Codon optimized complement factor I

Abstract

An isolated polynucleotide comprising a nucleotide sequence encoding a codon optimized complement factor I (CFI).

Description

[0001] This application is a divisional application of the Chinese patent application with application number 201980089811.4, application date December 20, 2019, and invention name “Codon-optimized complement factor I”. Technical Field

[0002] The present invention relates to agents for gene therapy. In particular, the present invention relates to polynucleotides encoding complement factor I (CFI) or complement factor H-like protein 1 (FHL1), vectors comprising the polynucleotides, and their use in the treatment or prevention of complement-mediated and complement-related disorders, including eye diseases, such as age-related macular degeneration (AMD). Background Art

[0003] The macula is a small area in the retina of the eye, approximately 3 to 5 millimeters in size, adjacent to the optic nerve. The macula is the most sensitive area of ​​the retina and contains the fovea, a sunken area that allows for high visual acuity and contains a dense concentration of cones, the photoreceptors responsible for color vision.

[0004] Age-related macular degeneration (AMD) is the most common cause of functional blindness in people over 50 years old in developed countries (Seddon, JM, Epidemiology of age-related macular degeneration. In: Ogden, TE et al., editor Ryan SJ editor. Retina Vol. II. 3rd edition. St. Louis, Mo.: Mosby; 2001: 1039-1050). AMD is related to the formation of new blood vessels originating from the choroidal vasculature and extending to the subretinal space. In addition, AMD is characterized by the progressive degeneration of the retina, retinal pigment epithelium (RPE) and the underlying choroid (the highly vascularized tissue below the RPE, between the retina and the sclera).

[0005] Multiple factors, including oxidative stress, inflammation that may have an autoimmune component, genetic background (eg, mutations), and environmental or behavioral factors (such as smoking and diet) may contribute to the development of AMD.

[0006] The clinical progression of AMD is characterized by stages based on changes in the macula. The hallmark of early AMD is the appearance of drusen, which is an accumulation of extracellular debris under the retina and appears as yellow spots in the retina on clinical examination and fundus photographs. Drusen are classified as small (<63 μM), medium (63-124 μM) and large (>124 μM) based on size. They are also considered hard or soft based on the appearance of their edges during ophthalmic examination. Hard drusen have clearly defined edges, while soft drusen have fluid edges that are not so clearly defined. The Age-Related Eye Disease Study (AREDS) Fundus Photography Severity Scale is one of the main classification systems used for this condition.

[0007] AMD is divided into " dry " and " wet " (exudative or neovascular) forms. Dry AMD is more common than wet AMD, but the dry form can develop into the wet form, and both occur simultaneously in a large number of cases. The typical characteristics of dry AMD are the progressive apoptosis of the bottom cells in the cell, overlying photoreceptor cells, more often the choriocapillaris layer in the RPE layer. The confluent area of ​​the RPE cell death with the atrophy of overlying photoreceptors is referred to as map-like atrophy. The patient who suffers from this form of AMD can experience the slow and progressive deterioration of central vision.

[0008] Wet AMD is characterized by hemorrhage and / or fluid leakage of abnormal blood vessels growing from the choroidal blood vessels (choriocapillaris) below the RPE and macula, which may be the reason causing sudden and disabled vision loss. It is estimated that most of the vision loss experienced by patients is due to this choroidal neovascularization (CNV) and secondary complications thereof. A subtype of neovascular AMD is referred to as retinal angiomatous proliferation (RAP). Here, angiomatous proliferation originates from retina and extends backwards into the subretinal space, finally communicating with choroidal neovascularization in some cases.

[0009] Based on the identification of complement system (CS) components in drusen of AMD patients' eyes, CS has been associated with the early onset of AMD. In AMD, at least 129 types of drusen deposited proteins have been identified, including different apolipoprotein types (E, B, or Al), several amyloid peptides (P, Aβ, or SA-1), TIMP-3, serum albumin, and certain proteins related to cellular function (e.g., ATP synthase β subunit, scavenger receptor B2, and retinol dehydrogenase). AMD-derived drusen also contain almost 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 MAC components in isolated and complex forms (i.e., 5, 6, 8 (α, β, and γ), and 9). Accumulated drusen may activate CS, trigger the local production of inflammatory mediators, and attract leukocytes, thereby increasing the local inflammatory state present in AMD.

[0010] Current treatment options for AMD include photodynamic therapy using benzoporphyrins (Arch Ophthalmol (1999) 117: 1329-1345) and a number of 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 (bevacizumab) (BMJ (2010) 340: c2459). However, not all patients respond to treatment with anti-VEGF antibodies, either failing to restore vision or progressing to registered blindness.

[0011] A treatment for geographic atrophy has been developed and is in Phase III clinical studies. Lampalizumab, a humanized monoclonal inhibitory antibody against complement factor D, is administered by intravitreal injection to block the rate of progression of geographic atrophy. However, in a Phase III randomized clinical trial involving 906 participants, after 48 weeks, lambalizumab failed to reduce GA enlargement when compared with sham treatment.

[0012] Therefore, there is a great need in the art for new methods of treating ocular diseases such as AMD.

[0013] Because the complement system is ubiquitous, an overactive or dysfunctional complement system is implicated in the pathology of many chronic inflammatory conditions for which therapeutic options are either nonexistent or require years of regular intervention to control symptoms. Thus, there is a general need to develop gene therapy treatments to provide new or alternative treatments for complement-mediated and complement-related disorders, particularly chronic inflammatory conditions, and even more particularly disorders associated with an overactive complement C3b feedback loop ( Figure 1 ). Summary of the invention

[0014] Applicants have identified codon-optimized sequences for complement factor I (CFI) and complement factor H-like protein 1 (FHL1) that provide significantly increased expression of the encoded CFI and FHL1 proteins compared to the wild-type sequences.

[0015] The improved CFI and FHL1 coding sequences developed by the applicant are capable of delivering higher doses of the corresponding proteins to patients without increasing the amount of vector administered. Therefore, the present invention provides improvements in manufacturing yield (i.e., protein delivery can be achieved with a smaller amount of vector produced), efficacy of the drug, and safety. Specifically, since a higher dose of the encoded protein can be achieved by delivering the same amount (e.g., volume) of vector, the risk of damage to the tissue to which the vector is administered is reduced. For example, when the vector is delivered to the eye by subretinal injection, the risk of retinal damage or detachment caused by injecting a larger volume of the drug is reduced. In addition, the risk of off-target effects caused by diffusing a larger volume of the drug to adjacent tissues is also reduced. In addition, the use of the claimed nucleotide sequences in gene therapy has the potential to deliver treatment with a single dose, thereby allowing long-term stable expression of the protein and avoiding the need for monthly or regular injections. Therefore, the nucleotide sequences and compositions of the present invention have the additional advantage that they have the potential to provide a one-time or "single-shot" therapy to avoid repeated or regular surgical interventions.

[0016] 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.

[0017] In some embodiments, the nucleotide sequence encoding CFI is at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:10.

[0018] In a preferred embodiment, the nucleotide sequence encoding CFI is SEQ ID NO:10.

[0019] 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.

[0020] In some embodiments, the nucleotide sequence encoding FHL1 is at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:12.

[0021] In a preferred embodiment, the nucleotide sequence encoding FHL1 is SEQ ID NO:12.

[0022] In some embodiments, the polynucleotide comprises one or more adeno-associated virus (AAV) inverted terminal repeats (ITRs). In preferred embodiments, the polynucleotide comprises an AAV ITR at its 5' end and an AAV ITR at its 3' end.

[0023] In some embodiments, the AAV ITR is an AAV2 or AAV8 ITR. In preferred embodiments, the AAV ITR is an AAV2 ITR.

[0024] In another aspect, the present invention provides a vector comprising the polynucleotide of the present invention.

[0025] In some embodiments, the vector is an adeno-associated virus (AAV), retroviral, lentiviral, or adenoviral vector.

[0026] In a preferred embodiment, the vector is an AAV vector.

[0027] In some embodiments, the vector is in the form of a viral vector particle.

[0028] In some embodiments, the AAV vector particle comprises an AAV2 or AAV8 genome.

[0029] In some embodiments, the AAV vector particle comprises an AAV2 or AAV8 capsid protein.

[0030] In some embodiments, the AAV vector particles comprise an AAV2 genome and an AAV2 capsid protein (AAV2 / 2). In some embodiments, the AAV vector particles comprise an AAV2 genome and an AAV2 capsid protein (AAV2 / 8). In other embodiments, the AAV vector particles comprise an AAV8 genome and an AAV8 capsid protein (AAV8 / 8).

[0031] 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 a preferred embodiment, the WPRE regulatory element is a WPRE3 regulatory element. In some embodiments, the nucleotide sequence encoding CFI is operably linked to a polyadenylation (poly-A) signal, such as a bovine growth hormone poly-A signal.

[0032] 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 poly-A signal.

[0033] 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 poly-A signal, such as a bovine growth hormone poly-A signal.

[0034] 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 poly-A signal.

[0035] In another aspect, the invention provides a cell comprising a polynucleotide of the invention.

[0036] In another aspect, the invention provides a cell transduced with a vector of the invention.

[0037] In another aspect, the present invention provides a pharmaceutical composition comprising a polynucleotide, vector or cell of the present invention, in combination with a pharmaceutically acceptable carrier, diluent or excipient.

[0038] In certain embodiments, the pharmaceutical composition is suitable for systemic administration, such as by peripheral intravenous infusion.

[0039] In certain embodiments, the pharmaceutical composition is suitable for topical administration, such as intrathecal administration.

[0040] In a preferred embodiment, the pharmaceutical composition is for intraocular administration, such as by intravitreal injection, suprachoroidal injection, or subretinal injection.

[0041] In another aspect, the invention provides a polynucleotide, vector or cell of the invention for use in therapy.

[0042] In certain embodiments, the polynucleotides, vectors or cells of the invention are used to treat complement-mediated disorders, particularly chronic inflammatory conditions.

