Codon-optimized complement factor I

Abstract

Gene therapy treatments are provided for complement-mediated and complement-associated disorders, particularly chronic inflammatory conditions, and more particularly those associated with an overactive complement C3b feedback cycle. [Solution] An isolated polynucleotide is provided comprising a nucleotide sequence encoding complement factor I (CFI), wherein the nucleotide sequence has at least 85% sequence identity to a specific sequence.

Description

[Technical Field]

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

[0002] The macula is a small area in the retina of the eye, approximately 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 the age of 50 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 arising from the choroidal vasculature and extending into the subretinal space. Furthermore, 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, 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 or behavioral factors such as smoking and diet.

[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 and 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 ophthalmologic examination. Hard drusen have a well-defined margin, while soft drusen have a less defined, fluid margin. The Age-Related Eye Disease Study (AREDS) Fundus Photography Severity Scale is one of the primary classification systems used for this condition.

[0006] 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, the overlying photoreceptor cells, and often the underlying cells of the choriocapillaris. Confluent areas of RPE cell death with overlying photoreceptor atrophy are called geographic atrophy. Patients with this form of AMD experience a slow, progressive deterioration in central vision.

[0007] Wet AMD is characterized by bleeding and / or fluid leakage from abnormal blood vessels growing from the RPE and choroidal vasculature (choriocapillaris) under the macula, which can cause sudden vision loss. It is believed 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 begins in the retina and spreads posteriorly into the subretinal space, eventually connecting with new blood vessels in the choroid.

[0008] The complement system (CS) is implicated in early AMD pathogenesis based on the identification of CS components in drusen from AMD patient eyes. At least 129 drusen-deposited proteins have been identified in AMD, including various apolipoprotein types (E, B, or AI), several amyloid peptides (P, Aβ, or SA-1), TIMP-3, serum albumin, and specific proteins related to cellular function (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., components 5, 6, 8 (α, β, and γ), and 9). Accumulating drusen can activate CS, trigger the local production of inflammatory mediators, and attract leukocytes, which in turn can potentiate the local inflammatory state present in AMD.

[0009] 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 the aptamer pegaptanib (N Engl J Med (2004) 351:2805-2816) and antibodies such as ranibizumab (N Engl J Med (2006) 355:1432-1444) and bevacizumab (BMJ (2010) 340:c2459). However, not all patients respond to treatment with anti-VEGF antibodies, and vision does not improve or progress to recognized blindness.

[0010] A therapy for the treatment of geographic atrophy has been developed and used in a phase III clinical trial. Lampalizumab is a humanized monoclonal inhibitor antibody against complement factor D, administered by intravitreal injection, to halt the rate of progression of geographic atrophy. However, in a phase III randomized clinical trial involving 906 participants, lampalizumab failed to reduce the expansion of GA when compared with sham over 48 weeks.

[0011] Thus, there is a significant need in the art for new approaches to treating ocular diseases such as AMD.

[0012] Due to the ubiquitous nature of the complement system, an overactive or improperly functioning complement system is implicated in the pathology of many chronic inflammatory conditions for which no treatment options exist or for which symptom management requires years of periodic intervention. Thus, there is a general need to develop gene therapy treatments that provide novel or alternative treatments for complement-mediated and complement-associated disorders, particularly chronic inflammatory conditions, and more particularly those associated with an overactive complement C3b feedback cycle (Figure 1). Summary of the Invention [Problem to be solved by the invention]

[0013] Applicants have identified codon-optimized sequences for complement factor I (CFI) and complement factor H-like protein 1 (FHL1) that provide substantially increased expression of the encoded CFI and FHL1 proteins compared to the wild-type sequences.

[0014] The improved CFI- and FHL1-encoding sequences developed by the applicant allow higher doses of each protein to be delivered to patients without increasing the amount of administered vector. The present invention therefore provides improvements in manufacturing output (i.e., protein delivery can be achieved with fewer vectors produced), drug efficacy, and safety. In particular, because higher doses of encoded protein can be achieved with the same amount (e.g., volume) of vector delivered, the risk of damage to the tissue into which the vector is administered is reduced. For example, when the vector is delivered to the eye via subretinal injection, the risk of retinal damage or retinal detachment caused by injection of a large amount of drug is reduced. Furthermore, the risk of off-target effects caused by diffusion of large amounts of drug to adjacent tissues is mitigated. Furthermore, the use of the claimed nucleotide sequences in gene therapy has the potential to deliver therapy with a single administration, allowing for long-term, stable expression of the protein and avoiding the need for monthly or periodic injections. The nucleotide sequences and compositions of the present invention have additional advantages; therefore, they have the potential to provide a one-time or "one-shot" therapy that avoids repeated or periodic surgical interventions.

[0015] In one aspect, the present invention provides an isolated polynucleotide comprising a nucleotide sequence encoding complement factor I (CFI), wherein the nucleotide sequence has at least 85% sequence identity to SEQ ID NO:10.

[0016] In some embodiments, the nucleotide sequence encoding CFI has at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 10. In a preferred embodiment, the nucleotide sequence encoding CFI is SEQ ID NO: 10.

[0017] In another aspect, the present invention provides an isolated polynucleotide comprising a nucleotide sequence encoding complement factor H-like protein 1 (FHL1), wherein the nucleotide sequence has at least 75% sequence identity to SEQ ID NO:12.

[0018] In some embodiments, the nucleotide sequence encoding FHL1 has at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:12.

[0019] In a preferred embodiment, the nucleotide sequence encoding FHL1 is SEQ ID NO:12.

[0020] In some embodiments, the polynucleotide comprises one or more adeno-associated virus (AAV) inverted terminal repeats (ITRs). In a preferred embodiment, the polynucleotide comprises an AAV ITR at its 5' end and an AAV ITR at its 3' end.

[0021] In some embodiments, the AAV ITRs are AAV2 or AAV8 ITRs. In preferred embodiments, the AAV ITRs are AAV2 ITRs.

[0022] In another aspect, the present invention provides a vector comprising a polynucleotide of the present invention.

[0023] In some embodiments, the vector is an adeno-associated viral (AAV), retroviral, lentiviral, or adenoviral vector.

[0024] In a preferred embodiment, the vector is an AAV vector.

[0025] In some embodiments, the vector is in the form of a viral vector particle.

[0026] In some embodiments, the AAV vector comprises an AAV2 or AAV8 genome.

[0027] In some embodiments, the AAV vector particle comprises AAV2 or AAV8 capsid proteins.

[0028] In some embodiments, the AAV vector particle comprises an AAV2 genome and AAV2 capsid proteins (AAV2 / 2). In other embodiments, the AAV vector particle comprises an AAV2 genome and AAV8 capsid proteins (AAV2 / 8). In other embodiments, the AAV vector particle comprises an AAV8 genome and AAV8 capsid proteins (AAV8 / 8).

[0029] In some embodiments, the nucleotide sequence encoding CFI is operably linked to a CMV promoter. In some embodiments, the nucleotide sequence encoding CFI is operably linked to a regulatory element, such as a WPRE regulatory element. In preferred embodiments, the WPRE regulatory element is a WPRE3 regulatory element. In some embodiments, the nucleotide sequence encoding CFI is operably linked to a polyadenylation (polyA) signal, such as a bovine growth hormone polyA signal.

[0030] In a preferred embodiment, the nucleotide sequence encoding CFI is operably linked to a CMV promoter, a WPRE regulatory element (preferably a WPRE3 regulatory element); and a bovine growth hormone polyA signal.

[0031] In some embodiments, the nucleotide sequence encoding FHL1 is operably linked to a CMV promoter. In some embodiments, the nucleotide sequence encoding FHL1 is operably linked to a regulatory element, such as a WPRE regulatory element. In a preferred embodiment, the WPRE regulatory element is a WPRE3 regulatory element. In some embodiments, the nucleotide sequence encoding FHL1 is operably linked to a polyA signal, such as a bovine growth hormone polyA signal.

[0032] In a preferred embodiment, the nucleotide sequence encoding FHL1 is operably linked to a CMV promoter, a WPRE regulatory element (preferably a WPRE3 regulatory element); and a bovine growth hormone polyA signal.

[0033] In another aspect, the present invention provides a cell comprising a polynucleotide of the present invention.

[0034] In another aspect, the invention provides a cell transduced with a vector of the invention.

[0035] In another aspect, the invention provides a pharmaceutical composition comprising a polynucleotide, vector or cell of the invention in combination with a pharmaceutically acceptable carrier, diluent or excipient.

[0036] In certain embodiments, the pharmaceutical composition is suitable for systemic administration (eg, by injection into a peripheral vein).

[0037] In certain embodiments, the pharmaceutical composition is suitable for local administration (eg, intrathecal administration).

[0038] In a preferred embodiment, the pharmaceutical composition is for intraocular administration, for example, by intravitreal, suprachoroidal, or subretinal injection.

[0039] In another aspect, the invention provides a polynucleotide, vector or cell of the invention for use in therapy.

