 1: HEK293 cells were dissociated and counted using a ViCell. Cells were plated at 1x10 in 400μL DMEM / Glutamax+10%FBS per well. 5 Cells were seeded into 24-well plates. Day 2: Add the required amount of viral vector to 2x102 A multiplicity of infection (MOI) of 100 was achieved. Day 3: Media was removed and replaced with 300 μL serum-free DMEM / Glutamax. Day 5: The supernatant was collected and centrifuged at 14000 rpm at 4° C. The clarified supernatant was transferred to a new tube and ready for analysis. Western blot

[0418] Supernatants from transductions were analyzed by Western blot (using primary antibodies against CFI and FHL-1; and ECL prime Western blotting detection reagents).

[0419] The results of the Western blot analysis are shown in FIG. CFI ELISA

[0420] Supernatants from transductions were analyzed by CFI ELISA using the following procedure: Day 1: ELISA plates were coated with 50 μL per well of sheep anti-CFI polyclonal antibody diluted 1 in 4000 in 1× coating buffer. Plates were stored at 4° C. overnight. Day 2: Plates were washed 3 times with 200 μL per well of PBS-Tween (0.05%) and blotted onto tissue.

[0421] 200 μL of 1% BSA Fraction V in PBS-Tween (0.05%) was applied to each well and blocked for 2 h at room temperature.

[0422] Samples and standard curves were generated during the blocking incubation. Standard curves were generated using purified CFI protein (Sigma C5938-1MG) diluted in DMEM 2% FBS. Samples were diluted 1:5 and 1:10 in DMEM 2% FBS.

[0423] After 2 h of blocking, the plates were washed three times as above, then 50 μL of sample or standard was loaded into each well and incubated for 1 h at room temperature.

[0424] After 1 hour, the plates were washed as above, then anti-CFI (0x21) antibody was diluted 1 in 2000 in DMEM 5% FBS, of which 50 μL was applied to each well and incubated for 1 hour at room temperature.

[0425] After 1 hour, the plates were washed as above and then donkey anti-mouse-HRP antibody was diluted 1 in 5000 in DMEM 5% FBS and 50 μL of this was applied to each well and incubated for 1 hour at room temperature.

[0426] After 1 hour, the plates were washed as above, then 100 μL of TMB reagent was applied to each well and incubated at room temperature in the dark for approximately 15 minutes. Once a sufficient blue color was obtained, 100 μL of 1 M sulfuric acid was added to each well to stop the reaction.

[0427] The A450 was then recorded and the data was processed and transferred to Microsoft Excel for analysis. FHL1 ELISA

[0428] Supernatants from transductions were analyzed by ELISA for FHL1 using the following procedure: Day 1: ELISA plates were coated with 50 μL per well of 3 μg / mL anti-FHL-1 antibody (Biorad, AbD33594.1) in 100 mM carbonate / bicarbonate buffer, pH 9.6. Plates were stored at 4° C. overnight. Day 2: Plates were washed 3 times with 200 μL TBS-Tween (0.05%) per well and blotted onto tissue.

[0429] 200 μL of 1% BSA Fraction V in PBS-Tween (0.05%) was applied to each well and blocked for 2 h at room temperature.

[0430] Samples and standard curves were generated during the blocking incubation. Standard curves were generated using FHL1 purified protein diluted in DMEM 2% FBS. Samples were diluted 1:5 and 1:10 in blocking buffer.

[0431] After 2 hours of blocking, the plates were washed three times as above, then 50 μL of sample or standard was loaded into each well and incubated for 1 hour at room temperature.

[0432] After 1 hour, plates were washed as above and then anti-CFH antibody (0x24, Santa Cruz Biotechnologies, sc-53067) was added at 0.33 μg / mL in blocking buffer, of which 50 μL was applied to each well and incubated for 1 hour at room temperature.