[0043] In a preferred embodiment, the polynucleotides, vectors or cells of the invention are used to treat disorders associated with an overactive complement C3b feedback loop.

[0044] In another aspect, the invention provides a polynucleotide, vector or cell of the invention for use in treating or preventing an ocular disorder.

[0045] In another aspect, the invention provides a polynucleotide, vector or cell of the invention for use in treating or preventing a complement-mediated ocular disorder.

[0046] In another aspect, the present invention provides a method of treating or preventing a complement-mediated ocular disorder comprising administering a polynucleotide, vector or cell of the present invention to a subject in need thereof.

[0047] 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 an eye of the subject.

[0048] In some embodiments, the disorder is associated with an overactive complement C3b feedback loop and / or an underactive C3b degradation loop (see Figure 1 ).

[0049] In some embodiments, the disorder is a chronic complement-mediated inflammatory condition of the eye.

[0050] In some embodiments, the condition is age-related macular degeneration (AMD) or diabetic retinopathy. In other embodiments, the condition is glaucoma, Stargardt's disease, central serous chorioretinopathy, or retinitis pigmentosa.

[0051] In preferred embodiments, the disorder is AMD.In some embodiments, AMD is dry AMD.

[0052] In some embodiments, the subject has been diagnosed with or is at risk for developing AMD.

[0053] In some embodiments, the use is for treating or preventing a condition in a subject who:

[0054] (a) having a lower than normal complement factor I activity or concentration in the eye and / or serum, preferably having a concentration of 0-30, 0-20 or 0-10 μg / mL in serum, or equivalent activity; and / or

[0055] (b) being heterozygous or homozygous for a SNP associated with age-related macular degeneration (AMD), preferably a rare complement factor I variant.

[0056] In some embodiments, the use is for treating or preventing a condition in a subject who:

[0057] (a) having normal levels of complement factor I activity or concentration in the eye and / or serum, preferably at least 30 μg / mL in serum, such as 30-40 μg / mL; and / or

[0058] (b) Not carrying rare complement factor I variant alleles.

[0059] In another aspect, the 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.

[0060] In another aspect, the present invention provides a polynucleotide, vector or cell of the present invention for use in treating or preventing diabetic retinopathy.

[0061] In some embodiments, the development of geographic atrophy is prevented or reduced, and / or the amount of geographic atrophy is reduced.

[0062] In some embodiments, the progression of geographic atrophy is slowed.

[0063] In some embodiments, the increase in the area of ​​geographic atrophy is at least 10% reduced after 12 months after administration to the treated eye of the subject relative to the untreated eye over the same time period. In other embodiments, the increase in the area of ​​geographic atrophy is at least 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90% reduced after 12 months after administration to the treated eye of the subject relative to the untreated eye over the same time period.

[0064] In some embodiments, administration of the polynucleotide, vector, or cell increases the level of C3b inactivation and iC3b degradation activity in a subject or the subject's eye, such as the retinal pigment epithelium (RPE), optionally to levels that exceed normal levels in the subject or its eye or RPE.

[0065] In another aspect, the invention provides polynucleotides, vectors or cells of the invention for use in improving or restoring vision or visual acuity in a subject, e.g., suffering from an ocular disorder, such as an ocular disorder disclosed herein. In another aspect, the invention provides polynucleotides, vectors or cells of the invention for use in alleviating loss of vision or visual acuity, e.g., loss of vision or visual acuity associated with an ocular disorder, such as an ocular disorder disclosed herein.

[0066] In another aspect, the invention provides polynucleotides, vectors or cells of the invention for use in increasing or restoring the reading speed of a subject, e.g., a subject suffering from an ocular disorder, such as an ocular disorder disclosed herein. In another aspect, the invention provides polynucleotides, vectors or cells of the invention for use in alleviating a decrease in the reading speed of a subject, e.g., a decrease in the reading speed associated with an ocular disorder, such as an ocular disorder disclosed herein.

[0067] In another aspect, the invention provides polynucleotides, vectors or cells of the invention for use in reducing or preventing loss of photoreceptors and / or retinal pigment epithelium (RPE), e.g., loss of photoreceptors and / or RPE associated with an ocular disorder, such as an ocular disorder disclosed herein.

[0068] In some embodiments, the polynucleotide, vector or cell is administered intraocularly.

[0069] In some embodiments, the polynucleotide, vector or cell is administered to the eye of the subject by subretinal, direct retinal, suprachoroidal or intravitreal injection.

[0070] In some embodiments, the polynucleotide, vector or cell is administered to the eye of the subject by subretinal injection.

[0071] In some embodiments, the polynucleotides or vectors of the invention do not comprise a hAAT promoter. In some embodiments, the polynucleotides or vectors of the invention do not comprise an ApoR enhancer. In other embodiments, the polynucleotides or vectors of the invention do not comprise two ApoR enhancers.

[0072] In some embodiments, the vector of the present invention does not comprise an AAV2 genome and an AAV8 capsid protein, ie, the vector of the present invention is not an AAV2 / 8 vector.

[0073] In some embodiments, the polynucleotides, vectors or cells of the invention are not administered systemically. In other embodiments, the polynucleotides, vectors or cells of the invention are not administered intravenously. BRIEF DESCRIPTION OF THE DRAWINGS

[0074] Figure 1

[0075] The C3b feedback (amplification) and degradation (downregulation) cycle of the vertebrate complement alternative pathway ("I" = complement factor I, "H" = complement factor H, "B" = complement factor B, and "D" = complement factor D).

[0076] Figure 2

[0077] Western blot analysis of supernatants from codon-optimized CFI and FHL1 plasmid transfections of ARPE19 cells.

[0078] Figure 3

[0079] ELISA analysis of supernatants from codon-optimized CFI plasmid transfection of ARPE19 cells.

[0080] Figure 4

[0081] ELISA analysis of supernatants from codon-optimized FHL1 plasmid transfection of ARPE19 cells.

[0082] Figure 5

[0083] ELISA analysis of supernatants from ARPE19 cells transduced with codon-optimized CFI AAV vectors.

[0084] Figure 6

[0085] ELISA analysis of supernatants from ARPE19 cells transduced with codon-optimized FHL1 AAV vectors. DETAILED DESCRIPTION

[0086] As used herein, the terms "comprising," "comprises," and "comprised of" are synonymous with "including," "includes," or "containing," "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."

[0087] Complement system

[0088] The complement system is a component of the humoral immune system and is involved in tissue inflammation, cell opsonization, and cytolysis. It provides protection against microorganisms and mediates the clearance of exogenous and endogenous cellular debris from host tissues.

[0089] The complement system cascade contains four activation pathways. All pathways ultimately end with the central cleavage of the C3 factor and the production of its active fragments C3a and C3b. C3a is an anaphylatoxin that triggers a series of chemotactic and proinflammatory responses, such as recruiting inflammatory cells and increasing microvascular permeability, while C3b is responsible for the opsonization of foreign surfaces covalently linked to C3b. The opsonization of activated C3 fragments (C3b and iC3b) achieves three main functions: (i) elimination of cell debris by phagocytes (e.g., macrophages or microglia) and stimulation of the adaptive immune system (B and T cells); (ii) amplification of complement activation by the formation of surface-bound C3 convertase; and (iii) assembly of C5 convertase.

[0090] The assembly of C5 convertase is responsible for C5 cleavage, resulting in the formation of a cytolytic membrane attack complex (MAC) that can produce perforations on the cell membrane, thereby promoting cell lysis and the elimination of unnecessary cells. Through all these activities, the innate complement cascade supports and promotes the function of the downstream mechanism of the protection host tissue integrity of the immune system. In general, complement system pathway activation leads to proinflammatory reactions, including MAC production, which mediates cell lysis, releases chemokines to attract inflammatory cells to the site of injury, and enhances capillary permeability to promote the extravasation of infiltrating leukocytes. Under physiological conditions, complement activation is effectively controlled by the coordination of soluble and membrane-related complement regulatory molecules (CRMs). Soluble complement regulators, such as C1 inhibitors, anaphylatoxin inhibitors, C4b binding proteins (C4BPs), complement factor H (CFH), complement factor I (CFI), clustering proteins and vitronectin, limit the role of complement in human tissues at multiple sites of the cascade reaction. In addition, each individual cell is protected from attack by homologous complement 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. Of note, host cells and tissues that are not adequately protected from complement attack may be susceptible to bystander cell lysis.

[0091] The present invention relates to the treatment or prevention of complement-mediated disorders of the eye. For example, a complement-mediated disorder may be a disorder associated with defective regulation of the alternative pathway, and in particular a disorder associated with an overactive complement C3b feedback loop and / or an underactive C3b degradation loop.

[0092] In some embodiments, prior to administration of the polynucleotides, vectors, cells or pharmaceutical compositions of the invention, the subject has a low level (e.g., below normal) of complement factor I activity, such as low levels of complement factor I activity in the eye and / or low serum levels of complement factor I activity. Complement factor I activity below normal levels may be due to below normal expression of a functionally normal complement factor I, or at least partial (e.g., heterozygous) expression (at normal or below normal levels) of a non- or subfunctional variant of complement factor I. (Such a subject may carry one or more copies of an AMD-associated SNP, e.g., a subject may be homozygous or heterozygous for one of the rare complement factor I variants discussed further below). Thus, the subject may have a low concentration (e.g., below normal concentration) of complement factor I in the eye and / or serum. For human subjects, normal levels of complement factor I activity (C3b inactivation and iC3b degradation activity) may be equivalent to the level provided by 30-40 μg / mL complement factor I in the serum of the subject. Thus, in a subject with low complement factor I activity, the complement factor I activity in the serum may correspond to less than 30 μg / mL and greater than 0 μg / mL of complement factor I, such as 0-20 or 0-10 μg / mL (these are ranges of complement factor I serum concentrations that may encompass subjects with low complement factor I concentrations).

[0093] Therefore, the subject to be treated of the present invention may suffer from a complement-mediated eye disorder, such as AMD, more specifically dry AMD (e.g., characterized by geographic atrophy), or may have a risk of developing such a disorder. For example, the subject may be homozygous or heterozygous sensitive to one or more SNPs associated with a complement-mediated disorder.