[0040] In a particular embodiment, the polynucleotides, vectors or cells of the invention are used to treat complement-mediated disorders, particularly chronic inflammatory conditions.

[0041] In a preferred embodiment, the polynucleotide, vector or cell of the invention is used to treat a disorder associated with an overactive complement C3b feedback cycle.

[0042] In another aspect, the invention provides a polynucleotide, vector or cell of the invention for use in treating or preventing an eye disorder.

[0043] 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.

[0044] In another aspect, the present invention provides a method for treating or preventing a complement-mediated disorder of the eye, comprising administering to a subject in need thereof a polynucleotide, vector, or cell of the present invention.

[0045] In another aspect, the present invention provides a method of providing complement factor I (CFI) and / or complement factor H-like protein 1 (FHL1) to a subject, comprising delivering a polynucleotide, vector, or cell of the present invention to the eye of the subject.

[0046] In some embodiments, the disorder is associated with overactivity of the complement C3b feedback cycle and / or underactivity of the C3b degradation cycle (see Figure 1).

[0047] In some embodiments, the disorder is a complement-mediated chronic inflammatory condition of the eye.

[0048] In some embodiments, the disorder is age-related macular degeneration (AMD) or diabetic retinopathy, hi other embodiments, the disorder is glaucoma, Stargardt's disease, central serous chorioretinopathy, or retinitis pigmentosa.

[0049] In preferred embodiments, the disease is AMD. In some embodiments, the AMD is dry AMD.

[0050] In some embodiments, the subject has been diagnosed with or is at risk for acquiring AMD.

[0051] 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 serum concentration of 0-30, 0-20, or 0-10 μg / mL or equivalent activity; and / or (b) heterozygous or homozygous for an age-related macular degeneration (AMD)-associated SNP, preferably a rare complement factor I variant.

[0052] 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., 30-40 μg / mL in serum; and / or (b) not carrying a rare complement factor I mutant allele;

[0053] In another aspect, the present invention provides a polynucleotide, vector, or cell of the invention for use in treating or preventing age-related macular degeneration (AMD). In a preferred embodiment, the AMD is dry AMD.

[0054] In another aspect, the invention provides a polynucleotide, vector or cell of the invention for use in treating or preventing diabetic retinopathy.

[0055] In some embodiments, the formation of geographic atrophy is prevented or reduced and / or the amount of geographic atrophy is reduced.

[0056] In some embodiments, the progression of geographic atrophy is slowed.

[0057] 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, 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, compared to the untreated eye over the same period.

[0058] 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 above normal levels in the subject, or in the eye, or RPE.

[0059] 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 afflicted with 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 vision or visual acuity loss, for example, vision or visual acuity loss associated with an ocular disorder, such as, for example, an ocular disorder disclosed herein.

[0060] 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 ocular disorder, e.g., an ocular disorder disclosed herein. In another aspect, the invention provides a polynucleotide, vector, or cell of the invention for use in alleviating reduced reading speed in a subject, e.g., reduced reading speed associated with an ocular disorder, e.g., an ocular disorder disclosed herein.

[0061] 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.

[0062] In some embodiments, the polynucleotide, vector, or cell is administered intraocularly.

[0063] In some embodiments, the polynucleotide, vector, or cell is administered to the subject's eye by subretinal, direct retinal, suprachoroidal, or intravitreal injection.

[0064] In some embodiments, the polynucleotide, vector, or cell is administered to the subject's eye by subretinal injection.

[0065] In some embodiments, the polynucleotide or vector of the present invention does not comprise an hAAT promoter. In some embodiments, the polynucleotide or vector of the present invention does not comprise an ApoR enhancer. In other embodiments, the polynucleotide or vector of the present invention does not comprise two ApoR enhancers.

[0066] In some embodiments, a vector of the invention does not comprise an AAV2 genome and an AAV8 capsid protein, i.e., the vector of the invention is not an AAV2 / 8 vector.

[0067] 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. [Brief explanation of the drawings]

[0068] [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). [Figure 2] Western blot analysis of supernatants from codon-optimized CFI and FHL1 plasmid transfections of ARPE19 cells. [Figure 3] ELISA analysis of supernatants from codon-optimized CFI plasmid transfection of ARPE19 cells. [Figure 4] ELISA analysis of supernatants from codon-optimized FHL1 plasmid transfection of ARPE19 cells. [Figure 5] ELISA analysis of supernatants from codon-optimized CFI AAV vector transduction of ARPE19 cells. [Figure 6] ELISA analysis of supernatants from codon-optimized FHL1 AAV vector transduction of ARPE19 cells. DETAILED DESCRIPTION OF THE INVENTION

[0069] 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

[0070] 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.

[0071] The complement cascade consists of four activation pathways. All pathways ultimately terminate with central cleavage of factor C3 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 microvascular permeability, while C3b is responsible for opsonizing foreign surfaces covalently bound to it. Opsonization by activated C3 fragments (C3b and iC3b) fulfills three major functions: (i) clearance of cellular debris by phagocytes (e.g., macrophages or microglia) and stimulation of the adaptive immune system (B cells and T cells), (ii) amplification of complement activation via the formation of surface-bound C3 convertase, and (iii) assembly of C5 convertase.

[0072] The assembly of C5 convertases is responsible for C5 cleavage, which leads to the formation of the cytolytic membrane attack complex (MAC), which can generate perforations in cell membranes, 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 leads to 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 increased capillary permeability to promote the extravasation of infiltrating leukocytes. Under physiological conditions, complement activation is effectively controlled by the coordinated action of soluble and membrane-bound complement regulatory molecules (CRMs). Soluble complement regulatory factors, such as C1 inhibitor, anaphylatoxin inhibitor, C4b-binding protein (C4BP), complement factor H (CFH), complement factor I (CFI), clusterin, and vitronectin, limit complement action in human tissues at multiple sites in the cascade. 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.

[0073] The present invention relates to the treatment or prevention of ocular complement-mediated disorders. For example, the complement-mediated disorder may be a disorder associated with defects in alternative pathway regulation, particularly hyperactivity of the complement C3b feedback cycle and / or hypoactivity of the C3b degradation cycle.

[0074] In some embodiments, before administration of the 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 have 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 described further below). Thus, the subject may have a low (e.g., lower-than-normal) concentration of complement factor I in the eye and / or serum. In a human subject, a normal level of complement factor I activity (activity to inactivate C3b and degrade iC3b) can be equivalent to that provided by 30-40 μg / mL of complement factor I in the subject's serum. Thus, in a subject with low activity of complement factor I, serum complement factor I activity can 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 can include subjects with low concentrations of complement factor I).

[0075] Thus, the subject to be treated by the present invention may be suffering from or at risk of developing a complement-mediated ocular disorder (e.g., characterized by geographic atrophy), such as AMD, more particularly dry AMD. For example, the subject may be homozygous or heterozygous for susceptibility to one or more SNPs associated with a complement-mediated disorder.

[0076] In some embodiments, the subject is at risk of developing AMD. For example, the subject may be homozygous or heterozygous for one or more SNPs associated with AMD, such as rare mutations in complement factor I that are generally associated with advanced AMD, leading to decreased 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).

[0077] The present invention may further include determining whether a subject is at risk for developing a complement-mediated disorder (e.g., AMD), for example, 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).

[0078] Alternatively, the 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.

[0079] In some embodiments, administration of a polynucleotide, vector, cell, or pharmaceutical composition of the present invention thereby increases the level of C3b inactivating and iC3b degrading activity in the subject's eye. In other embodiments, administration of a polynucleotide, vector, cell, or pharmaceutical composition of the present invention thereby increases the level of C3b inactivating and iC3b degrading activity in the subject's eye to a level above normal in the eye. More specifically, the level of C3b inactivating and iC3b degrading activity is increased in the RPE of the eye.

[0080] It will be understood that the C3b-inactivating and iC3b-degrading activity in a subject after expression of complement factor I from a polynucleotide or vector of the present invention may include C3b-inactivating and iC3b-degrading activity from the subject's endogenous complement factor I (i.e., the subject's complement factor I not produced by expression from a polynucleotide or vector), as well as C3b-inactivating and iC3b-degrading activity produced by expression from a polynucleotide or vector of the present invention, such that the total level of C3b-inactivating and iC3b-degrading activity in the subject is greater than normal.

[0081] 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.

[0082] In other embodiments, the level of C3b inactivating and iC3b degrading activity in the subject, e.g., in the eye, is increased to a level of up to twice the normal level, or up to 80%, 60%, 40%, or 20% of the normal level.

[0083] 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 the normal level.

[0084] In some embodiments, administration of a polynucleotide, vector, cell, or pharmaceutical composition of the invention does not detectably increase the level of C3b inactivating and iC3b degrading activity in the subject's plasma / serum, hi other embodiments, administration of a polynucleotide, vector, cell, or pharmaceutical composition of the invention does not detectably increase the level of C3b inactivating and iC3b degrading activity in the subject's plasma / serum to a level higher than normal.