[0433] After 1 hour, plates were washed as above and then donkey anti-mouse HRP antibody was added at 0.2 μg / mL in blocking buffer, of which 50 μL was applied to each well and incubated for 1 hour at room temperature.

[0434] After 1 hour, the plates were washed as above, then 100 μL of TMB reagent was applied to each well and incubated at room temperature in the dark for approximately 15 minutes. When a sufficient blue color was obtained, 50 μL of 1 M sulfuric acid was added to each well to stop the reaction.

[0435] The A450 was then recorded and the data was processed and transferred to Microsoft Excel for analysis. conclusion

[0436] The results of the Western blot and ELISA studies are shown in Figures 3 and 4, respectively.

[0437] From the Western blot and ELISA analyses it can be concluded that: All candidates produce both CFI and FHL1 following transduction. CFI and FHL1 codon optimization increases protein levels. RC206 and RC212 are optimal for expression of CFI, while RC212 achieves the optimal CFL FHL1 molar ratio (>1:2). RC212 is the best candidate based on CFI / FHL1 expression. Vector packaging Alkaline gel analysis

[0438] 24 μL of each undiluted sample and SRM control (2.45x10 11 vg / mL; SRM#16-048) was loaded onto a 0.8% alkaline gel, which was run in alkaline running buffer (40 ml 50x alkaline buffer + 1960 mL MilliQ water) at 20 V for 19 hours in a refrigerator.

[0439] The gel was then incubated in 3 gel volumes of 0.1 M Tris pH 8.0 for 1 hour at room temperature, then incubated in 1 gel volume of 0.1 M NaCl containing 4x SYBR Gold nucleic acid stain for 2 hours at room temperature (protected from light exposure), then rinsed twice with MilliQ water.

[0440] The gel was then visualized using a SYBR Gold UV transilluminator set on a Chemidoc with an exposure time of 10 seconds.

[0441] The results are shown in FIG. 5 (top panel). Full:empty particle ratio

[0442] The ratio of full to empty virus particles was analyzed by comparing the titers calculated from qPCR (DNA titer) and capsid ELISA.

[0443] The results are shown in FIG. 5 (lower panel). conclusion

[0444] From these analyses it can be concluded that: The only efficient packaging is RC212. Packaging of genomes larger than 4.7 kb appears to be incomplete. The full-empty particle ratio of RC212 is comparable to that of the control monocistronic vector. RC212 is the best candidate based on the package analysis. C3b cleavage assay

[0445] For the C3b cleavage assay, 1 mg of plasma purified C3b is incubated with transduced HEK293 supernatant samples for 1 hour at 37°C.

[0446] Analysis was performed using ELISA or Western blot analysis (described below). Reactions were stopped by adding 4xLaemmli buffer containing p-mercaptoethanol. Samples were diluted and loaded onto 10% precast polyacrylamide SDS PAGE gels (Bio-Rad). After transfer to PVDF membranes (Bio-Rad) and blocking in blocking buffer (1xTBS pH8 [Sigma] / 0.05% Tween-20 and 5% dry nonfat powdered milk [Marvel]), C3b cleavage was detected using a goat anti-human C3 antibody (Biorad).

[0447] The results are shown in Figure 6. From this it can be concluded that RC212 is the most potent. Example 3 - Additional Complement Downregulation with Addition of Multiple Complement Regulators method