[0094] 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 associated with late AMD, which typically result in reduced serum complement factor I levels (Kavanagh et al. (2015) Hum Mol Genet 24: 3861-3870). Specifically, the subject may carry one or two copies of one or more of the following rare complement factor I variants: rs144082872 (encoding P50A), 4:110687847 (encoding P64L), rs141853578 (encoding G119R), 4:110685721 (encoding V152M), 4:110682846 (encoding G162D), 4:110682801 (encoding N177I), rs146444258 (encoding A240G), rs182078921 (encoding G287R), rs41278047 (encoding K441R) and rs121964913 (encoding R474).

[0095] The invention can also 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 rare complement factor I variants associated with AMD listed above).

[0096] 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.

[0097] In some embodiments, the administration of the polynucleotides, vectors, cells or pharmaceutical compositions of the invention increases the level of C3b inactivation and iC3b degradation activity in the eye of a subject. In other embodiments, the administration of the polynucleotides, vectors, cells or pharmaceutical compositions of the invention increases the level of C3b inactivation and iC3b degradation activity in the eye of a subject to a level that exceeds normal levels in the eye. More specifically, the level of C3b inactivation and iC3b degradation activity is increased in the RPE of the eye.

[0098] It should be understood that following expression of complement factor I from a polynucleotide or vector of the invention, the C3b inactivation and iC3b degradation activity in a subject can include C3b inactivation and iC3b degradation activity from the subject's endogenous complement factor I (i.e., the subject's complement factor I that is not produced from expression of the polynucleotide or vector), as well as C3b inactivation and iC3b degradation activity produced by expression from a polynucleotide or vector of the invention, such that the total level of C3b inactivation and iC3b degradation activity in the subject exceeds normal levels.

[0099] In some embodiments, the level of C3b inactivation and iC3b degradation activity in a subject, such as in the eye, is increased to a level at least 5%, 10%, 15%, 20%, or 25% above normal levels.

[0100] In other embodiments, the level of C3b inactivation and iC3b degradation activity in a subject, such as in the eye, is increased to up to twice the normal level or up to 80%, 60%, 40%, or 20% above normal levels.

[0101] For example, the level of C3b inactivation and iC3b degradation activity can be increased to levels 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% above normal levels in a subject, e.g., in the eye.

[0102] In some embodiments, administration of the polynucleotides, vectors, cells or pharmaceutical compositions of the invention does not significantly increase the level of C3b inactivation and iC3b degradation activity in the plasma / serum of a subject. In other embodiments, administration of the polynucleotides, vectors, cells or pharmaceutical compositions of the invention does not significantly increase the level of C3b inactivation and iC3b degradation activity in the plasma / serum of a subject to a level above normal levels.

[0103] In the above sections, unless clearly inapplicable, references to complement factor I and C3b inactivation and iC3b degradation activity may be replaced with complement factor H or complement factor H-like protein 1, as well as the ability to act as a cofactor for complement factor I-mediated C3b cleavage and increase the dissociation rate of C3 convertase and C5 convertase, respectively. In some embodiments, prior to administration of the polynucleotides, vectors, cells or pharmaceutical compositions of the invention, the subject has a low level (e.g., below normal) of complement factor H, such as a low level of complement factor H in the eye and / or a low serum level of complement factor H. For human subjects, the normal level of complement factor H in the serum of a subject may be about 200-500 μg / mL. Thus, in subjects with low levels of complement factor H, the level in serum may be less than 200 μg / mL and greater than 0 μg / mL, such as 0-100 μg / mL. Alternatively, the subject may have a normal level of endogenous complement factor H, for example in the eye and / or serum.

[0104] Complement Factor I (CFI)

[0105] Complement factor I (factor I, CFI), also known as C3b / C4b inactivator, is a protein that is encoded by the CFI gene in humans.

[0106] Complement factor I is a serine protease that is expressed at a concentration of ~35 μg / mL (Nilsson et al. (2011) Mol Immunol 48:1611-1620)(2011) PNAS 108:(Roversi et al. (2011) PNAS 108:1611-1620). The complement factor I protein is a heavily N-glycosylated heterodimer consisting of two polypeptide chains linked by a disulfide bond. The heavy chain (50 kDa) contains an N-terminal region, a Fl membrane attack complex (FIMAC) domain, a CD5-like domain or a scavenger receptor cysteine-rich (SRCR) domain, two low-density lipoprotein receptor (LDLr) domains, and a C-terminal region of unknown function that is the site of sequence variation 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).

[0107] Complement factor I inactivates C3b by cleaving it into iC3b, C3d, and C3d,g and similarly cleaves C4b into C4c and C4d. To properly 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) (De gn et al. (2011) Am J Hum Genet 88:689-705).

[0108] iC3b is unable to associate with factor B and, therefore, is unable to perpetuate complement cascade amplification or activation via the alternative pathway. Thus, once C3b is cleaved to iC3b, neither the initial nor the terminal complement cascade activation via the alternative pathway can occur.

[0109] iC3b can provide pro-inflammatory effects by binding and activating complement receptor 3 (CR3) (CD11b / CD18) on polymorphonuclear leukocytes (mainly neutrophils), NK cells, and mononuclear phagocytes (such as macrophages).

[0110] Complement factor I is able to process iC3b into C3d,g through a protease activity that requires the cofactor CR1. C3d,g cannot bind to CR3. Since the reaction of iC3b with the complement receptor CR3 is the main mechanism of inflammation caused by complement activation, the breakdown of iC3b into C3d,g is crucial to reduce complement-induced inflammation (Lachmann (2009) Adv. Immunol. 104: 115-149).

[0111] The unique ability of complement factor I to promote the cleavage of C3b to iC3b and to accelerate the breakdown of iC3b - combined with its relatively low concentration in human serum, which has an impact on the amount required to be delivered to achieve a therapeutic effect - makes it a particularly advantageous target.

[0112] In some embodiments, the complement factor I polypeptide is capable of cleaving C3b into inactive degradation products. For example, the complement factor I polypeptide may be capable of cleaving C3b into iC3b.

[0113] In some embodiments, the complement factor I polypeptide is capable of processing iC3b into inactive degradation products. For example, the complement factor I polypeptide may be capable of processing iC3b into C3d,g.

[0114] In preferred embodiments, the complement factor I polypeptide is capable of cleaving C3b to iC3b and processing iC3b to C3d,g.

[0115] Suitably, the fragment or derivative of complement factor I may retain at least 50%, 60%, 70%, 80%, 90%, 95% or 100% of the C3b inactivation and iC3b degradation activities of native complement factor I.

[0116] The C3b inactivation and iC3b degradation activity of complement factor I or its fragments or derivatives can be determined using any suitable method known to the skilled person. For example, the measurement of complement factor I proteolytic activity is described in Hsiung et al. (Biochem. J. (1982) 203: 293-298). The hemolysis and conglutinating assays of CFI activity are described in Lachmann PJ & Hobart MJ (1978) "Complement Technology" in Handbook of Experimental Immunology, 3rd edition, DM Weir Blackwells, Scientific Publications, Chapter 5A, page 17. A more detailed description is given by Harrison RA (1996) in "Weir's Handbook of Experimental Immunology" 5th edition; Herzenberg Leonore A'Weir DM, Herzenberg Leonard A & Blackwell C Blackwells eds., Scientific Publications, Chapter 75, pages 36-37, also including proteolytic assays. The coaggregation assay is highly sensitive and can be used to detect the first (double) clip that converts fixed C3b to iC3b and acquires reactivity with conglutinin; and the last clip that detects C3dg by starting with fixed iC3b and looking for loss of reactivity with conglutinin. The hemolytic assay is used to convert C3b to iC3b, and the proteolytic assay detects all clips.

[0117] In some embodiments, the complement factor I is human complement factor I.

[0118] An exemplary 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 (as disclosed in SEQ ID NO: 1), of which amino acids 1 to 18 form a signal sequence.

[0119] 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.

[0120]

[0121] 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.

[0122]

[0123] An exemplary wild-type nucleotide sequence encoding complement factor I is the nucleotide sequence having NCBI Accession No. NM_000204, disclosed herein as SEQ ID NO:2.

[0124]

[0125] The nucleotide sequence of complement factor I used in the present invention is preferably codon optimized. Different cells differ in the use of specific codons. This codon preference corresponds to the preference of the relative abundance of a specific tRNA in the cell type. By changing the codons in the sequence so that they are tailored to match the relative abundance of the corresponding tRNA, expression can be increased. For the same reason, expression can be reduced by intentionally selecting codons that are known to be rare in the corresponding tRNA in a specific cell type. Therefore, an additional degree of translational control can be obtained.

[0126] A preferred nucleotide sequence encoding complement factor I is the nucleotide sequence disclosed as SEQ ID NO:10.

[0127]

[0128] In some embodiments, the nucleotide sequence encoding complement factor I is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 10. Preferably, the protein encoded by the nucleotide sequence substantially retains the amino acid sequence of the protein represented by SEQ ID NO: 1 or 9.

[0129] In other embodiments, the nucleotide sequence encoding complement factor I is SEQ ID NO:10.

[0130] In other embodiments, the nucleotide sequence encoding complement factor I is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 95%, 99%, 97%, 98% or 99% identical to positions 55 to 1752 of SEQ ID NO: 10. Preferably, the protein encoded by the nucleotide sequence substantially retains the amino acid sequence of the protein represented by SEQ ID NO: 1 or 9.

[0131] In other embodiments, the nucleotide sequence encoding complement factor I is positions 55 to 1752 of SEQ ID NO:10.

[0132] Another exemplary codon-optimized nucleotide sequence encoding complement factor I is SEQ ID NO:8.