[0085] In the preceding sections, unless clearly applicable, references to complement factor I and its activity in inactivating C3b and degrading iC3b may be substituted with complement factor H or complement factor H-like protein 1, and its ability to act as a cofactor for complement factor I-mediated C3b cleavage and increase the dissociation rates of C3 convertase and C5 convertase, respectively. In some embodiments, prior to administration of the polynucleotide, vector, cell, or pharmaceutical composition of the invention, the subject has low (e.g., lower than normal) levels of complement factor H, e.g., low levels of complement factor H in the eye and / or low serum levels of complement factor H. For human subjects, normal levels of complement factor H may be approximately 200-500 μg / mL in the subject's serum. Thus, in subjects with low levels of complement factor H, serum levels may be less than 200 μg / mL, greater than 0 μg / 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)

[0086] Complement factor I (factor I, CFI), also known as C3b / C4b inactivator, is a protein that in humans is encoded by the CFI gene.

[0087] 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 FI membrane attack complex (FIMAC) domain; a CD5-like domain or scavenger receptor cysteine-rich (SRCR) domain; two low-density lipoprotein receptor (LDLr) domains; and a C-terminal region of unknown function, which is a site of sequence diversity between 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).

[0088] 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 function, 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).

[0089] 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.

[0090] 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.

[0091] Complement factor I can process iC3b,g to C3d,g through a protease activity that requires the cofactor CR1. C3d,g cannot bind to CR3. Because iC3b reacts with the complement receptor CR3, which is the primary mechanism by which complement activation causes inflammation, degradation of iC3b to C3d,g is essential for reducing complement-induced inflammation (Lachmann (2009) Adv. Immunol. 104:115-149).

[0092] 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.

[0093] In some embodiments, a complement factor I polypeptide can cleave C3b into inactive breakdown products, for example, a complement factor I polypeptide can cleave C3b into iC3b.

[0094] In some embodiments, the complement factor I polypeptide can process iC3b into inactive degradation products. For example, the complement factor I polypeptide can process iC3b into C3d,g.

[0095] In a preferred embodiment, the complement factor I polypeptide is capable of cleaving C3b into iC3b and processing iC3b into C3d,g.

[0096] 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.

[0097] The C3b-inactivating and iC3b-degrading activity of complement factor I, or its fragments or derivatives, can be determined using any suitable method known to those skilled in the art. For example, 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 for 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 provided by Harrison RA (1996) in "Weir's Handbook of Experimental Immunology" (5th Edition, eds. Herzenberg Leonard 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) clipping, which converts fixed C3b to iC3b and acquires reactivity with conglutinin, and the final clipping to C3dg, starting with fixed iC3b and looking for loss of reactivity with conglutinin. Hemolytic assays are used for the conversion of C3b to iC3b, and proteolytic assays detect all clippings.

[0098] In some embodiments, the complement factor I is human complement factor I.

[0099] 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 the signal sequence.

[0100] In some embodiments, the amino acid sequence of complement factor I is SEQ ID NO: 1. In other embodiments, the amino acid sequence of complement factor I is the sequence disclosed as positions 19 to 583 of SEQ ID NO: 1. [ka] (SEQ ID NO: 1)

[0101] 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] (SEQ ID NO: 9)

[0102] An exemplary wild-type nucleotide sequence encoding complement factor I is the nucleotide sequence having NCBI accession number NM_000204, disclosed herein as SEQ ID NO:2. [ka] (SEQ ID NO: 2)

[0103] The nucleotide sequence of complement factor I used in the present invention is preferably codon-optimized. Different cells have different codon usage patterns. This codon bias corresponds to the relative abundance bias of certain tRNAs in cell types. By changing the codons in the sequence so that they are adjusted to match the relative abundance of the corresponding tRNAs, expression can be increased. 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 can be utilized.

[0104] A preferred nucleotide sequence encoding complement factor I is the nucleotide sequence disclosed as SEQ ID NO:10. [ka] (SEQ ID NO: 10)

[0105] In some embodiments, the nucleotide sequence encoding complement factor I has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homology to 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.

[0106] In some embodiments, the nucleotide sequence encoding complement factor I is SEQ ID NO:10.

[0107] In other embodiments, the nucleotide sequence encoding complement factor I has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to 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.

[0108] In another embodiment, the nucleotide sequence encoding complement factor I is positions 55 to 1752 of SEQ ID NO:10.

[0109] A further exemplary codon-optimized nucleotide sequence encoding complement factor I is SEQ ID NO:8. [ka] (SEQ ID NO: 8)

[0110] An advantage of the present invention is that complement factor I is particularly difficult to prepare in the form of a purified protein. Therefore, the inventors have devised a method of modulating the complement system to allow for the treatment of, for example, 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 I. The AAV vector can be administered to a site of interest, for example, the eye, to allow in situ translation of the complement factor I polypeptide. Complement factor H (CFH)

[0111] Complement factor H (factor H, CFH) is a complement regulatory protein.

[0112] 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.

[0113] Complement factor H provides cofactor activity for complement factor I-mediated cleavage of C3b. 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.

[0114] 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 CCP module consists of approximately 60 amino acids with four cysteine ​​disulfides linked in a 1–3 2–4 configuration and a hydrophobic core built around a nearly invariant tryptophan residue. CCP modules are numbered 1–20 (from the N-terminus of the protein). CCPs 1–4 and CCPs 19–20 bind C3b, while CCPs 7 and 19–20 bind GAGs and sialic acids (Schmidt et al., (2008) Journal of Immunology 181:2610–2619).

[0115] Gene therapy using complement factor H has been shown 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.

[0116] In some embodiments, the complement factor H polypeptide, or a fragment or derivative thereof, can act as a cofactor for complement factor I-mediated cleavage of C3b. In some embodiments, the complement factor H polypeptide, or a fragment or derivative thereof, can increase the dissociation rate of C3 convertase and C5 convertase.

[0117] In a preferred embodiment, the complement factor H polypeptide or fragment or derivative thereof is capable of acting as a cofactor for complement factor I-mediated cleavage of C3b and increasing the rate of dissociation of C3 and C5 convertases.

[0118] In some embodiments, the complement factor H is human complement factor H.

[0119] An example of a human complement factor H protein is the human complement factor H protein having UniProtKB accession number P08603. This exemplary sequence is 1231 amino acids in length (disclosed as SEQ ID NO:3), of which amino acids 1-18 form the signal sequence.

[0120] 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 to 1231 of SEQ ID NO: 3. [ka] (SEQ ID NO: 3)

[0121] An example of a nucleotide sequence encoding complement factor H is the nucleotide sequence having NCBI accession number NM_000186.

[0122] In some embodiments, the nucleotide sequence encoding complement factor H is SEQ ID NO:4. [ka] TIFF2026041876000008.tif112161 (SEQ ID NO: 4)

[0123] 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.

[0124] In another embodiment, the nucleotide sequence encoding complement factor H is SEQ ID NO:4.

[0125] 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.

[0126] In another embodiment, the nucleotide sequence encoding complement factor H is positions 55 to 3696 of SEQ ID NO:4.

[0127] 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.

[0128] In another embodiment, the nucleotide sequence encoding complement factor H encodes the amino acid sequence of SEQ ID NO:3.

[0129] 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 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.

[0130] 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)

[0131] 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 four amino acid carboxy-terminal tail (Clark, SJ et al. (2015) J Clin Med 4:18-31).

[0132] In some embodiments, the FHL1 is human FHL1.

[0133] In some embodiments, the amino acid sequence of FHL1 is SEQ ID NO:11. [ka] (SEQ ID NO: 11)

[0134] The nucleotide sequence of FHL1 used in the present invention is preferably codon optimized.

[0135] A preferred nucleotide sequence encoding FHL1 is SEQ ID NO:12. [ka] (SEQ ID NO: 12)

[0136] 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. Preferably, the protein encoded by the nucleotide sequence substantially retains the functional activity of the protein represented by SEQ ID NO: 11.

[0137] In another embodiment, the nucleotide sequence encoding FHL1 is SEQ ID NO:12. Polynucleotides

[0138] The polynucleotide 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 due to the degeneracy of the genetic code, many different polynucleotides can encode 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 specific host organism in which the polypeptide of the present invention is expressed.

[0139] 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 lifespan of polynucleotides of the invention.

[0140] 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.

[0141] 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 animal or human cells, 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. Primers may be designed to contain suitable restriction enzyme recognition sites so that the amplified DNA can be cloned into a suitable vector. Eye structure

[0142] 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).

[0143] 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

[0144] The retina is a multilayered 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 the neurosensory retina and the retinal pigment epithelium layer, with the choroid lying outside the retinal pigment epithelium. Neurosensory retina and photoreceptor cells

[0145] The neurosensory retina contains the photoreceptor cells that directly sense light. It includes 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 (photoreceptor nuclei), the outer limiting membrane (ELM), and the photoreceptors (inner and outer segments of rods and cones).

[0146] 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. Photoreceptor cells include rod cells and cone cells, and are distributed differently throughout the retina.

[0147] Rod cells are found primarily in the outer retina. They are highly sensitive and provide vision at low light levels. On average, a normal human retina contains approximately 125 million rod cells.