[0448] To measure the functional activity of complement regulators, an LPS deposition assay was performed. Nunc Maxisorp plates were coated overnight at 4°C with 1 μg / mL LPS (Sigma, Escherichia coli O26:B6) diluted in ELISA coating buffer (Bio Rad, BUF030B). Plates were washed with PBS-0.05% Tween20. 25% serum in alternative pathway buffer (PBS, 2 mM MgCl2 and 10 mM EGTA, pH 7.2) was prepared and supplemented with complement regulators. Dilutions were added to the LPS-coated plates and incubated for 1 h at 37°C. 10 mM EDTA was added to a separate tube containing serum to prevent complement activation, and this sample was used to measure background signal in the assay. Plates were washed as before, and complement activation was measured by detecting C3 deposition on the plates (goat anti-C3d Abeam, ab17453; 1:20,000). After 1 h incubation at ambient temperature, the plates were washed and incubated with donkey anti-mouse HRP-conjugated antibody (Jackson Immunoresearch, 715-035-150; 1:1,000) for an additional 1 h. After 4 washes, the plates were incubated with 1-Step Ultra TMB-ELISA Substrate (Life Technologies) and the reaction was quenched with 1 M H2SO4. OD at 450 nm was measured using a Varioskan plate reader (Thermo Fisher) and IC50 was determined from 4PL fit curves using GraphPad Prism. Complement-preserved female human serum was used for these experiments. result

[0449] The IC50 concentrations of the complement regulator proteins, complement factor I, soluble complement receptor 1, complement factor H or factor H-like 1, were determined in separate experiments using the same assay platform. Here, these results show that the alternative pathway (as measured by a reduction in C3 deposition) is quenched (shaded columns) when the IC50 concentrations of the regulator proteins are added (Figure 8). When complement factor I is co-supplemented with one of its cofactors (soluble complement receptor 1, complement factor H or factor H-like 1), additional quenching is observed (white columns), indicating that increasing the concentration of two complement regulators is superior to adding either one alone. These results indicate that conditions caused by a hyper-reactive complement system may benefit from dual administration of multiple complement regulators.

[0450] Although the IC50 concentrations of the individual regulators vary widely between those tested (sCR1 is approximately 10-fold more potent than FHL1 and approximately 50-fold more potent than CFH and CFI) and have different molecular weights (sCR1=213 kDa, CFI=88 kDa, CFH=155 kDa, and FHL1=49 kDa), molar concentrations were used for this direct comparison. Endogenous levels of the proteins also contribute to the observed potency differences, with factor H having a much higher plasma concentration than FHL1 or sCR1. ​​Comparing the ability of regulators at IC50 concentrations to quench the alternative pathway demonstrated that sCR1 was the most potent regulator, as it required the lowest molar concentration to achieve IC50. CR1 is a membrane-bound receptor on red blood cells that is abundant in blood but not in serum / plasma, and since only trace amounts of liquid CR1 (i.e., sCR1) are present in serum or plasma, this implies that sCR1 is a very potent complement regulator and CFI cofactor in sites where red blood cells are separated from plasma, such as the choroidal space, Bruch's membrane subretinal space, and glomeruli. Example 4 - Expression of complement factor I and factor H-like 1 in vitro method

[0451] HEK-293 cells were transduced with one of the following rAAV vectors: AAV expressing CFI (GT005), AAV expressing FHL1 (RC001), or AAV expressing CFI and FHL1 (GT007). Supernatants were analyzed by non-reducing Western blot to determine relative expression of proteins. Goat anti-human CFI (Comptech) and goat anti-human FH (Quidel, A312) were used as primary antibodies to detect CFI and FHL1 proteins. result

[0452] Western blot analysis showed that CFI and FHL1 proteins were expressed and secreted into the medium from all three constructs (Figure 9A and Figure 9B). To confirm this expression pattern, the supernatants were analyzed by immunoblotting to visualize CFI protein and determine correct processing and secretion in the supernatant. As shown in Figure 9B, heavy and light chains CFI and proCFI were secreted from HEK-293 cells after transduction with GT007, confirming that the CFI protein was translated and that proteolytic processing had occurred. In mammalian cells transfected with a plasmid encoding the CFI cDNA, not all of the recombinant proCFI protein undergoes cleavage, resulting in the secretion of proCFI (88 kDa) and the processed mature CFI consisting of a 50 kDa heavy chain and a 38 kDa light chain. Example 5 - Functionality of Complement Factor I and Factor H-like 1 upon in vitro expression method