[0133]

[0134] One advantage of the present invention is that complement factor I is particularly difficult to prepare as a purified protein. Therefore, the inventors have designed a way to modulate the complement system by administering complement factor I in the form of an AAV vector comprising a nucleotide sequence encoding complement factor I, for example to achieve treatment of age-related macular degeneration (AMD). The AAV vector can be administered to a site of interest, such as the eye, to achieve in situ translation of complement factor I polypeptides.

[0135] Complement Factor H (CFH)

[0136] Complement factor H (factor H, CFH) is a complement control protein.

[0137] 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 main function of complement factor H is to regulate the alternative pathway of the complement system.

[0138] Complement factor H provides cofactor activity for the cleavage of C3b mediated by complement factor I. Complement factor H also increases the dissociation rate of the C3bBb complex (C3 convertase) and the (C3b)NBB complex (C5 convertase), thereby reducing the activity of the alternative complement pathway.

[0139] Complement factor H is composed of 20 complement control protein (CCP) modules (also referred to as short common repeats or sushi domains), which are interconnected by short linkers (3 to 8 amino acid residues) and arranged in a head-to-tail extension. Each CCP module consists of approximately 60 amino acids, wherein there are four cysteine ​​residues bonded by disulfide bonds arranged in 1-3 2-4, and a hydrophobic core built around almost unchanged tryptophan residues. CCP modules are numbered 1-20 (from the N-terminal of the protein). CCP1-4 and CCP19-20 engage with C3b, while CCP7 and CCP19-20 bind to GAG and sialic acid (Schmidt et al. (2008) Journal of Immunology 181: 2610-2619).

[0140] Gene therapy using complement factor H has been shown to improve AMD-like pathology induced in mice (Cashman et al. (2015) J. Gene Med. 17: 229-243). Mice were subretinal co-injected with: (i) an adenoviral vector expressing complement component C3, which has previously been shown to reproduce many pathological features of human AMD; and (ii) an adenoviral vector expressing complement factor H. Complement factor H-transduced mice showed a 91% reduction in endothelial cell proliferation and a 69% reduction in RPE atrophy relative to control animals receiving GFP instead of complement factor H. Electroretinograms showed improved retinal function in mice receiving complement factor H, and immunocytochemistry of rhodopsin and RPE65 was consistent with the rescue of photoreceptors and RPE in such animals.

[0141] In some embodiments, 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. In some embodiments, the complement factor H polypeptide or fragment or derivative thereof is capable of increasing the dissociation rate of C3 convertase and C5 convertase.

[0142] In preferred embodiments, 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 dissociation rate of C3 convertase and C5 convertase.

[0143] In some embodiments, the complement factor H is human complement factor H.

[0144] An exemplary 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 (as disclosed in SEQ ID NO: 3), of which amino acids 1 to 18 form a signal sequence.

[0145] 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.

[0146]

[0147] An exemplary nucleotide sequence encoding complement factor H is the nucleotide sequence having NCBI Accession No. NM_000186.

[0148] In some embodiments, the nucleotide sequence encoding complement factor H is SEQ ID NO:4.

[0149]

[0150] In some embodiments, the nucleotide sequence encoding complement factor H is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical 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.

[0151] In some embodiments, the nucleotide sequence encoding complement factor H is SEQ ID NO:4.

[0152] In other embodiments, the nucleotide sequence encoding complement factor H is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical 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.

[0153] In other embodiments, the nucleotide sequence encoding complement factor H is positions 55 to 3696 of SEQ ID NO:4.

[0154] In other embodiments, the nucleotide sequence encoding complement factor H encodes an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 3. Preferably, the amino acid sequence substantially retains the functional activity of the protein represented by SEQ ID NO: 3.

[0155] In other embodiments, the nucleotide sequence encoding complement factor H encodes the amino acid sequence of SEQ ID NO:3.

[0156] In other embodiments, the nucleotide sequence encoding complement factor H encodes an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to positions 19 to 1231 of SEQ ID NO: 3. Preferably, the amino acid sequence substantially retains the functional activity of the protein represented by SEQ ID NO: 26.

[0157] In other embodiments, the nucleotide sequence encoding complement factor H encodes the amino acid sequence of positions 19 to 1231 of SEQ ID NO:3.

[0158] Complement factor H-like protein 1 (FHL1)

[0159] 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 carboxyl-terminal tail (Clark, SJ et al. (2015) J Clin Med 4:18-31).

[0160] In some embodiments, FHL1 is human FHL1.

[0161] In some embodiments, the amino acid sequence of FHL1 is SEQ ID NO:11.

[0162]

[0163] The nucleotide sequence of FHL1 used in the present invention is preferably codon-optimized.

[0164] A preferred nucleotide sequence encoding FHL1 is SEQ ID NO:12.

[0165]

[0166] In some embodiments, the nucleotide sequence encoding FHL1 is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical 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.

[0167] In other embodiments, the nucleotide sequence encoding FHL1 is SEQ ID NO:12.

[0168] Polynucleotide

[0169] The polynucleotides of the present invention may comprise DNA or RNA, preferably DNA. They may be single-stranded or double-stranded. The skilled person will appreciate that, due to the degeneracy of the genetic code, a plurality of different polynucleotides can encode the same polypeptide. In addition, it should be appreciated that the skilled person may use conventional techniques to perform nucleotide substitutions that do not affect the polypeptide sequence encoded by the polynucleotides of the present invention, to reflect the codon usage of any particular host organism in which the polypeptide of the present invention will be expressed.

[0170] The polynucleotides may be modified by any method available in the art. Such modifications may be performed to enhance the in vivo activity or lifespan of the polynucleotides of the invention.

[0171] Polynucleotides such as DNA polynucleotides can be produced recombinantly, synthetically, or by any means available to those skilled in the art. They can also be cloned by standard techniques.

[0172] Longer polynucleotides are usually produced using recombinant methods, such as using polymerase chain reaction (PCR) cloning techniques. This will involve making a pair of primers (e.g., about 15 to 30 nucleotides) flanking the target sequence you want to clone, contacting the primers with mRNA or cDNA obtained from animal or human cells, performing a polymerase chain reaction under conditions that cause amplification of the desired region, isolating the amplified fragments (e.g., by purifying the reaction mixture with an agarose gel) and recovering the amplified DNA. The primers can be designed to contain suitable restriction enzyme recognition sites so that the amplified DNA can be cloned into a suitable vector.

[0173] The structure of the eye

[0174] The medicaments disclosed herein may be delivered to a mammal, preferably a human eye, in connection with the treatment or prevention of an eye disease, such as age-related macular degeneration (AMD).

[0175] Those skilled in the art of treating eye diseases will have a detailed and thorough understanding of the structure of the eye. However, the following structures are described that are particularly relevant to the present invention.

[0176] Retina

[0177] The retina is a multi-layered membrane that lines the inner posterior chamber of the eye and perceives images of the visual world that communicates with the brain through the optic nerve. In order from the inside to the outside of the eye, the retina includes the retinal neurosensory layer and the retinal pigment epithelium, with the choroid located outside the retinal pigment epithelium.

[0178] Retinal neurosensory layers and photoreceptor cells

[0179] The neurosensory layers of the retina contain photoreceptor cells that directly sense light. It consists of the following layers: inner limiting membrane (ILM), nerve fiber layer, ganglion cell layer, inner plexiform layer, inner nuclear layer, outer plexiform layer, outer nuclear layer (nuclei of photoreceptors), external limiting membrane (ELM), and photoreceptors (inner and outer segments of rods and cones).

[0180] Technicians will have a detailed understanding of photoreceptor cells. In short, photoreceptor cells are special neurons located in the retina that convert light into biological signals. Photoreceptor cells include rod cells and cone cells, which are distributed differently on the retina.

[0181] Rods are mainly found in the outer part of the retina. They are highly sensitive and provide vision at low light levels. There are an average of about 125 million rods in a normal human retina.

[0182] Cones are found throughout the retina, but are particularly highly concentrated in the fovea, a pit in the retina's neural sensory layer that is responsible for central, high-resolution vision. Cones are less sensitive than rods. There are an average of about 6-7 million cones in a normal human retina.

[0183] Retinal Pigment Epithelium

[0184] The retinal pigment epithelium (RPE) is a layer of pigmented cells just outside the neurosensory layer of the retina. The RPE performs a variety of functions, including transporting nutrients and other substances to photoreceptor cells and absorbing scattered light to improve vision.

[0185] Choroid

[0186] The choroid is a vascular layer located between the RPE and the outer sclera of the eye. The vasculature of the choroid is able to supply oxygen and nutrients to the retina.

[0187] Age-related macular degeneration (AMD)

[0188] The clinical progression of age-related macular degeneration (AMD) is characterized in stages based on changes in the macula. The hallmark of early AMD is the appearance of drusen, which are accumulations of extracellular debris beneath the retina and appear as yellow spots in the retina on clinical examination and fundus photographs. Drusen are classified as small (<63 μm), medium (63-124 μm), and large (>124 μm) based on size. They are also considered hard or soft based on their appearance at the edge on ophthalmological examination. Hard drusen have clearly defined edges, while soft drusen have less clearly defined fluid edges. The Age-Related Eye Disease Study (AREDS) Fundus Photography Severity Scale is one of the main classification systems used for this condition.

[0189] AMD is divided into " dry " and " wet " (exudative or neovascular) forms. Dry AMD is more common than wet AMD, but the dry form can develop into the wet form, and both occur simultaneously in a large number of cases. The typical characteristics of dry AMD are the progressive apoptosis of the bottom cells in the cell, overlying photoreceptor cells, more often the choriocapillaris layer in the RPE layer. The confluent area of ​​the RPE cell death with the atrophy of overlying photoreceptors is referred to as map-like atrophy (GA). The patient suffering from this form of AMD can experience the slow and progressive deterioration of central vision.

[0190] Wet AMD is characterized by bleeding and / or fluid leakage from abnormal blood vessels that grow from the RPE and choroidal vessels (choriocapillaris) beneath the macula, which can be the cause of sudden and disabling vision loss. It is estimated that most of the vision loss experienced by patients is due to this choroidal neovascularization (CNV) and its secondary complications.