[0148] 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. A normal human retina contains an average of 6-7 million cone cells. retinal pigment epithelium

[0149] 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

[0150] The choroid is a vascular layer located 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)

[0151] The clinical progression of age-related macular degeneration (AMD) is characterized by stages depending on changes in the macula. A hallmark of early AMD is the appearance of drusen, which are accumulations of extracellular debris beneath the retina and appear as yellowish 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 ophthalmologic examination. Hard drusen have clearly defined margins, while soft drusen have a less defined, fluid margin. The Age-Related Eye Disease Study (AREDS) Fundus Photography Severity Scale is one of the primary classification systems used for this condition.

[0152] 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 underlying cells of the choriocapillaris. Confluent areas of RPE cell death with overlying photoreceptor atrophy are called geographic atrophy (GA). Patients with this form of AMD experience a slow, progressive deterioration in central vision.

[0153] Wet AMD is characterized by bleeding and / or fluid leakage from abnormal blood vessels growing from the RPE and choroidal vessels (choriocapillaris) beneath the macula, which can cause sudden vision loss. It is believed that much of the vision loss experienced by patients is due to such choroidal neovascularization (CNV) and its secondary complications.

[0154] The treatment or prevention of AMD described herein may reduce or prevent the appearance of the above-mentioned AMD phenotypes. Preferably, the treatment of AMD allows for the maintenance or improvement of visual function.

[0155] 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 causes at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90% reduction in the increase of GA area after administration to the treated eye of the subject for 12 months, compared with the untreated eye for 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.

[0156] In some embodiments, treating or preventing AMD results in the prevention or reduction of drusen formation, hi 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.

[0157] 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.

[0158] 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.

[0159] 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.

[0160] In some embodiments, treating or preventing AMD results in a reduction or prevention of photoreceptor and / or retinal pigment epithelium (RPE) loss. diabetic retinopathy

[0161] 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.

[0162] Although subjects with mild diabetic retinopathy may have good vision, two types of diabetic retinopathy, diabetic macular edema (DMO) and proliferative diabetic retinopathy (PDR), can threaten a subject's vision.

[0163] Diabetic macular edema is characterized by fluid leakage from damaged blood vessels at the back of the eye. The fluid accumulates in the macula, causing swelling and blurred vision. This ultimately leads to a decline in central vision, which may make reading or driving impossible. Side vision usually remains normal.

[0164] Proliferative diabetic retinopathy is characterized by the closure of retinal blood vessels, which leads to the growth of abnormal, destructive blood vessels on the surface of the retina, which can result in permanent loss of vision due to bleeding into the eye, scarring, and retinal detachment. vector

[0165] A vector is a tool that allows or facilitates the transfer of entities from one environment to another. Adeno-associated virus (AAV) vectors

[0166] In one aspect, the invention provides an AAV vector comprising a polynucleotide of the invention.

[0167] Preferably, the AAV vector is in the form of an AAV vector particle.

[0168] Methods for preparing and modifying viral vectors and viral vector particles, such as those derived from AAV, are well known in the art.

[0169] An AAV vector may comprise the AAV genome or a fragment or derivative thereof.

[0170] 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).

[0171] AAV genome is a polynucleotide sequence, which can encode the functions necessary for the production of AAV particles.These functions include the functions that are operated in the AAV replication and packaging cycle in host cells, including the encapsidation of AAV genome into AAV particles.Naturally occurring AAV is replication-defective and depends on the provision of trans helper functions to complete the replication and packaging cycle.Therefore, the AAV genome of the AAV vector of the present invention is typically replication-defective.

[0172] The AAV genome can be in a single-stranded form, either positive-sense or negative-sense, or in a double-stranded form. Use of the double-stranded form can bypass the DNA replication step in the target cell, thereby facilitating transgene expression.

[0173] 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.

[0174] Generally, AAV is referred to from the viewpoint of its serotype.Serotype corresponds to the variant subspecies of AAV, and it has a unique reactivity that can be used to distinguish it from other variant subspecies according to the expression profile of capsid surface antigen.Usually, the virus with a certain AAV serotype does not effectively cross-react with the neutralizing antibody specific for any other AAV serotype.

[0175] AAV serotypes include AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, and AAV11, as well as recombinant serotypes recently identified from primate brains, such as Rec2 and Rec3. Any of these AAV serotypes can be used in the present invention.

[0176] 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.

[0177] In some embodiments, the AAV may be an AAV1, AAV2, AAV5, AAV7, or AAV8 serotype.

[0178] In some embodiments, the AAV may be of the AAV2 or AAV8 serotype.

[0179] In some embodiments, the AAV may be of the AAV2 serotype, while in other embodiments, the AAV may be of the AAV8 serotype.

[0180] The capsid protein may be a mutant capsid protein as disclosed in WO 2008 / 124724, which is incorporated herein by reference.

[0181] In some embodiments, the AAV vector comprises an AAV8 capsid with a Y733F mutation.

[0182] 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 can 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.

[0183] AAVs can also be referred to in terms of clades or clones. This refers to the phylogenetic relationships of naturally occurring AAVs, typically referring to a phylogenetic group of AAVs that can be traced back to a common ancestor and includes all of its descendants. Additionally, AAVs can be referred to in terms of specific isolates, i.e., specific genetic isolates of AAVs found in nature. The term genetic isolate refers to a population of AAVs that has undergone limited genetic mixing with other naturally occurring AAVs, thereby defining distinct populations that can be recognized at the genetic level.

[0184] Those skilled in the art can select appropriate serotypes, clades, clones, or isolates of AAV for use in the present invention based on general knowledge. For example, AAV5 capsids have been shown to efficiently transduce cone photoreceptors in primates, as evidenced by the successful correction of inherited color blindness (Mancuso et al., (2009) Nature 461:784-7).

[0185] The AAV serotype determines the tissue specificity of infection (or tropism) of the AAV. Thus, preferred AAV serotypes for use in AAV administered to patients according to the present invention are those that have a natural tropism or high efficiency for infecting target cells within the eye. In some embodiments, AAV serotypes for use in the present invention transduce cells of the neurosensory retina, retinal pigment epithelium, and / or choroid.

[0186] 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 for integration and excision of the vector from the cellular genome. In a preferred embodiment, one or more ITR sequences flank a nucleotide sequence encoding complement factor I or FHL1. The AAV genome also typically contains packaging genes, such as the rep gene and / or cap gene, which encode the packaging function of AAV particles. 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.

[0187] A promoter is operably linked to each of the packaging genes. Specific examples of such promoters include the p5, p19, and p40 promoters (Laughlin et al., (1979) Proc. Natl. Acad. Sci. USA 76:5567-5571). For example, the p5 and p19 promoters are generally used to express the rep gene, and the p40 promoter is generally used to express the cap gene.

[0188] As described above, the AAV genome used in the AAV vectors of the present invention can therefore be the complete genome of a naturally occurring AAV. For example, a vector containing the complete AAV genome can be used to prepare an AAV vector or vector particle in vitro. However, while such a vector could 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 it 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, as well as 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, Choi et al., and Wu et al. (see above).

[0189] The derivative of the AAV genome includes any shortened or modified form of the AAV genome that allows the AAV vector of the present invention to express a transgene in vivo. Typically, the AAV genome can be significantly shortened to contain minimal viral sequences and still retain the above-mentioned functions. This is preferable for safety reasons, as it reduces the risk of vector recombination with wild-type viruses and avoids the induction of cellular immune responses due to the presence of viral gene proteins in target cells.

[0190] 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 of different serotypes, or can be chimeric or mutant ITRs. A preferred mutant ITR is one that has a trs (terminal resolution site) deletion. This deletion allows the genome to continue replicating, generating a single-stranded genome containing both the coding sequence and the complementary sequence, i.e., a self-complementary AAV genome. This allows bypass of DNA replication in target cells, thus promoting the expression of the transgene.

[0191] The one or more ITRs will preferably flank the nucleotide sequence encoding complement factor I or FHL1 at either end. 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, e.g., after conversion of single-stranded vector DNA to double-stranded DNA by the action of host cell DNA polymerases. Formation of such episomal concatemers protects the vector construct for the life of the host cell, thereby enabling long-term expression of the transgene in vivo.

[0192] In a preferred embodiment, the ITR element is the only sequence that is retained from the natural 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 sequences 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.

[0193] Thus, the following portions 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 comprise 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, with only a small random integration frequency, and therefore, retention of integration capacity in the vector may be acceptable in a therapeutic setting.

[0194] 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 vector).

[0195] Chimeric, shuffled, or capsid-modified derivatives are typically selected to provide one or more desired functions to the AAV vector. Thus, compared to AAV vectors containing naturally occurring AAV genomes, such as AAV2, these derivatives may exhibit improved gene delivery efficiency, reduced immunogenicity (humoral or cellular), altered tropism, and / or improved targeting of specific cell types. Improved gene delivery efficiency can be influenced by improved receptor or co-receptor binding on the cell surface, improved internalization, improved intracellular and nuclear transport, improved uncoating of viral particles, and improved conversion of single-stranded genomes to double-stranded forms. Improved efficiency may also be related to changes in the tropism or targeting of specific cell populations, thereby preventing the vector dose from being diluted by administration to tissues where it is not needed.