[0453] To analyze the functional activity of CFI and FHL1 secreted from transduced cells, conditioned supernatants from HEK-293 cells transduced with AAV expressing only CFI (GT005), AAV2 expressing only FHL1 (RC001); co-transduced with both GT005 and RC001; or transduced with CFI and FHL1 expressing AAV (GT007) were tested in a C3b cleavage assay (Figure 10). In this assay, C3b is mixed with a source of CFI and FHL1 and incubated at 37°C for 4 hours. The incubation time was optimized for the concentration of transgene expressed from the transduced cells. The principle of this assay is based on the ability of CFI to cleave C3b into iC3b and C3f in the presence of FHL1. The assays are analyzed by C3b Western blot, which stains for C3b cleavage products, and by iC3b ELISA, which quantifies the amount of C3b degradation product iC3b. result

[0454] Both the C3 Western blot and iC3b ELISA results correlate and show the functional activity of CFI and FHL1 expressed in vitro. In the C3b Western blot (Figure 10A), lane 1 shows C3b alone, lane 2 shows C3b mixed with CFI and FHL1 (positive control), and lane 3 shows C3b mixed with conditioned supernatant from non-transduced cells (UTD, negative control). Lane 4 shows C3b degradation in conditioned supernatant of cells co-transduced with GT005 (CFI) and RC001 (FHL1). Lane 5 shows conditioned supernatant of cells transduced with GT007 (expressing CFI and FHL1). This assay confirms that conditioned supernatant of GT007-transduced cells contains active CFI and FHL1 that degrade C3b to iC3b.

[0455] iC3b ELISA (Figure 10B) was performed using the same supernatants used for C3b Western blot to quantify the amount of iC3b as a direct function of protein function. As before, C3b incubated with CFI and FHL1 acts as a positive control, as does conditioned supernatant of GT005 and RC001 co-transduced cells. Supernatant of GT007 transduced cells shows C3b cleavage activity, confirming the presence of active CFI and FHL1. Example 6 - In vivo efficacy in a mouse model of choroidal neovascularization (CNV) method

[0456] A laser-induced choroidal neovascularization model was performed in mice. Mice (n=12–14 / group) received unilateral subretinal injection of AAV vectors 4 weeks prior to CNV induction or aflibercept (positive control) immediately after CNV induction. The contralateral eye served as a control. Mice were followed using in vivo imaging, fluorescein angiography (FA), and spectral domain optical coherence tomography (SD-OCT) on days 4 and 7. At the end of the study period on day 7 of follow-up after CNV induction, mice were sacrificed by anesthesia overdose, serum was collected, and eyes were enucleated. Neural retinas were excised and fresh frozen in liquid nitrogen. Choroids were postfixed and choroidal flatmounts were prepared. Histological analysis from choroidal flatmounts was used to quantify the area of ​​isolectin B4 staining in CNV lesions. result

[0457] The aflibercept group significantly affected the extent of CNV leakage compared with the null vector control group at 4 days after CNV induction (P<0.0001 compared with all other groups), but this was lost by day 7, likely due to drug washout over time. The area of ​​CNV leakage was significantly reduced in aflibercept-treated eyes at both day 4 (P<0.0001 for all group comparisons) and day 7 (P=0.019 vs. null vector group).

[0458] Choroidal flat mounts were co-stained with fluorescein-labeled isolectin. Isolectin B4 stains endothelial cells and is used to visualize CNV lesions. Because data were non-normally distributed as assessed by the Kolmogorov-Smirnov test (P<0.05), the statistical significance of observed differences was determined using generalized linear model (GLM) analysis. All treatment groups showed a statistically significant reduction in isolectin B4 stained area compared to the null treatment group (GLM, P<0.05 for all). Co-administration of CFI-expressing vector and FHL1-expressing vector (GT005:RC001) produced the most significant reduction.