[0191] The treatment or prevention of AMD described herein can reduce or prevent the occurrence of the above-mentioned AMD phenotypes.Preferably, the treatment of AMD can maintain or improve visual function.

[0192] In some embodiments, the treatment or prevention result of AMD is the prevention or reduction of the formation of map-like atrophy. In other embodiments, the treatment or prevention result of AMD is the progress of slowing down map-like atrophy. For example, relative to the untreated eyes through the same time period, after applying to the treated eyes of the experimenter, through 12 months, the result is that the increase of GA area is at least reduced by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90%. In other embodiments, the treatment or prevention result of AMD is the treatment of map-like atrophy, for example the reduction of map-like atrophy amount.

[0193] In some embodiments, the treatment or prevention of AMD results in the prevention or reduction of drusen formation. In other embodiments, the treatment or prevention of AMD results in the reduction of existing drusen, such as a reduction in the size and / or number of existing drusen.

[0194] In some embodiments, the outcome of treating or preventing AMD is the prevention or reduction of complement deposition. In other embodiments, the outcome of treating or preventing AMD is the reduction of existing complement deposition.

[0195] In some embodiments, the treatment or prevention of AMD results in improvement or restoration of vision or visual acuity. In other embodiments, the treatment or prevention of AMD reduces the loss of vision or visual acuity.

[0196] In some embodiments, the treatment or prevention of AMD results in an increase or restoration of a subject's reading speed. In other embodiments, the treatment or prevention of AMD reduces a decrease in a subject's reading speed.

[0197] In some embodiments, the outcome of treating or preventing AMD is the reduction or prevention of photoreceptor and / or retinal pigment epithelium (RPE) loss.

[0198] Diabetic retinopathy

[0199] Diabetic retinopathy is a condition characterized by damage to the blood vessels in the retina, caused by high blood sugar levels associated with diabetes. If left untreated, diabetic retinopathy can lead to blindness.

[0200] Although subjects with mild diabetic retinopathy may have good vision, two types of diabetic retinopathy, diabetic macular edema (DMO) and proliferative diabetic retinopathy (PDR), may threaten a subject's vision.

[0201] Diabetic macular edema is characterized by fluid leakage from damaged blood vessels at the back of the eye. The leaking fluid accumulates in the macula, which causes swelling and blurred vision. This eventually results in poor central vision and an inability to read or drive. Side vision usually remains normal.

[0202] Proliferative diabetic retinopathy is characterized by the closure of retinal blood vessels, which leads to abnormal growth of fragile blood vessels on the surface of the retina. This can lead to permanent vision loss due to bleeding in the eye, scarring, and retinal detachment.

[0203] Carrier

[0204] A vector is a tool that allows or facilitates the transfer of an entity from one environment to another.

[0205] Adeno-associated virus (AAV) vector

[0206] In one aspect, the invention provides an AAV vector comprising a polynucleotide of the invention.

[0207] Preferably, the AAV vector is in the form of an AAV vector particle.

[0208] Methods for making and modifying viral vectors and viral vector particles, such as those derived from AAV, are known in the art.

[0209] The AAV vector may comprise the AAV genome or a fragment or derivative thereof.

[0210] AAV is known to be able to package genomes up to 5.2 kb in size (Dong, J.-Y. et al. (1996) Human Gene Therapy 7:2101-2112).

[0211] The AAV genome is a polynucleotide sequence that encodes the functions required for the production of AAV particles. These functions include those that play a role in the replication and packaging cycle of AAV in host cells, including encapsulation of the AAV genome into AAV particles. Naturally occurring AAV is replication-defective and relies on the provision of trans-acting helper functions to complete the replication and packaging cycle. Therefore, the AAV genome of the AAV vector of the present invention is generally replication-defective.

[0212] The AAV genome can be in single-stranded form, either sense or antisense, or in double-stranded form. The use of the double-stranded form allows bypassing the DNA replication step in the target cell, thus accelerating transgene expression.

[0213] The AAV genome may be from any naturally derived AAV serotype, isolate or clade. Thus, the AAV genome may be the full genome of a naturally occurring AAV. As known to the skilled artisan, AAVs present in nature may be classified according to various biological systems.

[0214] Typically, AAV is referred to by its serotype. A serotype corresponds to a variant subspecies of AAV that has a unique reactivity due to its expression profile of capsid surface antigens that can be used to distinguish it from other variant subspecies. Typically, viruses with a particular AAV serotype do not cross-react efficiently with neutralizing antibodies specific for any other AAV serotype.

[0215] AAV serotypes include AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, and AAV11, as well as recombinant serotypes recently identified from primate brain, such as Rec2 and Rec3. Any of these AAV serotypes may be used in the present invention.

[0216] 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.

[0217] In some embodiments, the AAV can be an AAV1, AAV2, AAV5, AAV7, or AAV8 serotype.

[0218] In some embodiments, the AAV can be of the AAV2 or AAV8 serotype.

[0219] In some embodiments, the AAV may be of the AAV2 serotype. In other embodiments, the AAV may be of the AAV8 serotype.

[0220] The capsid protein may be a mutant capsid protein, such as disclosed in WO 2008 / 124724, which reference is hereby incorporated by reference.

[0221] In some embodiments, the AAV vector comprises an AAV8 capsid having a Y733F mutation.

[0222] AAV serotypes are reviewed in Choi et al. (2005) Curr. Gene Ther. 5:299-310 and Wu et al. (2006) Molecular Therapy 14:316-27. The sequences of the AAV genome or AAV genome elements used in the present invention, including ITR sequences, rep or cap genes, can be derived from the accession numbers of the following AAV complete genome sequences: 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.

[0223] AAV may also be referred to in terms of clades or clones. This refers to the phylogenetic relationships of AAVs of natural origin, and generally refers to a phylogenetic group of AAVs that can be traced back to a common ancestor, and includes all of its descendants. Additionally, AAV may be referred to in terms of specific isolates, i.e., genetic isolates of a particular AAV found in nature. The term "genetic isolate" describes an AAV population that has limited genetic admixture with other naturally occurring AAVs, thereby defining a distinct population that is identifiable at the genetic level.

[0224] The skilled person will be able to select the appropriate AAV serotype, clade, clone or isolate for use in the present invention based on their common general knowledge. For example, the AAV5 capsid has been shown to be able to efficiently transduce primate cone photoreceptors, as demonstrated by the successful correction of inherited color vision defects (Mancuso et al. (2009) Nature 461:784-7).

[0225] The AAV serotype determines the tissue specificity (or tropism) of AAV infection. Therefore, the preferred AAV serotypes of AAV for administration to patients according to the present invention are those with natural tropism or high infection efficiency for intraocular target cells. In some embodiments, the AAV serotypes used in the present invention are those AAV serotypes that transduce cells of the retinal neurosensory layer, retinal pigment epithelium and / or choroid.

[0226] Typically, the AAV genome of a natural source serotype, isolate or clade of AAV contains at least one inverted terminal repeat (ITR). The ITR sequence acts in cis to provide a functional replication origin and allow integration of the vector with the cell genome and excision from the cell genome. In a preferred embodiment, one or more ITR sequences are located on the flank of the nucleotide sequence encoding complement factor I or FHL1. The AAV genome also typically contains packaging genes, such as rep and / or cap genes that encode the packaging function of AAV particles. The rep gene encodes one or more of the proteins Rep78, Rep68, Rep52 and Rep40 or their variants. The cap gene encodes one or more capsid proteins, such as VP1, VP2 and VP3 or their variants. These proteins constitute the capsid of the AAV particles. Capsid variants are discussed below.

[0227] The promoter will be operably linked to each packaging gene. 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, while the p40 promoter is generally used to express the cap gene.

[0228] As discussed above, the AAV genome used in the AAV vector of the present invention can therefore be a full genome of a naturally occurring AAV. For example, a vector comprising a full AAV genome can be used to prepare an AAV vector or vector particle in vitro. However, although such a vector can be administered to a patient in principle, this is rarely done in practice. Preferably, the AAV genome will be derivatized for administration to a patient. Such derivatization is standard in the art, and the present invention encompasses the use of any known AAV genome derivative, as well as the use of derivatives that can be produced by applying techniques known in the art. The derivatization of the AAV genome and AAV capsid is reviewed in Coura and Nardi (2007) Virology Journal 4:99 and in Choi et al. and Wu et al. cited above.

[0229] Derivatives of the AAV genome include any truncated or modified form of the AAV genome that allows expression of transgenes from the AAV vector of the present invention in vivo. Generally, the AAV genome can be significantly truncated to include minimal viral sequences, but still retain the above functions. This is preferred for safety reasons to reduce the risk of vector recombination with wild-type viruses, and also to avoid triggering cellular immune responses due to the presence of viral gene proteins in target cells.

[0230] Typically, the derivative will include at least one inverted terminal repeat (ITR), preferably more than one ITR, such as two ITRs or more. One or more ITRs may be derived from an AAV genome having a different serotype, or may be a chimeric or mutant ITR. A preferred mutant ITR is a mutant ITR with a trs (terminal resolution site) deletion. This deletion allows continued replication of the genome to produce a single-stranded genome comprising both the coding sequence and the complementary sequence, i.e., a self-complementary AAV genome. This allows DNA replication to be avoided in the target cell, and thus can accelerate transgene expression.

[0231] One or more ITRs will preferably flank either end of the nucleotide sequence encoding complement factor I or FHL1. The inclusion of one or more ITRs is preferred to assist the vector of the invention in forming concatamers in the host cell nucleus, for example, after the single-stranded vector DNA is converted to double-stranded DNA by the action of a host cell DNA polymerase. The formation of such episomal concatamers protects the vector construct during the life of the host cell, thereby allowing for extended in vivo expression of the transgene.

[0232] In a preferred embodiment, the ITR element will be the only sequence retained from the natural AAV genome in the derivative. Therefore, the derivative will preferably not include the rep and / or cap genes of the natural genome and any other sequence of the natural genome. This is preferred for the reasons mentioned above, and is also to reduce the possibility of vector integration into the host cell genome. In addition, reducing the size of the AAV genome allows for increased flexibility in incorporating other sequence elements (such as regulatory elements) in addition to transgenes into the vector.