[0196] Chimeric capsid proteins include those generated by recombination between two or more capsid-coding sequences of naturally occurring AAV serotypes. This can be achieved, 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 directed selection is used to select for capsid sequences with desired properties. Capsid sequences of different serotypes can be altered by homologous recombination in cells to generate novel chimeric capsid proteins.

[0197] Chimeric capsid proteins also include those generated by manipulating capsid protein sequences 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.

[0198] Shuffled or chimeric capsid proteins can also be generated by DNA shuffling or error-prone PCR. Hybrid AAV capsid genes can be created by randomly fragmenting related AAV gene sequences, 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. Libraries of hybrid AAV genes 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 then be selected for desired properties.

[0199] 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.

[0200] The unrelated protein or peptide may advantageously act as a ligand for a specific cell type, thereby providing improved binding to target cells or improving the specificity of targeting the vector to a specific cell population. An example would be the use of RGD peptides to block uptake in the retinal pigment epithelium, thereby facilitating transduction into surrounding retinal tissues (Cronin et al., (2008) ARVO Abstract:D1048). The unrelated protein may also aid in the purification of viral particles as part of the production process, i.e., as an epitope or affinity tag. The insertion site is typically selected so as not to interfere with other functions of the viral particle (such as internalization or transport of the viral particle). Those skilled in the art can identify suitable sites for insertion based on general knowledge. Specific sites are disclosed in Choi et al., referenced above.

[0201] 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 with 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 from different viral species.

[0202] An AAV vector of the invention may take the form of a nucleotide sequence comprising an AAV genome or a derivative thereof and a sequence encoding a complement factor I, or FHL1 transgene or a derivative thereof.

[0203] The AAV particles of the present invention include transcapsid forms, in which an AAV genome or derivative with ITRs of one serotype is packaged in a 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 of the present invention also include chemically modified forms that carry ligands adsorbed on the capsid surface.For example, such ligands can include antibodies for targeting specific cell surface receptors.

[0204] 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.

[0205] AAV vectors may contain multiple copies (e.g., 2, 3, etc.) of the nucleotide sequences referred to herein. Promoters and Regulatory Sequences

[0206] The polynucleotides or vectors of the present invention may also contain elements that allow for expression of the complement factor I or FHL1 transgene in vitro or in vivo. These may also be referred to as expression control sequences. Thus, the polynucleotides or vectors typically contain an expression control sequence (e.g., a promoter sequence) operably linked to the nucleotide sequence encoding the transgene.

[0207] Any suitable promoter can be used, and its selection can be easily performed by one 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 targeted expression of the transgene in a specific cell type (e.g., tissue-specific promoter). The promoter can also exhibit inducible expression in response to the presence of another factor, for example, a factor present in the host cell. In either event, when the vector is administered for therapeutic purposes, it is preferable that the promoter be functional in the background of the target cell.

[0208] In some embodiments, the promoter preferably exhibits retinal cell-specific expression, so that the transgene can be expressed only in a population of retinal cells. Thus, expression from the promoter can be retinal cell-specific, for example, restricted to only cells of the neurosensory retina and retinal pigment epithelium.

[0209] Preferred promoters that are not retinal cell-specific include the chicken beta actin (CBA) promoter, optionally combined with the cytomegalovirus (CMV) enhancer element. An exemplary promoter for use in the present invention is the CAG promoter, such as the promoter used in the rAVE expression cassette (GeneDetect.com).

[0210] In a preferred embodiment, the polynucleotide or vector comprises a CMV promoter.

[0211] An example of a CMV promoter sequence is: [ka] (SEQ ID NO: 13)

[0212] 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.

[0213] In other embodiments, the polynucleotide or vector comprises a promoter having the nucleotide sequence of SEQ ID NO:13.

[0214] Further examples of promoter sequences are as follows: [ka] (SEQ ID NO: 5)

[0215] 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.

[0216] In other embodiments, the polynucleotide or vector comprises a promoter having the nucleotide sequence of SEQ ID NO:5.

[0217] 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).

[0218] The polynucleotide or vector of the present invention may also contain one or more additional regulatory sequences that can act before or after transcription. The regulatory sequences may be part of the natural transgene locus or may be heterologous regulatory sequences. The polynucleotide or vector of the present invention may contain a portion of the 5'-UTR or 3'-UTR from the natural transgene transcript.

[0219] 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, post-transcriptional regulatory elements, and polyadenylation sites.

[0220] A preferred polyadenylation site is the bovine growth hormone polyA (bGH polyA) signal.

[0221] An example of a bovine growth hormone polyA (bGH polyA) signal is: [ka] (SEQ ID NO: 14)

[0222] A further example of a bovine growth hormone polyA (bGH polyA) signal is as follows: [ka] (SEQ ID NO: 6)

[0223] 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, the nucleotide sequence substantially retains the functional activity of the polyadenylation signal represented by SEQ ID NO: 14 or 6.

[0224] In other embodiments, the polynucleotide or vector comprises a polyadenylation signal having the nucleotide sequence of SEQ ID NO: 14 or 6.

[0225] 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.

[0226] 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.

[0227] An example of a WPRE is: [ka] (SEQ ID NO: 7)

[0228] The WPRE is a tripartite element containing 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) containing only minimal gamma and alpha elements may be used in the present invention.

[0229] An example of a WPRE3 sequence is as follows: [ka] (SEQ ID NO: 15)

[0230] 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, the nucleotide sequence substantially retains the functional activity of the post-transcriptional regulatory element represented by SEQ ID NO: 15 or 7.

[0231] In other embodiments, the polynucleotide or vector comprises a post-transcriptional regulatory element having the nucleotide sequence of SEQ ID NO: 15 or 7.

[0232] Another regulatory sequence that can be used in the polynucleotides or vectors of the present invention is a scaffold attachment region (SAR). Additional regulatory sequences can be readily selected by those skilled in the art. Administration method

[0233] The polynucleotides or vectors of the invention can be administered systemically (e.g., by peripheral intravenous injection) and locally (e.g., to the CNS system by intrathecal injection). In a preferred embodiment, the polynucleotides or vectors are administered intraocularly.

[0234] 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.

[0235] In some embodiments, the polynucleotide or vector is administered to the subject's eye by subretinal, direct retinal, suprachoroidal, or intravitreal injection, hi some embodiments, the administration is performed robotically.

[0236] The volume of the pharmaceutical composition to be injected may be, for example, about 10 to 500 μL, e.g., 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.

[0237] Those skilled in the art will be familiar with and able to perform individual subretinal, direct retinal, suprachoroidal or intravitreal injections.

[0238] Preferably, the polynucleotide or vector is administered by subretinal injection.

[0239] In some embodiments, the polynucleotide, vector, or pharmaceutical composition is administered no more than once, or no more than twice, during the subject's lifetime. subretinal injection

[0240] 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.

[0241] If the injection is made through a small retinotomy, retinal detachment can occur. The detached raised layer of retina created by the injected material is called a "bleb."

[0242] The hole created by the subretinal injection must be small enough so that the injected solution does not significantly backflow back into the vitreous cavity after administration. Such backflow may be particularly problematic when a drug is injected, as the drug's effect 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 little or no injected material is released from the hole.

[0243] 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), which are needles designed to perform subretinal injections.

[0244] As long as no damage to the retina occurs during injection and 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 little injected material usually escapes into the vitreous. The bleb may dissipate over a longer time frame as the injected material is absorbed.

[0245] Visualization of the eye, and particularly the retina, can be performed preoperatively using, for example, optical coherence tomography.

[0246] The volume of the pharmaceutical composition injected may be, for example, about 10 to 500 μL, e.g., 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 injected is 100 μL. A larger volume may increase the risk of retinal elongation, while a smaller volume may result in poorer vision. Two-stage subretinal injection

[0247] The 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 polynucleotide or vector. A second subretinal injection is then used to deliver a drug containing the polynucleotide or vector into the subretinal fluid of the bleb created by the first subretinal injection. Because the injection that delivers the drug is not used to detach the retina, a specific volume of solution can be injected in this second step.

[0248] In some embodiments, the subretinal injection of the vector comprises: (a) administering a solution to a subject by subretinal injection in an amount effective to at least partially detach the retina and form a subretinal bleb, wherein the solution does not contain a polynucleotide or vector; (b) administering a pharmaceutical composition by subretinal injection into the bleb formed by step (a), wherein the pharmaceutical comprises a polynucleotide or a vector; Includes.

[0249] The volume of the solution injected in step (a) to at least partially detach the retina may be, for example, about 10 to 1000 μL, e.g., about 50 to 1000, 100 to 1000, 250 to 1000, 500 to 1000, 10 to 500, 50 to 500, 100 to 500, or 250 to 500 μL. The volume may be, for example, about 10, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 μL.