[0459] All publications mentioned in the above specification are incorporated herein by reference.Various modifications and variations of the disclosed agents, compositions, uses and methods of the present invention will be apparent to those skilled in the art without departing from the scope and spirit of the present invention.Although the present invention is disclosed in connection with certain preferred embodiments, it should be understood that the invention claimed should not be unduly limited to such specific embodiments.Indeed, various modifications of the disclosed modes for carrying out the present invention that are apparent to those skilled in the art are intended to be within the scope of the following claims.

[0460] The invention is further described in the following numbered paragraphs: [Item 1] A product comprising (i) complement factor H-like protein 1 (FHL1) or complement factor H (CFH) and (ii) complement factor I (CFI), or a nucleotide sequence encoding same, as a combined preparation for simultaneous, separate or sequential use in therapy. [Item 2] A product comprising (i) complement factor H-like protein 1 (FHL1) or complement factor H (CFH), and (ii) complement factor I (CFI) or a nucleotide sequence encoding same, as a combined preparation for simultaneous, separate or sequential use in treating or preventing complement-mediated disorders of the eye. [Item 3] The product for use according to Item 2, wherein the complement-mediated disorder is age-related macular degeneration (AMD) or diabetic retinopathy, preferably AMD. [Item 4] The product for use according to Item 3, wherein the AMD is dry AMD. [Item 5] The product for use according to any one of items 1 to 4, wherein the product provides (i) and (ii) to a subject in a molar ratio of (i):(ii) of at least 2:1, preferably at least 3:1, more preferably at least 8:1, more preferably at least 15:1. [Item 6] The product for use according to any one of Items 1 to X, which provides (i) and (ii) to a subject in a molar ratio of (i):(ii) of 2:1 to 12:1, preferably 3:1 to 10:1. [Item 7] An isolated polynucleotide comprising a nucleotide sequence encoding (i) complement factor H-like protein 1 (FHL1) or complement factor H (CFH) and (ii) cofactor I (CFI). [Item 8] The isolated polynucleotide according to Item 7, (a) a CMV promoter, optionally upstream of a nucleotide sequence encoding (i) and (ii); and (b) a WPRE regulatory element, optionally downstream of the nucleotide sequence encoding (i) and (ii), and / or (c) optionally downstream of the nucleotide sequence encoding (i) and (ii), a polyA signal, optionally a bovine growth hormone polyA signal; The polynucleotide further comprises a nucleotide sequence encoding [Item 9] An isolated polynucleotide according to item 7 or 8, wherein the nucleotide sequence encoding (i) is upstream of the nucleotide sequence encoding (ii). [Item 10] The isolated polynucleotide of any one of Items 7 to 9, further comprising one or more adeno-associated virus (AAV) inverted terminal repeats (ITRs). [Item 11] The isolated polynucleotide according to any one of Items 7 to 10, comprising an AA VITR at its 5' end and an AAV ITR at its 3' end. [Item 12] (a) 5′ AAV ITR; (b) CMV promoter; (c) a nucleotide sequence encoding FHL1 or CFH, preferably FHL1; (d) a linker optionally comprising or defined by a furin cleavage site, a GSG, an 11aa1D sequence and an F2A sequence; (e) a nucleotide sequence encoding CFI; (f) a WPRE regulatory element, optionally a WPRE3 regulatory element; (g) bovine growth hormone polyA signal; and (h) 3′ AAV ITRs, 12. The isolated polynucleotide according to any one of items 7 to 11, comprising: [Item 13] The isolated polynucleotide described in any one of Items 7 to 12, wherein the AAV ITR is an AAV2 or AAV8 ITR. [Item 14] The isolated polynucleotide of any one of Items 7 to 13, wherein the nucleotide sequence encoding FHL1 or CFH and CFI is codon-optimized. [Item 15] An isolated polynucleotide described in any one of Items 7 to 14, wherein (a) the nucleotide sequence encoding FHL1 has at least 75% sequence identity to SEQ ID NO: 12; and / or (b) the