[0233] Thus, the following parts can be removed in the derivatives of the present invention: an inverted terminal repeat (ITR) sequence, a replication (rep) gene, and a capsid (cap) gene. However, in some embodiments, the derivatives may additionally include one or more rep and / or cap genes or other viral sequences of the AAV genome. Naturally occurring AAV integrates at a high frequency at a specific site on human chromosome 19 and exhibits a negligible frequency of random integration, making the retention of integration capacity in the vector acceptable in a therapeutic setting.

[0234] In the case where the derivative comprises capsid proteins, i.e., VP1, VP2 and / or VP3, the derivative may be a chimeric, shuffled or capsid-modified derivative of one or more naturally occurring AAVs. Specifically, the present invention encompasses providing capsid protein sequences from different serotypes, clades, clones or isolates of AAV in the same vector (i.e., pseudotyped vectors).

[0235] Typically, chimeric, reorganized or capsid-modified derivatives will be selected to provide one or more desired functions for AAV vectors. Therefore, compared with AAV vectors containing naturally occurring AAV genomes (such as AAV2 genomes), these derivatives can exhibit increased gene delivery efficiency, reduced immunogenicity (humoral or cellular), altered tropism ranges and / or improved targeting of specific cell types. The increased gene delivery efficiency can be affected by the following: improvements in cell surface receptor or co-receptor binding, improvements in internalization, intracellular and intracellular transport into the nucleus, improvements in viral particle uncoating and single-stranded genome conversion to double-stranded forms. The increased efficiency can also be related to the tropism range of the changes or the targeting of specific cell populations, so that the carrier dose is not diluted due to application to tissues that do not need it.

[0236] Chimeric capsid proteins include those produced by recombination between two or more capsid coding sequences of naturally occurring AAV serotypes. This can be performed, 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 a capsid sequence with desired properties. These capsid sequences of different serotypes can be altered by homologous recombination within the cell to produce new chimeric capsid proteins.

[0237] Chimeric capsid proteins also include those produced by engineering of the capsid protein sequence to transfer specific capsid protein domains, surface loops, or specific amino acid residues between two or more capsid proteins, such as between capsid proteins of two or more different serotypes.

[0238] Shuffled or chimeric capsid proteins can also be produced by DNA shuffling or by error-prone PCR. Hybrid AAV capsid genes can be produced by randomly fragmenting the sequences of related AAV genes, such as those encoding a variety of different serotype capsid proteins, and then reassembling these fragments in a self-priming polymerase reaction, which can also lead to exchanges in sequence homology regions. Hybrid AAV gene libraries generated in this way by shuffling the capsid genes of several serotypes can be screened to identify viral clones with desired functionality. Similarly, error-prone PCR can be used to randomly mutate AAV capsid genes to produce a variety of different variant libraries, which can then be selected for desired properties.

[0239] The sequence of the capsid gene can also be genetically modified to introduce specific deletions, substitutions or insertions relative to the natural wild-type sequence. Specifically, the capsid gene can be modified by inserting a 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.

[0240] The non-related protein or peptide can advantageously be a protein or peptide that acts as a ligand for a specific cell type, thereby conferring improved binding to the target cell or improving the specificity of the vector targeting to a specific cell population. An example may include the use of an RGD peptide to block uptake by the retinal pigment epithelium, thereby enhancing transduction of the surrounding retinal tissue (Cronin et al. (2008) ARVO Abstract: D1048). The non-related protein can also be a protein that assists in the purification of viral particles (as part of the production process), i.e., an epitope or affinity tag. The insertion site will generally be selected so as not to interfere with other functions of the viral particles, such as internalization, transport of viral particles. Technicians are able to identify suitable insertion sites based on their common knowledge. Specific sites are disclosed in Choi et al. cited above.

[0241] The present invention further encompasses providing AAV genome sequences in an order and configuration different from 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 more than one virus. Such chimeric genes may be composed of sequences of related viral proteins from two or more different viral species.

[0242] The AAV vector of the present invention may take the form of a nucleotide sequence comprising the AAV genome or a derivative thereof and a sequence encoding a complement factor I or FHL1 transgene or a derivative thereof.

[0243] The AAV particles of the present invention include transcapsid forms, wherein an AAV genome or derivative having one serotype ITR is packaged in a capsid of a different serotype. The AAV particles of the present invention also include mosaic forms, wherein a mixture of unmodified capsid proteins from two or more different serotypes constitutes the viral capsid. AAV particles also include chemically modified forms with ligands adsorbed to the capsid surface. For example, such ligands may include antibodies for targeting specific cell surface receptors.

[0244] Thus, for example, AAV particles of the 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 more than one serotype.

[0245] An AAV vector may contain multiple copies (eg, 2, 3, etc.) of a nucleotide sequence mentioned herein.

[0246] Promoter and regulatory sequences

[0247] The polynucleotides or vectors of the invention may also include elements that allow for expression of a complement factor I or FHL1 transgene in vitro or in vivo. These may be referred to as expression control sequences. Thus, a polynucleotide or vector typically comprises an expression control sequence (e.g., comprising a promoter sequence) operably linked to a nucleotide sequence encoding a transgene.

[0248] Any suitable promoter may be used, the selection of which can be readily performed by a skilled artisan. The promoter sequence may be constitutively active (i.e., operable in any host cell background), or may be active only in a specific host cell environment, thereby allowing targeted expression of a transgene in a specific cell type (e.g., a tissue-specific promoter). The promoter may display inducible expression in response to the presence of another factor, such as a factor present in the host cell. In any case, when the vector is administered for therapy, it is preferred that the promoter should function in the target cell background.

[0249] In some embodiments, it is preferred that the promoter exhibits retinal cell-specific expression so that the transgene is expressed only in a retinal cell population. Thus, expression from the promoter can be retinal cell-specific, for example, limited to cells of the retinal neurosensory layer and retinal pigment epithelium.

[0250] Preferred promoters that are not retinal cell specific include the chicken beta-actin (CBA) promoter, optionally combined with a cytomegalovirus (CMV) enhancer element. An exemplary promoter for use in the present invention is the CAG promoter, such as that used in the rAVE expression cassette (GeneDetect.com).

[0251] In a preferred embodiment, the polynucleotide or vector comprises a CMV promoter.

[0252] An example CMV promoter sequence is:

[0253]

[0254] In some embodiments, the polynucleotide or vector comprises a promoter having a nucleotide sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 13. Preferably, the nucleotide sequence substantially retains the functional activity of the promoter represented by SEQ ID NO: 13.

[0255] In other embodiments, the polynucleotide or vector comprises a promoter having the nucleotide sequence of SEQ ID NO:13.

[0256] Another example promoter sequence is:

[0257]

[0258] In some embodiments, the polynucleotide or vector comprises a promoter having a nucleotide sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 5. Preferably, the nucleotide sequence substantially retains the functional activity of the promoter represented by SEQ ID NO: 5.

[0259] In other embodiments, the polynucleotide or vector comprises a promoter 5 having a nucleotide sequence of SEQ ID NO:5.

[0260] Examples of promoters based on human sequences that can induce retina-specific gene expression include rhodopsin kinase for rods and cones (Allocca et al. (2007) J. Virol. 81: 11372-80), PR2.1 for cones only (Mancuso et al. (2009) Nature 461: 784-7), and / or RPE65 for the retinal pigment epithelium (Bainbridg et al. (2008) N. Engl. J. Med. 358: 2231-9) or VMD2 (Esumi et al. (2004) J. Virol. Chem. 279: 19064-73).

[0261] The polynucleotides or vectors of the present invention may also contain one or more additional regulatory sequences that may function before or after transcription. The regulatory sequences may be part of the native transgenic locus or may be heterologous regulatory sequences. The polynucleotides or vectors of the present invention may contain portions of the 5'-UTR or 3'-UTR from the native transgenic transcript.

[0262] A regulatory sequence is any sequence that promotes transgene expression, i.e., acts to increase transcript expression, improve nuclear export of mRNA, or enhance its stability. Such regulatory sequences include, for example, enhancer elements, post-transcriptional regulatory elements, and polyadenylation sites.

[0263] A preferred polyadenylation site is the bovine growth hormone poly-A (bGH poly-A) signal.

[0264] An example bovine growth hormone poly-A (bGH poly-A) signal is:

[0265]

[0266] Another example bovine growth hormone poly-A (bGH poly-A) signal is:

[0267]

[0268] In some embodiments, the polynucleotide or vector comprises a polyadenylation signal having a nucleotide sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical 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.

[0269] In other embodiments, the polynucleotide or vector comprises a polyadenylation signal having the nucleotide sequence of SEQ ID NO: 14 or 6.

[0270] In the context of the polynucleotides or vectors of the invention, such regulatory sequences will be cis-acting. However, the invention also encompasses the use of trans-acting regulatory sequences located on additional gene constructs.

[0271] A preferred post-transcriptional regulatory element for use in the AAV vectors of the present invention is the woodchuck hepatitis post-transcriptional regulatory element (WPRE) or a variant thereof.

[0272] An example WPRE is:

[0273]

[0274] WPRE is a ternary element that contains gamma, alpha, and beta elements in the given order. A shortened version of WPRE containing only minimal gamma and alpha elements (called WPRE3; Choi, J.-H. et al. (2014) Molecular Brain 7:17) can also be used in the present invention.

[0275] An example WPRE3 sequence is:

[0276]

[0277] In some embodiments, the polynucleotide or vector comprises a post-transcriptional regulatory element having a nucleotide sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical 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.

[0278] In other embodiments, the polynucleotide or vector comprises a post-transcriptional regulatory element having a nucleotide sequence of SEQ ID NO: 15 or 7.

[0279] Another regulatory sequence that can be used in the polynucleotides or vectors of the present invention is the scaffold attachment region (SAR). Additional regulatory sequences can be readily selected by the skilled artisan.