[0250] The volume of the pharmaceutical composition injected in step (b) may be, for example, about 10 to 500 μL, e.g., 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 injected in step (b) is 100 μL. A larger volume may increase the risk of retinal elongation, while a smaller volume may result in poorer visualization.

[0251] The drug-free solution (i.e., the "solution" of step (a)) can 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 retinal visualization

[0252] 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®, Dorc) 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.

[0253] 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

[0254] The polynucleotides or vectors of the present invention can be delivered to the suprachoroidal space using an externa 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 (e.g., iScience Interventional's iTrack250A, 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 manipulating the tip of the microcatheter to the desired location, injection of the polynucleotide or vector forms a bleb in the retina and choroid.

[0255] Thus, in some embodiments, the polynucleotide or vector is delivered suprachoroidally by a method comprising: (i) introducing a microcatheter into the suprachoroidal space; (ii) advancing the microcatheter within the suprachoroidal space until the tip is near the affected area of ​​the retina; and (iii) injecting the polynucleotide or vector through the tip of the microcatheter to form a bleb.

[0256] In some embodiments, the above administration steps are performed directly by the robot. Pharmaceutical compositions and injection solutions

[0257] The agent of the present invention, for example, polynucleotide or vector, can be formulated into pharmaceutical compositions.These compositions can contain, in addition to the agent, pharmaceutically acceptable carriers, diluents, excipients, buffers, stabilizers, or other materials 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 carriers 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.

[0258] Pharmaceutical compositions are typically in liquid form. Liquid pharmaceutical compositions generally contain a liquid carrier such as water, petroleum, animal or vegetable oil, mineral oil, or synthetic oil. Physiological saline, magnesium chloride, dextrose or other sugar solution, or glycols such as ethylene glycol, propylene glycol, or polyethylene glycol may be included. In some cases, a surfactant such as pluronic acid (PF68) 0.001% may be used.

[0259] For injection at the 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, for example, by 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.

[0260] 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

[0261] It will be understood that all references herein to treatment include curative, palliative and prophylactic treatment, and that in the context of the present invention reference to prevention more generally relates to prophylactic treatment. Treatment may also include preventing the progression of disease severity.

[0262] Mammalian, and particularly human, treatment is preferred, however, both human and veterinary treatment is within the scope of the present invention. Variants, derivatives, analogs, homologs and fragments

[0263] 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. In the context of the present invention, a variant of any given sequence is a sequence in which a specific sequence of residues (whether amino acid or nucleic acid residues) has been modified so that the polypeptide or polynucleotide in question substantially retains its function. Variant sequences can be obtained by addition, deletion, substitution, modification, substitution, and / or alteration of at least one residue present in the naturally occurring protein.

[0264] The term "derivative" as used herein in reference to a protein or polypeptide of the invention includes any substitution, alteration, modification, substitution, deletion and / or addition 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.

[0265] 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 that it mimics.

[0266] Typically, amino acid substitutions can be made, for example, from 1, 2, or 3 to 10 or 20 substitutions, provided that the altered sequence substantially retains the required activity or ability. Amino acid substitutions can include the use of non-naturally occurring analogs.

[0267] The proteins used in the present invention may also have deletions, insertions, or substitutions of amino acid residues, which result in silent changes and result in functionally equivalent proteins. As long as the intrinsic function is maintained, deliberate amino acid substitutions can be made based on similarities in the polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphipathic properties of the residues. 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.

[0268] 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]

[0269] The term "homologue" as used herein refers to 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."

[0270] A homologous sequence may comprise an amino acid sequence that is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90% identical to the subject sequence, preferably at least 95%, 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.

[0271] A homologous sequence may comprise a nucleotide sequence that is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85% or 90% identical to the subject sequence, preferably at least 95%, 97% or 99% identical. Although homology can also be considered in terms of similarity, in the context of the present invention it is preferred to express homology in terms of sequence identity.

[0272] 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 referenced SEQ ID NO.

[0273] 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.

[0274] The percentage of homology can be calculated across adjacent sequences. That is, one sequence is aligned with the other sequence, and each amino acid in one sequence is directly compared with the corresponding amino acid in the other sequence, one residue at a time. This is called "ungapped" alignment. Typically, such ungapped alignment is only performed over a relatively short number of residues.

[0275] While this is a very simple and consistent method, it does not take into account, for example, that 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 significantly reduce the 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 attempt to maximize local homology.

[0276] However, these more complex methods assign a "gap penalty" to 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 existence 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, it is preferable to use the default values ​​when using such software for sequence comparisons. 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.

[0277] Calculation of maximum percentage homology therefore first requires the creation of an optimal alignment, taking into account gap penalties. A suitable computer program for performing such an alignment is the GCG Wisconsin Bestfit package (University of Wisconsin, USA; Devereux et al., (1984) Nucleic Acids Res. 12:387). Other examples of software capable of performing 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., pp. 7-58 to 7-60). However, for some applications, it is preferable 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).

[0278] 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, which 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 typically 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.

[0279] 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.

[0280] A "fragment" of full-length complement factor I or FHL1 is also a variant, and this term typically refers to a selected section of a polypeptide or polynucleotide that is of functional or, for example, assay interest. A "fragment" thus refers to an amino acid or nucleic acid sequence that is a portion of the full-length polypeptide or polynucleotide.

[0281] Such variants can be prepared using standard recombinant DNA techniques, such as site-directed mutagenesis. Where insertion is to be made, synthetic DNA encoding the insert can be made, with 5' and 3' flanking regions corresponding to the naturally occurring sequence on either side of the insertion site. The flanking regions contain convenient restriction sites corresponding 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 sites. The DNA is then expressed in accordance with 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; other known techniques may also be used.

[0282] 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.

[0283] Preferred features and embodiments of the present invention will now be described by way of non-limiting examples.

[0284] The practice of the present invention will employ, unless otherwise indicated, conventional techniques of chemistry, biochemistry, molecular biology, microbiology and immunology which are within the capabilities of those skilled in the art and 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. Example Example 1 Codon optimization

[0285] The nucleotide sequences encoding complement factor I (CFI) and complement factor H-like protein 1 (FHL1) were codon-optimized using a series of approaches summarized in Table 1. [Table 2]

[0286] For "basic" codon optimization, the sequences of CFI or FHL-1 were input into five online 10-codon optimization tools. 1.GeneArt(https: / / www.thermofisher.com / uk / en / home / life-science / cloning / gene-synthesis / geneart-gene-synthesis / geneoptimizer.html) 2.GenScript(https: / / www.genscript.com / quick_order / gene_services_gene_synthesis) 3.IDT(https: / / eu.idtdna.com / CodonOpt) 4. JCat (http: / / www.jcat.de / ) 5. COOL (http: / / cool.syncti.org / setup_input_sequence_create_wf1.php?=Start+Using+Codon+ Optimization+On-Line+%3E%3E%3E)

[0287] The standard human genetic code was used for all tools.

[0288] For the above tools 1 to 4, one sequence was generated from each tool.

[0289] In Tool 5, default settings were used, and the target expression host was set to Homo sapiens. Additionally, 39 genes highly expressed in RPE (Table 2) were entered into the tool. (Based on Table 4 in Booij, JC et al. (2010) PLoS One 5:e9341). [Table 3]

[0290] Tool 5 generated 70 optimized sequences for CFI and 55 optimized sequences for FHL-1, and the top sequence was used.

[0291] In the "manual" codon optimization, the five basic CFI and FHL-1 codon-optimized sequences generated above were manually optimized to eliminate cryptic splice sites, microRNA binding sites, remove tandem duplicate codons, and confirm GC content. Cryptic splice site removal

[0292] Cryptic splice sites were identified using the www.Fruitfly.org tool. A cutoff value of 0.4 was used in the analysis, but only sequences scoring above 0.75 were altered.

[0293] Splice sites were removed by changing the donor site to GT or the acceptor site to AG where possible, and when this was not possible (such as in sequences encoding valine), the 5' flanking base was changed.

[0294] All altered sequences were then analyzed with the www.Fruitfly.org tool to ensure that all splice sites were removed or reduced below a threshold of 0.75. Removal of microRNA binding sites

[0295] MicroRNAs were identified using the www.Genecards.org tool.

[0296] For CFI, the following miRNA binding sites were identified: hsa-mir-335-5p, hsa-mir-181a-5p, and hsa-mir-26b-5p.

[0297] For FHL-1, the following miRNA binding sites were identified (based on the sequence of complement factor H, CFH): hsa-mir-146a-5p.

[0298] Each codon-optimized sequence (after removing splice sites where necessary) was then passed through the STarMir tool (http: / / sfold.wadsworth.org / cgi-bin / starmirtest2.pl) to verify that miRNA sites were still present. Any miRNA sites identified with a logistic probability greater than 0.75 were modified. Removal of tandem duplicated codons

[0299] All sequences were manually checked for tandemly duplicated codons, and if these were found, the second codon was changed to the next most commonly used codon in Homo sapiens (using the SnapGene codon usage table).