nucleotide sequence encoding CFI has at least 75% sequence identity to SEQ ID NO: 10. [Item 16] The isolated polynucleotide according to any one of Items 7 to 15, wherein (a) the nucleotide sequence encoding FHL1 is SEQ ID NO: 12 and / or (b) the nucleotide sequence encoding CFI is SEQ ID NO: 10. [Item 17] An isolated polynucleotide according to any one of Items 7 to 16, comprising a nucleotide sequence of SEQ ID NO: 22 or 23, or a nucleotide sequence having at least 75% sequence identity to SEQ ID NO: 22 or 23. [Item 18] The isolated polynucleotide according to any one of Items 7 to 17, which is 4.7 kb or less. [Item 19] A vector comprising the polynucleotide described in any one of Items 7 to 18. [Item 20] The vector described in Item 19, which is an adeno-associated virus (AAV) vector. [Item 21] The vector described in Item 19 or 20, which is in the form of a viral vector particle. [Item 22] The vector described in Item 21, wherein the AAV vector particle comprises an AAV2 or AAV8 genome and AAV2 or AAV8 capsid proteins. [Item 23] A cell comprising the polynucleotide described in any one of Items 7 to 18. [Item 24] A cell transduced with the vector described in any one of Items 19 to 22. [Item 25] A pharmaceutical composition comprising the polynucleotide, vector or cell according to any one of Items 7 to 24 in combination with a pharma- ceutically acceptable carrier, diluent or excipient. [Item 26] The polynucleotide, vector or cell according to any one of Items 7 to 24 for use in treatment. [Item 27] ​​A polynucleotide, vector, or cell described in any one of Items 7 to 24 for use in treating or preventing a complement-mediated disorder of the eye. [Item 28] The polynucleotide, vector or cell for use according to Item 27, wherein the complement-mediated disorder is age-related macular degeneration (AMD) or diabetic retinopathy, preferably AMD. [Item 29] The polynucleotide, vector or cell for use according to Item 28, wherein AMD is dry AMD. [Item 30] A polynucleotide, vector or cell for use according to any one of Items 26 to 29, wherein the formation of geographic atrophy is prevented or reduced, and / or the amount of geographic atrophy is reduced. [Item 31] The polynucleotide, vector, or cell for use according to any one of Items 26 to 30, wherein the progression of geographic atrophy is slowed. [Item 32] A polynucleotide, vector or cell for use according to any one of Items 26 to 31, which reduces the increase in geographic atrophy area by at least 10% over a 12 month period following administration to the treated eye of the subject, compared to the untreated eye over the same period. [Item 33] A polynucleotide, vector or cell for use according to any one of Items 26 to 32, wherein administration of the polynucleotide, vector or cell increases the level of C3b inactivation and iC3b degrading activity in a subject or in the subject's eye (such as the retinal pigment epithelium (RPE)), optionally to a level above normal levels for the subject, or for that eye or for that RPE. [Item 34] The polynucleotide, vector, or cell for use according to any one of Items 26 to 33, wherein the polynucleotide, vector, or cell is administered intraocularly. [Item 35] The polynucleotide, vector or cell for use according to any one of Items 26 to 34, wherein the polynucleotide, vector or cell is administered to the subject's eye by subretinal, direct retinal, suprachoroidal or intravitreal injection. [Item 36] The polynucleotide, vector or cell for use according to any one of Items 26 to 35, wherein the polynucleotide, vector or cell is administered to the subject's eye by subretinal injection. [Item 37] A method for treating or preventing a complement-mediated eye disorder, comprising administering to a subject in need thereof the polynucleotide, vector, or cell described in any one of Items 7 to 24. [Item 38] A method for providing a subject with (i) complement factor H-like protein 1 (FHL1) or complement factor H (CFH), and (ii) complement factor I (CFI), comprising delivering to the eye of the subject a polynucleotide, vector or cell described in any one of items 7 to 24.