[0280] Application method

[0281] The polynucleotides or vectors of the invention may be administered systemically (eg, by peripheral intravenous infusion) and may be administered locally (eg, by intrathecal injection into the CNS system). In a preferred embodiment, the polynucleotide or vector is administered intraocularly.

[0282] The term "intraocular" refers to the interior of the eye, and thus intraocular administration involves administration to the interior of the eye of a subject.

[0283] In some embodiments, the polynucleotide or vector is administered to the subject's eye by subretinal, direct retinal, suprachoroidal, or intravitreal injection. In some embodiments, administration is performed by a robot.

[0284] The volume of the injected pharmaceutical composition can be, for example, about 10-500 μL, such as about 50-500, 100-500, 200-500, 300-500, 400-500, 50-250, 100-250, 200-250 or 50-150 μL. The volume can be, for example, about 10, 50, 100, 150, 200, 250, 300, 350, 400, 450 or 500 μL. Preferably, the volume of the injected pharmaceutical composition is 100 μL.

[0285] The technician will be familiar with and well-equipped to perform isolated subretinal, direct retinal, suprachoroidal, or intravitreal injections.

[0286] Preferably, the polynucleotide or vector is administered by subretinal injection.

[0287] In some embodiments, the polynucleotide, vector, or pharmaceutical composition comprising the same is administered no more than once or no more than twice in the lifetime of the subject.

[0288] Subretinal injection

[0289] Subretinal injections are injections into the subretinal space, just below the neurosensory layer of the retina. During a subretinal injection, the injected material is directed into the photoreceptor cells and the retinal pigment epithelium (RPE) layer and creates a space between the two.

[0290] When injection is done through a small retinotomy, retinal detachment may occur. The detached, raised layer of retina created by the injected material is called a "bleb."

[0291] The hole created by the subretinal injection must be small enough so that the injected solution does not significantly flow back into the vitreous cavity after administration. Such backflow can be particularly problematic when injecting drugs, as the effects of the drug will be directed away from the target area. Preferably, the injection creates a self-sealing entry point in the retinal neurosensory layer, i.e., once the injection needle is removed, the hole created by the needle reseals so that little or substantially no injected material is released through the hole.

[0292] To facilitate this process, specialized subretinal injection needles are commercially available (eg, DORC 41G Teflon subretinal injection needle, Dutch Ophthalmic Research Center International BV, Zuidland, The Netherlands). These are needles designed for performing subretinal injections.

[0293] Unless retinal damage occurs during the injection process, and as long as a sufficiently small needle is used, substantially all injected materials remain between the detached retinal neurosensory layer and the RPE at the local retinal detachment site (i.e., will not flow back into the vitreous cavity). In fact, the typical persistence of blisters in a short period of time shows that the injected material usually rarely escapes into the vitreous. Along with the injected material being absorbed, the blisters can dissipate over a longer period of time.

[0294] Visualization of the eye, specifically the retina, can be performed prior to surgery, for example using optical coherence tomography.

[0295] The volume of the injected pharmaceutical composition can be, for example, about 10-500 μL, such as about 50-500, 100-500, 200-500, 300-500, 400-500, 50-250, 100-250, 200-250, or 50-150 μL. The volume can be, for example, about 10, 50, 100, 150, 200, 250, 300, 350, 400, 450, or 500 μL. Preferably, the volume of the injected pharmaceutical composition is 100 μL. Larger volumes may increase the risk of stretching the retina, while smaller volumes may be difficult to see.

[0296] Two-step subretinal injection

[0297] The polynucleotides or vectors of the present invention can be delivered with increased accuracy and safety by using a two-step method in which a localized retinal detachment is produced by subretinal injection of a first solution. The first solution does not contain a polynucleotide or vector. A second subretinal injection is then used to deliver a drug containing a polynucleotide or vector to the subretinal fluid of the blister produced by the first subretinal injection. Because the injection delivering the drug is not used to detach the retina, a specific volume of solution can be injected in this second step.

[0298] In some embodiments, subretinal injection of the vector comprises the following steps:

[0299] (a) administering to a subject a solution by subretinal injection in an amount effective to at least partially detach the retina to form a subretinal bleb, wherein the solution does not comprise a polynucleotide or a vector; and

[0300] (b) administering a pharmaceutical composition by subretinal injection into the bleb formed in step (a), wherein the drug comprises a polynucleotide or a vector.

[0301] The volume of solution injected in step (a) to at least partially detach the retina can be, for example, about 10-1000 μL, such as about 50-1000, 100-1000, 250-1000, 500-1000, 10-500, 50-500, 100-500, 250-500 μL. The volume can be, for example, about 10, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900 or 1000 μL.

[0302] The volume of the pharmaceutical composition injected in step (b) can be, for example, about 10-500 μL, such as about 50-500, 100-500, 200-500, 300-500, 400-500, 50-250, 100-250, 200-250 or 50-150 μL. The volume can be, for example, about 10, 50, 100, 200, 150, 200, 250, 300, 350, 400, 450 or 500 μL. Preferably, the volume of the pharmaceutical composition injected in step (b) is 100 μL. Larger volumes may increase the risk of stretching the retina, while smaller volumes may be difficult to see.

[0303] The solution not containing the drug (i.e., the "solution" of step (a)) can be prepared similarly to the solution containing the drug, as described below. The preferred solution not containing the drug is a balanced saline solution (BSS) or a similar buffered solution that matches the pH and osmotic pressure of the subretinal space.

[0304] Visualizing the retina during surgery

[0305] In some cases, such as during advanced retinal degeneration, identification of the retina is difficult because it is thin, transparent, and difficult to see against the damaged and heavily pigmented epithelium in which it rests. Geuder; Dorc) can be useful for identifying retinal breaks formed for retinal detachment surgery (i.e., step (a) in the two-step subretinal injection method of the present invention) so that the drug can be administered through the same break without the risk of reflux back into the vitreous cavity.

[0306] The use of blue vital dye can also identify any areas within the retina where a thickened internal limiting membrane or epiretinal membrane is present, as injection through either of these structures will preclude smooth access to the subretinal space. In addition, contraction of either of these structures in the immediate postoperative period may result in stretching of the retinal access break, which may result in reflux of medication into the vitreous cavity.

[0307] Suprachoroidal injection

[0308] The polynucleotide or vector of the present invention can be delivered to the suprachoroidal space using an ab externo method utilizing a microcatheter (see, e.g., Peden et al. (2011) PLoS One 6 (2): e17140). In this method, a limbal conjunctival peritomy is performed to expose the exposed sclera, followed by a sclerotomy to expose the exposed choroid. A microcatheter (such as the iTrack250A from iScience Interventional, optionally connected to an illumination system, such as a micro-illumination system based on an iLumin laser diode (iScience Interventional)) is introduced into the suprachoroidal space and advanced backward toward the optic disc. After the microcatheter tip is manipulated to the desired position, the injection of the polynucleotide or vector forms a bleb in the retina and choroid.

[0309] 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 in the space until the tip is close to the diseased area of ​​the retina; and (iii) injecting the polynucleotide or vector from the tip of the microcatheter to form a bleb.

[0310] In some embodiments, the above-described administration procedures are performed directly by a robot.

[0311] Pharmaceutical compositions and injection solutions

[0312] The drugs of the present invention, such as polynucleotides or vectors, can be formulated into pharmaceutical compositions. In addition to the drug, these compositions may also contain pharmaceutically acceptable carriers, diluents, excipients, buffers, stabilizers or other materials known in the art. Such materials should be non-toxic and should not interfere with the efficacy of the active ingredient. The exact nature of the carrier or other material can be determined by the technician according to the route of administration, such as subretinal, direct retinal, suprachoroidal or intravitreal injection.

[0313] Pharmaceutical compositions are generally in liquid form. Liquid pharmaceutical compositions generally include liquid carriers such as water, petroleum, animal or vegetable oils, mineral oils or synthetic oils. Physiological saline solutions, magnesium chloride, dextrose or other sugar solutions, or glycols, such as ethylene glycol, propylene glycol or polyethylene glycol, may be included. In some cases, a surfactant may be used, such as 0.001% pluronic acid (PF68).

[0314] For injections in the affected part, the active ingredient can be a pyrogen-free aqueous solution with suitable pH, isotonicity and stability. The technician can use well, for example, isotonic vehicles such as sodium chloride injection, Ringer's injection or lactated Ringer's injection to prepare suitable solutions. Preservatives, stabilizers, buffers, antioxidants and / or other additives can be included as needed.

[0315] For delayed release, the drug can be contained in a pharmaceutical composition formulated for slow release according to methods known in the art, such as in microcapsules formed from biocompatible polymers or in a liposomal carrier system.

[0316] Treatment

[0317] It should be understood that all references to treatment herein include curative treatment, palliative treatment and prophylactic treatment; although in the context of the present invention, references to prevention are more generally associated with prophylactic treatment. Treatment may also include arresting the progression of disease severity.

[0318] Treatment of mammals, particularly humans, is preferred. However, both human and veterinary treatments are within the scope of the present invention.

[0319] Variants, derivatives, analogs, homologs and fragments

[0320] 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.

[0321] In the context of the present invention, a variant of any given sequence is a sequence in which a particular sequence of residues (whether amino acid residues or nucleic acid residues) has been modified in such a way that the polypeptide or polynucleotide in question substantially retains its function. Variant sequences may be obtained by addition, deletion, substitution, modification, replacement and / or variation of at least one residue present in a naturally occurring protein.

[0322] As used herein, the term "derivative" in relation to a protein or polypeptide of the present invention includes any replacement, variation, 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.

[0323] As used herein, the term "analog" in relation to a polypeptide or polynucleotide includes any mimetic, that is, a compound that has at least one endogenous function of the polypeptide or polynucleotide that it mimics.

[0324] Generally, amino acid substitutions can be made, for example from 1, 2 or 3 to 10 or 20 substitutions, provided that the modified sequence substantially retains the desired activity or ability.Amino acid substitutions may include the use of non-naturally occurring analogs.