[0300] Details of the wild-type and codon-optimized sequences are shown below: GT005:CFI wild type sequence: [ka] (SEQ ID NO: 16) RC001:FHL-1 wild type sequence: [ka] (SEQ ID NO: 17) RC128:CFI GeneArt-Basic: [ka] (SEQ ID NO: 18) RC129:CFI GeneArt - Manually Optimized: [ka] (SEQ ID NO: 19) RC130:CFI Genscript-Basic: [ka] (SEQ ID NO: 20) RC131:CFI Genscript - Manually Optimized: [ka] (SEQ ID NO: 21) RC132:CFI IDT-Basic: [ka] (SEQ ID NO: 22) RC133:CFI IDT - Manually Optimized: [ka] (SEQ ID NO: 23) RC134:CFI JCat-Basic: [ka] (SEQ ID NO: 24) RC135:CFI JCat - Manually Optimized: [ka] (SEQ ID NO: 25) RC136: CFI COOL-Basic: See SEQ ID NO: 10 above. RC137:CFI COOL - Manually Optimized: [ka] (SEQ ID NO: 26) RC138:FHL-1 GeneArt-Basic: [ka] (SEQ ID NO: 27) RC139:FHL-1 GeneArt - Manually Optimized: [ka] (SEQ ID NO: 28) RC140:FHL-1 Genscript-Basic: [ka] (SEQ ID NO: 29) RC141:FHL-1 Genscript - Manually Optimized: [ka] (SEQ ID NO: 30) RC142:FHL-1 IDT-Basic: [ka] (SEQ ID NO: 31) RC143:FHL-1 IDT - Manually Optimized: [ka] (SEQ ID NO: 32) RC144:FHL-1 JCat-Basic: [ka] (SEQ ID NO: 33) RC145:FHL-1 JCat - Manually Optimized: [ka] (SEQ ID NO: 34) RC146: FHL-1 COOL-basic: See SEQ ID NO: 12 above. RC147:FHL-1 COOL - Manually Optimized: [ka] (SEQ ID NO: 35) Plasmid generation

[0301] All 10 codon-optimized sequences were synthesized and cloned into an AAV vector backbone. The vector also contained AAV-2 left and right inverted terminal repeats (ITRs) flanking a modified CBA / CAG promoter (chicken beta actin with a CMV enhancer; "CBA"). The promoter drives expression of the codon-optimized FHL1 or CFI. Additionally, downstream of the transgene was a modified woodchuck hepatitis virus posttranscriptional regulatory element (WPRE) sequence and a bovine growth hormone polyA (bGH polyA) sequence provided 3' to the cDNA. Transfection

[0302] All 20 plasmids were transfected into ARPE19 cells using the following procedure. Day 1: ARPE19 cells were dissociated and counted using ViCell. Cells were plated in 48-well plates at 6x10 cells per well in 500µL DMEM, 10% FBS. 4 cells / well. Day 2: Confluency was checked and found to be 70-80%. Cells were then transfected with 0.25 µg of plasmid DNA using PEI at a DNA:PEI ratio of 1:3 in duplicate. 1.2x0.25 μg of DNA was diluted in 2x5 μL of PBS. 2.2x 0.75 μL PEI was diluted with 2x 5 μL PBS. 3. The PEI mix was added dropwise to the DNA mix, mixed and then incubated at room temperature for 20 minutes. 4.2x250 μL DMEM / Glutamax / 10% FBS was added to the mixture. 5. The medium was removed from the cells and replaced with 250 μL / well of DNA / PEI complexes. Day 3: Media was removed and replaced with 125 μL of serum-free DMEM / Glutamax. Day 5: The medium was collected and centrifuged at 14000 rpm for 10 minutes at 4°C, and the supernatant was transferred to a new tube. Western blot

[0303] Transfection supernatants were analyzed by Western blot (using primary antibodies against CFI and FHL-1: goat antiserum CFI 1:3000; Quidel A312 1:3000; and secondary antibody rabbit anti-goat HRP 1:5000).

[0304] The results of the Western blot analysis are shown in FIG. CFI ELISA

[0305] Supernatants from transfections were analyzed by ELISA for CFI 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: The plates were washed three times with 200 μL of PBS-Tween (0.05%) per well and then blotted onto tissue. 200 μL of 1% BSA Fraction V in PBS-Tween (0.05%) was applied to each well and blocked for 2 hours at room temperature.

[0306] Samples and standard curves were prepared during the blocking incubation. The standard curve was prepared from purified CFI protein (Sigma C5938-1MG) diluted in DMEM 2% FBS. Samples were diluted 1:10, 1:20, and 1:40 in DMEM 2% FBS.

[0307] After 2 hours of blocking, the plates were washed three times as above, and then 50 μL of sample or standard was loaded into each well and incubated for 1 hour at room temperature.

[0308] After 1 hour, the plates were washed as above, and then anti-CFI (0x21) antibody was diluted 1:2000 in DMEM 5% FBS, and 50 μL was applied to each well and incubated at room temperature for 1 hour.

[0309] After 1 hour, the plates were washed as above, then donkey anti-mouse-HRP antibody was diluted 1 in 5000 in DMEM 5% FBS and 50 μL was applied to each well and incubated for 1 hour at room temperature.

[0310] After 1 hour, the plate was washed as above, and then 100 μL of TMB reagent was applied to each well and incubated for approximately 15 minutes in the dark at room temperature. Once a sufficient blue color was obtained, 100 μL of 1 M sulfuric acid was added to each well to stop the reaction.

[0311] The A450 was then recorded and the data was processed and transferred to Microsoft Excel for analysis.

[0312] The results of the CFI ELISA are shown in FIG. FHL1 ELISA

[0313] Supernatants from transductions were analyzed by ELISA for FHL1 using the following procedure: Day 1: ELISA plates were coated with 50 μL / well of anti-FHL-1 antibody (Biorad, AbD33594.1) diluted to 5 μg / mL in 100 mM carbonate / bicarbonate buffer (pH 9.6). Plates were stored at 4° C. overnight. Day 2: The plates were washed three times with 200 μL of PBS-Tween (0.05%) per well and then blotted onto tissue. 200 μL of 1% BSA Fraction V in PBS-Tween (0.05%) was applied to each well and blocked for 2 hours at room temperature.

[0314] Samples and standard curves were prepared during the blocking incubation. The standard curve was prepared from FHL1-His protein diluted in DMEM + 2% FBS. Samples were diluted 1:5, 1:10, and 1:30 in blocking solution. After 2 hours of blocking, the plates were washed three times as above, and then 50 μL of sample or standard was loaded into each well and incubated for 1 hour at room temperature.

[0315] After 1 hour, the plates were washed as described above, and then anti-CFH antibody (0x24, Santa Cruz Biotechnologies, sc-53067) was diluted 1:3000 in DMEM 5% FBS, and 50 μL was applied to each well and incubated at room temperature for 1 hour.

[0316] After 1 hour, the plates were washed as above, then anti-mouse-HRP antibody was diluted 1 in 5000 in DMEM 5% FBS, and 50 μL was applied to each well and incubated at room temperature for 1 hour.

[0317] After 1 hour, the plate was washed as above, and then 100 μL of TMB reagent was applied to each well and incubated for approximately 15 minutes in the dark at room temperature. Once a sufficient blue color was obtained, 100 μL of 1 M sulfuric acid was added to each well to stop the reaction.

[0318] The A450 was then recorded and the data was processed and transferred to Microsoft Excel for analysis.

[0319] The results of the FHL1 ELISA are shown in FIG. AAV2 vector production

[0320] The best four (CFI) and five (FHL1) sequences were carried forward into studies using AAV. HEK293 cells were transfected with the selected codon-optimized plasmid along with pRepCap and pHelper according to a typical triple transfection protocol. Day 1: HEK293 cells were dissociated and counted using ViCell. Cells were plated at 6x10 in a 10cm dish. 5 cells / cm 2 The cells were seeded in 10 mL of DMEM 10% FBS per dish. Day 2: Confluency was checked and found to be 70-80%.

[0321] The medium was replaced with 10 mL DMEM / Glutamax containing 5% FBS.

[0322] After 4 hours, cells were transfected with 5 μg of plasmid using PEI at a DNA:PEI ratio of 1:3. Day 3: 15 mM butyric acid was added to 11 mL medium in each plate. Day 5: The supernatant was collected and centrifuged at 1000 rpm for 10 minutes to remove cell debris.

[0323] The supernatant was transferred to a new tube and 1 / 5 volume of AAVanced (AAV110A-1, Cambridge Bioscience) reagent was added (2.75 mL in 11 mL).

[0324] The mixture was then stored at 4°C. Day 8: The supernatant / AAVanced mixture was centrifuged at 1000 rpm for 30 minutes at 4°C.

[0325] The supernatant was discarded and the pellet was resuspended in 500 μL of PBS, which was then transferred to a 1.5 mL tube and centrifuged at 1500 g for 3 minutes.