Claims

1. (i) a Complement Factor I (CFI) cofactor, (ii) Complement Factor I (CFI), or a product comprising a nucleotide sequence encoding same, as a combined preparation for simultaneous, separate or sequential use in therapy.

2. As a combined preparation for simultaneous, separate or sequential use in treating or preventing complement-mediated disorders, preferably complement-mediated disorders of the eye, (i) a complement factor I (CFI) cofactor, and (ii) a product comprising complement factor I (CFI) or a nucleotide sequence encoding same.

3. 3. The product for use according to claim 2, wherein the complement mediated disorder is age-related macular degeneration (AMD) or diabetic retinopathy, preferably AMD.

4. 4. The product for use according to claim 3, wherein the AMD is dry AMD.

5. 5. The product for use according to any one of claims 1 to 4, wherein the Complement Factor I (CFI) cofactor is selected from the group consisting of Complement Factor H-like protein 1 (FHL1), Complement Factor H (CFH); Complement Receptor 1 (CR1) or a fragment thereof; and Membrane Cofactor Protein (MCP) or a fragment thereof.

6. 6. A product for use according to any one of claims 1 to 5, providing (i) and (ii) to a subject in a molar ratio of (i):(ii) of at least 2:1, preferably at least 3:1, more preferably at least 8:1, more preferably at least 15:

1.

7. 7. A product for use according to any one of claims 1 to 6, providing (i) and (ii) to a subject in a molar ratio (i):(ii) of from 2:1 to 12:1, preferably from 3:1 to 10:

1.

8. (i) a complement factor I (CFI) cofactor; and (ii) an isolated polynucleotide comprising a nucleotide sequence encoding cofactor I (CFI).

9. (a) a CMV promoter, optionally upstream of the nucleotide sequence encoding (i) and (ii); and (b) a WPRE regulatory element, optionally downstream of the nucleotide sequence encoding (i) and (ii), and / or (c) optionally downstream of said nucleotide sequence encoding (i) and (ii), a polyA signal, optionally a bovine growth hormone polyA signal; 9. The isolated polynucleotide of claim 8, further comprising a nucleotide sequence encoding

10. 10. The isolated polynucleotide of claim 8 or 9, wherein the nucleotide sequence encoding (i) is upstream of the nucleotide sequence encoding (ii).

11. The isolated polynucleotide of any one of claims 8 to 10, wherein the polynucleotide further comprises one or more adeno-associated virus (AAV) inverted terminal repeats (ITRs).

12. The isolated polynucleotide of any one of claims 8 to 11, wherein the polynucleotide comprises an AAV ITR at its 5' end and an AAV ITR at its 3' end.

13. (a) 5'AAV ITR; (b) CMV promoter; (c) a nucleotide sequence encoding a complement factor I (CFI) cofactor; (d) a linker, optionally defined by a furin cleavage site, GSG, a 11aa1D sequence and a F2A sequence; (e) a nucleotide sequence encoding CFI; (f) a WPRE regulatory element, optionally a WPRE3 regulatory element; (g) bovine growth hormone polyA signal; and (h) 3'AAV ITR, The isolated polynucleotide according to any one of claims 8 to 12, comprising:

14. 14. The isolated polynucleotide of any one of claims 8 to 13, wherein the Complement Factor I (CFI) cofactor is selected from the group consisting of Complement Factor H-like protein (FHL1); Complement Factor H (CFH); Complement Receptor 1 (CR1) or a fragment thereof; and Membrane Cofactor Protein (MCP) or a fragment thereof.

15. The isolated polynucleotide of any one of claims 8 to 14, wherein the AAV ITR is an AAV2 or AAV8 ITR.

16. 16. The isolated polynucleotide of any one of claims 8 to 15, wherein the nucleotide sequence encoding the CFI cofactor and CFI is codon optimized.

17. 17. The isolated polynucleotide of any one of claims 8 to 16, wherein (a) the nucleotide sequence encoding FHL1 has at least 75% sequence identity to SEQ ID NO:12; and / or (b) the nucleotide sequence encoding CFI has at least 75% sequence identity to SEQ ID NO:

10.

18. 18. The isolated polynucleotide of any one of claims 8 to 17, wherein (a) the nucleotide sequence encoding FHL1 is SEQ ID NO:12; and / or (b) the nucleotide sequence encoding CFI is SEQ ID NO:

10.

19. 19. The isolated polynucleotide of any one of claims 8 to 18, wherein the polynucleotide comprises the nucleotide sequence of SEQ ID NO: 22 or 23, or a nucleotide sequence having at least 75% sequence identity to SEQ ID NO: 22 or 23.

20. 20. The isolated polynucleotide of any one of claims 8 to 19, wherein the polynucleotide is 4.7 kb or less.

21. A vector comprising the polynucleotide according to any one of claims 8 to 20.

22. 22. The vector of claim 21, which is an adeno-associated virus (AAV) vector.

23. 23. The vector of claim 21 or 22, in the form of a viral vector particle.

24. 24. The vector of claim 23, wherein the AAV vector particle comprises an AAV2 or AAV8 genome and AAV2 or AAV8 capsid proteins.

25. A cell comprising the polynucleotide according to any one of claims 8 to 20.

26. A cell transduced with a vector according to any one of claims 21 to 24.

27. A pharmaceutical composition comprising the polynucleotide, vector or cell according to any one of claims 8 to 26 in combination with a pharma- ceutically acceptable carrier, diluent or excipient.

28. A polynucleotide, vector or cell according to any one of claims 8 to 26 for use in therapy.

29. A polynucleotide, vector or cell according to any one of claims 8 to 26 for use in treating or preventing a complement mediated disorder, preferably a complement mediated disorder of the eye.

30. 30. The polynucleotide, vector or cell for use according to claim 29, wherein the complement mediated disorder is age-related macular degeneration (AMD) or diabetic retinopathy, preferably AMD.

31. 31. The polynucleotide, vector or cell for use according to claim 30, wherein the AMD is dry AMD.

32. A polynucleotide, vector or cell for use according to any one of claims 28 to 31, wherein the formation of geographic atrophy is prevented or reduced and / or the amount of geographic atrophy is reduced.

33. The polynucleotide, vector or cell for use according to any one of claims 28 to 32, wherein the progression of geographic atrophy is slowed down.

34. A polynucleotide, vector or cell for use according to any one of claims 28 to 33, in which there is at least a 10% reduction in the increase in area of ​​geographic atrophy over 12 months following administration to the treated eye of a subject compared to an untreated eye over the same period.

35. 35. The polynucleotide, vector or cell for use according to any one of claims 28 to 34, wherein administration of the polynucleotide, vector or cell increases the level of C3b inactivating and iC3b degrading activity in the subject or in the subject's eye (such as in the retinal pigment epithelium (RPE)), optionally to a level above normal levels in the subject, or in that eye or in that RPE.

36. The polynucleotide, vector or cell for use according to any one of claims 28 to 35, wherein said polynucleotide, vector or cell is administered intraocularly.

37. The polynucleotide, vector or cell for use according to any one of claims 28 to 36, wherein said polynucleotide, vector or cell is administered to the subject's eye by subretinal, direct retinal, suprachoroidal or intravitreal injection.

38. The polynucleotide, vector or cell for use according to any one of claims 28 to 37, wherein said polynucleotide, vector or cell is administered to the subject's eye by subretinal injection.

39. A method for treating or preventing a complement mediated disorder, preferably an ocular complement mediated disorder, comprising administering to a subject in need thereof a polynucleotide, vector or cell according to any one of claims 8 to 26.

40. 27. A method of providing a subject with (i) a complement factor I (CFI) cofactor, and (ii) complement factor I (CFI), comprising delivering to said subject, preferably to an eye of said subject, a polynucleotide, vector or cell according to any one of claims 8 to 26.

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