[0326] The supernatant was discarded and the remaining pellet was resuspended in 1 / 100 of the original volume (ie, 100 μL per 11 mL of supernatant). The vector was stored at -80°C. Transduction of ARPE19 cells

[0327] ARPE19 cells were transduced with a vector containing a codon-optimized transgene. Day 1: Dissociate ARPE19 cells, count ViCell, then incubate in 200 μL DMEM / Glutamax + 1x10 per well in 10% FBS 5 cells. Day 2: The vector was added to the cells. Day 3: The medium was changed to serum-free medium. Day 4: The supernatant was collected and centrifuged at 14000 rpm for 10 minutes at 4°C, then transferred to a new tube.

[0328] Total protein concentration was assessed by Bradford assay. CFI ELISA

[0329] Supernatants from transduction were analyzed by ELISA for CFI according to the protocol described above.

[0330] The results of the CFI ELISA are shown in FIG. FHL-1 ELISA

[0331] Supernatants from transduction were analyzed by ELISA for FHL1 according to the protocol described above.

[0332] The results of the FHL1 ELISA are shown in FIG. conclusion

[0333] RC136 (CFI; SEQ ID NO: 10) and RC146 (FHL-1; SEQ ID NO: 12) each confer higher transgene expression than the wild-type sequence and the other codon-optimized sequences tested.

[0334] 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 has been disclosed in connection with certain preferred embodiments, it should be understood that the claimed invention should not be excessively limited to such specific embodiments.In fact, 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.

Claims

1. An isolated polynucleotide comprising a nucleotide sequence encoding complement factor I (CFI), wherein the nucleotide sequence has at least 85% sequence identity to SEQ ID NO:

10.

2. 2. The isolated polynucleotide of claim 1, wherein the nucleotide sequence encoding CFI is SEQ ID NO:

10.

3. 3. The isolated polynucleotide of claim 1, wherein the polynucleotide comprises an adeno-associated virus (AAV) inverted terminal repeat (ITR) at its 5' end and an AAV ITR at its 3' end, preferably wherein the AAV ITR is an AAV2 or AAV8 ITR, and preferably wherein the AAV ITR is an AAV2 ITR.

4. 4. The isolated polynucleotide of claim 1, wherein the nucleotide sequence encoding the CFI is operably linked to a promoter, preferably the promoter is a CMV promoter or a CAG promoter.

5. 5. The isolated polynucleotide of claim 4, wherein the promoter has the nucleotide sequence of SEQ ID NO:5 or SEQ ID NO:13, or a nucleotide sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:5 or SEQ ID NO:

13.

6. 6. The isolated polynucleotide of any one of claims 1 to 5, wherein the nucleotide sequence encoding the CFI is operably linked to a WPRE regulatory element, preferably the WPRE regulatory element is a WPRE3 regulatory element.

7. The isolated polynucleotide of claim 6, wherein the WPRE regulatory element has a nucleotide sequence of SEQ ID NO:7 or SEQ ID NO:15, or a nucleotide sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:7 or SEQ ID NO:

15.

8. 8. The isolated polynucleotide of any one of claims 1 to 7, wherein the nucleotide sequence encoding CFI is operably linked to a polyA signal, preferably the polyA signal is a bovine growth hormone polyA signal.

9. 9. The isolated polynucleotide of claim 8, wherein the polyA signal has a nucleotide sequence of SEQ ID NO:6 or SEQ ID NO:14, or a nucleotide sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:6 or SEQ ID NO:

14.

10. 10. The isolated polynucleotide of any one of claims 1 to 9, wherein the nucleotide sequence encoding CFI is operably linked to a CMV promoter, a WPRE regulatory element, and a polyA signal.

11. A vector comprising the polynucleotide according to any one of claims 1 to 10.

12. The vector of claim 11 , wherein the vector is an adeno-associated virus (AAV) vector.

13. 13. The vector of claim 11 or 12, wherein the vector is in the form of a viral vector particle.

14. 14. The vector of claim 13, wherein the AAV vector particle comprises an AAV2 or AAV8 genome and AAV2 or AAV8 capsid proteins.

15. 14. The vector of claim 13, wherein the AAV vector particle comprises an AAV2 genome and AAV2 capsid proteins (AAV2 / 2), an AAV2 genome and AAV8 capsid proteins (AAV2 / 8), or an AAV8 genome and AAV8 capsid proteins (AAV8 / 8).

16. A cell comprising the polynucleotide of any one of claims 1 to 11.

17. A cell transduced with the vector according to any one of claims 12 to 15.

18. 18. A pharmaceutical composition comprising the polynucleotide of any one of claims 1 to 11, the vector of any one of claims 12 to 15, or the cell of claim 16 or 17, in combination with a pharmaceutically acceptable carrier, diluent or excipient.

19. Use of a polynucleotide according to any one of claims 1 to 11, a vector according to any one of claims 12 to 15, or a cell according to claim 16 or 17 in the manufacture of a medicament.

20. 18. Use of a polynucleotide according to any one of claims 1 to 11, a vector according to any one of claims 12 to 15, or a cell according to claim 16 or 17 in the manufacture of a medicament for treating or preventing a complement-mediated disorder of the eye.

21. 21. The use according to claim 20, wherein the disorder is age-related macular degeneration (AMD) or diabetic retinopathy, preferably AMD.

22. 22. The use according to claim 21, wherein the AMD is dry AMD.

23. Use according to any one of claims 20 to 22, wherein the formation of geographic atrophy is prevented or reduced and / or the amount of geographic atrophy is reduced.

24. The use according to any one of claims 20 to 23, wherein the progression of geographic atrophy is slowed down.

25. 25. The use of any one of claims 20 to 24, wherein 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 the subject compared to the untreated eye over the same period.

26. 26. The use of any one of claims 20 to 25, 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 RPE), optionally to a level above normal levels in the subject, or in the eye or retinal pigment epithelium (RPE) thereof.

27. 18. Use of a polynucleotide according to any one of claims 1 to 11, a vector according to any one of claims 12 to 15, or a cell according to claim 16 or 17 in the manufacture of a medicament for improving or restoring reading speed in a subject suffering from an eye disorder such as AMD and / or for alleviating the loss of reading speed associated with an eye disorder such as AMD.

28. Use of a polynucleotide according to any one of claims 1 to 11, a vector according to any one of claims 12 to 15, or a cell according to claim 16 or 17 in the manufacture of a medicament for reducing or preventing loss of photoreceptors and / or retinal pigment epithelium (RPE).

29. The use according to any one of claims 19 to 28, wherein the polynucleotide, vector or cell is administered intraocularly.

30. 30. The use according to any one of claims 19 to 29, wherein the polynucleotide, vector or cell is administered to the subject's eye by subretinal, direct retinal, suprachoroidal or intravitreal injection.

31. The use according to any one of claims 19 to 30, wherein the polynucleotide, vector or cell is administered to the subject's eye by subretinal injection.

32. A pharmaceutical composition for treating or preventing a complement-mediated ocular disorder, comprising the polynucleotide of any one of claims 1 to 11, the vector of any one of claims 12 to 15, or the cell of claim 16 or 17.

33. 33. The pharmaceutical composition of claim 32, wherein the disorder is age-related macular degeneration (AMD) or diabetic retinopathy, preferably AMD.

34. 34. The pharmaceutical composition of claim 33, wherein the AMD is dry AMD.

35. The pharmaceutical composition according to any one of claims 32 to 34, wherein the formation of geographic atrophy is prevented or reduced and / or the amount of geographic atrophy is reduced.

36. The pharmaceutical composition according to any one of claims 32 to 35, wherein the progression of geographic atrophy is slowed.

37. 37. The pharmaceutical composition of any one of claims 32-36, wherein there is at least a 10% reduction in the increase in area of ​​geographic atrophy over 12 months after administration to the treated eye of the subject compared to the untreated eye over the same period.

38. 38. The pharmaceutical composition of any one of claims 32-37, wherein administration of the pharmaceutical composition increases the level of C3b-inactivating and iC3b-degrading activity in the subject or in the subject's eye (such as in the RPE), optionally to a level above normal levels in the subject, or in the eye or retinal pigment epithelium (RPE) thereof.

39. A pharmaceutical composition for improving or restoring reading speed in a subject suffering from an eye disorder such as AMD, and / or for alleviating the decrease in reading speed associated with an eye disorder such as AMD, comprising a polynucleotide according to any one of claims 1 to 11, a vector according to any one of claims 12 to 15, or a cell according to claim 16 or 17.

40. A pharmaceutical composition for reducing or preventing loss of photoreceptors and / or retinal pigment epithelium (RPE), comprising a polynucleotide according to any one of claims 1 to 11, a vector according to any one of claims 12 to 15, or a cell according to claim 16 or 17.

41. The pharmaceutical composition according to any one of claims 32 to 40, wherein the pharmaceutical composition is administered intraocularly.

42. 42. The pharmaceutical composition of any one of claims 32 to 41, wherein the pharmaceutical composition is administered to the subject's eye by subretinal, direct retinal, suprachoroidal or intravitreal injection.

43. 43. The pharmaceutical composition of any one of claims 32 to 42, wherein the pharmaceutical composition is administered to the subject's eye by subretinal injection.

Images