[0325] The proteins used in the present invention may also have deletions, insertions or substitutions of amino acid residues that produce silent changes and result in functionally equivalent proteins. Deliberate amino acid substitutions may be made based on similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphipathic properties of the residues, as long as endogenous function is retained. For example, negatively charged amino acids include aspartic acid and glutamic acid; positively charged amino acids include lysine and arginine; and amino acids with uncharged polar head groups of similar hydrophilicity values ​​include asparagine, glutamine, serine, threonine, and tyrosine.

[0326] For example, conservative substitutions can be made according to the following table. Amino acids in the same block of the second column, and preferably in the same row of the third column, can be substituted with each other:

[0327]

[0328] As used herein, the term "homolog" refers to an entity having a certain homology with a wild-type amino acid sequence and a wild-type nucleotide sequence. The term "homology" can be equivalent to "identity".

[0329] Homologous sequences may include amino acid sequences that are at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85% or 90% identical to the subject sequence, preferably at least 95% or 97% or 99% identical. Typically, homologs will contain the same active sites, etc. as the subject amino acid sequence. Although homology can also be considered from the perspective of similarity (i.e., amino acid residues with similar chemical properties / functions), in the context of the present invention, it is preferred to express homology from the perspective of sequence identity.

[0330] Homologous sequences can include nucleotide sequences that are at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85% or 90% identical to the target sequence, preferably at least 95% or 97% or 99% identical. Although homology can also be considered from the perspective of similarity, in the context of the present invention, it is preferred to express homology from the perspective of sequence identity.

[0331] Preferably, reference to a sequence having a percentage identity to any one of the SEQ ID NOs detailed herein refers to the sequence having that percentage identity over the entire length of the referenced SEQ ID NO.

[0332] 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 homology or identity percentage between two or more sequences.

[0333] The percent homology can be calculated for consecutive sequences, i.e. one sequence is aligned with the other and each amino acid in one sequence is directly compared to the corresponding amino acid in the other sequence, one residue at a time. This is called a "ungapped" alignment. Typically, such ungapped alignments are performed only over a relatively short number of residues.

[0334] While this is a very simple and consistent approach, it does not take into account that, for example, an insertion or deletion in a nucleotide sequence in an otherwise identical sequence pair may cause subsequent codons to be out of alignment, thus potentially resulting in a substantial reduction in the homology percentage when a global alignment is performed. Therefore, most sequence comparison methods are designed to produce an optimal alignment that takes possible insertions and deletions into account without excessively penalizing the overall homology score. This is achieved by inserting "gaps" in the sequence alignment to try to maximize local homology.

[0335] However, these more complex methods assign a "gap penalty" to each gap that occurs in the alignment, so for the same number of identical amino acids, a sequence alignment with as few gaps as possible, reflecting a higher correlation between the two compared sequences, will receive a higher score than an alignment with many gaps. An "affine gap cost" is often used, which charges a relatively high cost for the presence of a gap and a smaller penalty for each subsequent residue in the gap. This is the most commonly used gap scoring system. A high gap penalty will of course produce an optimized alignment with fewer gaps. Most alignment programs allow modification of the gap penalty. However, when using such software for sequence comparison, it is preferred to use the default values. For example, when using the GCG WisconsinBestfit software package, the default gap penalty for amino acid sequences is -12 for a gap and -4 for each extension.

[0336] Therefore, considering the gap penalty, the calculation of the maximum homology percentage first needs to produce the best comparison. The suitable computer program for carrying out this comparison is the GCG Wisconsin Bestfit software package (University of Wisconsin, USA; Devereux et al. (1984) Nucleic Acids Res.14,4683-4690). 12:387). The examples of other software that can perform sequence comparison include, but are not limited to, BLAST software package (see Ausubel et al. (1999) ibid-Chapter 18), FASTA (Atschul et al. (1990) J.Mol.Biol.403-410) and GENEWORKS comparison tool suite. BLAST and FASTA can be used for offline and online search (see Ausubel et al. (1999) ibid, 7-58 pages to 7-60 pages). However, for some applications, it is preferred to use the GCG Bestfit program. Another tool called BLAST 2 Sequences can also be used to compare protein and nucleotide sequences (see FEMS Microbiol. Lett. (1999) 174:247-50; FEMS Microbiol. Lett. (1999) 177:187-8).

[0337] Although the final homology percentage can be measured according to identity, the alignment process itself is usually not based on an all-or-nothing pair comparison. On the contrary, a scaled similarity score matrix is ​​generally used, which is based on chemical similarity or evolutionary distance to assign scores to each paired comparison. An example of this matrix commonly used is the default matrix of the BLOSUM62 matrix-BLAST program suite. The GCG Wisconsin program generally uses public default values ​​or custom symbol comparison tables, if provided (for details, please refer to the user manual). For some applications, it is preferred to use the public default values ​​of the GCG software package, or in the case of other software, use a default matrix, such as BLOSUM62.

[0338] Once the software has produced an optimal alignment, the percent homology, preferably the percent sequence identity, can be calculated. The software typically does this as part of the sequence comparison and generates a numerical result.

[0339] A "fragment" of a full-length complement factor I or FHL1 is also a variant, and the term generally refers to a selected region of a polypeptide or polynucleotide that is of functional or, for example, interesting in an assay. Thus, a "fragment" refers to an amino acid or nucleic acid sequence that is a portion of a full-length polypeptide or polynucleotide.

[0340] Such variants can be prepared using standard recombinant DNA techniques such as site-directed mutagenesis. In the case of insertion, the synthetic DNA encoding the insertion can be prepared together with 5' and 3' flanking regions corresponding to the naturally occurring sequence on either side of the insertion site. The flanking regions will contain convenient restriction enzyme sites corresponding to the sites in the naturally occurring sequence so that the sequence can be cut with a suitable enzyme and the synthetic DNA can be connected to the nick. The DNA is then expressed according to the present invention to prepare the encoded protein. These methods are only examples of many standard techniques known in the art for manipulating DNA sequences, and other known techniques may also be used.

[0341] Those skilled in the art will understand that they can combine all features of the present invention disclosed herein without departing from the scope of the invention as disclosed.

[0342] Preferred features and embodiments of the invention will now be described by way of non-limiting examples.

[0343] 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 one of ordinary skill in the art and are explained in the literature. See, e.g., Sambrook, J., Fritsch, EF, and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press; Ausubel, FM et al. (1995 and regular 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, JM and McGee, J.O'D. (1990) In Situ Hybridization: Principles and Practice, Oxford University Press; Gait, MJ (1984) Oligonucleotide Synthesis: A Practical Approach, IRL Press; and Lilley, DM and Dahlberg, JE (1992) Methods in Enzymology: DNA Structures Part A: Synthesis and Physical Analysis of DNA, Academic Press. Each of these general texts is incorporated herein by reference.

[0344] Example

[0345] Example 1

[0346] Codon optimization

[0347] The nucleotide sequences encoding complement factor I (CFI) and complement factor H-like protein 1 (FHL1) were codon optimized using a series of methods summarized in Table 1.

[0348] Table 1

[0349]

[0350]

[0351] For “basic” codon optimization, the sequence of CFI or FHL-1 was input into 5 online codon optimization tools:

[0352] 1.GeneArt(https: / / www.thermofisher.com / uk / en / home / life-scienc e / cloning / gene-synthesis / geneart-gene-synthesis / geneoptimizer.html)

[0353] 2.GenScript(https: / / www.genscript.com / quick_order / gene_services_gene_synthesis)

[0354] 3.IDT(https: / / eu.idtdna.com / CodonOpt)

[0355] 4.JCat (http: / / www.jcat.de / )

[0356] 5.COOL(http: / / cool.syncti.org / setup_input_sequence_create_wf1.php?=Start+Using+Codon+Optimization+On-Line+%3E%3E%3E)

[0357] All tools use the standard human genetic code.

[0358] For tools 1-4 above, each tool generates a sequence.

[0359] For Tool 5, the default settings were used and the target expression host was set to Homo sapiens. In addition, 39 genes highly expressed in RPE (Table 2) were entered into the tool (based on Table 4 of Booij, JC et al. (2010) PLoS One 5: e9341).

[0360] Table 2

[0361]

[0362]

[0363] Tool 5 generated 70 optimized sequences for CFI and 55 optimized sequences for FHL-1, and the highest ranked sequences were used.

[0364] For “manual” codon optimization, the five base CFI and FHL-1 codon-optimized sequences generated above were manually optimized to eliminate cryptic splice sites, microRNA binding sites, remove tandem repeat codons and check GC content.

[0365] Cryptic splice site removal

[0366] Cryptic splice sites were identified using the www.Fruitfly.org tool. A cutoff of 0.4 was used for the analysis, but only sequences with a score > 0.75 were modified.

[0367] Splice sites were removed whenever possible by changing to GT for donor sites or AG for acceptor sites. If this was not possible (e.g., for sequences encoding valine), the 5' adjacent base was altered.

[0368] All modified sequences were then analyzed using the www.Fruitfly.org tool to confirm that all splice sites had been removed or reduced to below a 0.75 threshold.

[0369] MicroRNA binding site removal

[0370] MicroRNAs were identified using the www.Genecards.org tool.

[0371] For CFI, the following miRNA binding sites were identified: hsa-mir-335-5p, hsa-mir-181a-5p, hsa-mir-26b-5p.

[0372] For FHL-1, the following miRNA binding site was identified (based on the sequence of complement factor H, CFH): hsa-mir-146a-5p.

[0373] Each codon optimized sequence (after removing splice sites if necessary) was then passed through the STarMir tool (http: / / sfold.wadsworth.org / cgi-bin / starmirtest2.pl) to see if the miRNA site was still present. Any miRNA site identified with a logistic probability of >0.75 was modified.

[0374] Tandem repeat codon removal

[0375] All sequences were manually checked for tandemly repeated codons. Where these were found, the second codon was changed to the next most frequently used codon in Homo sapiens (using the SnapGene codon usage table).

[0376] The wild type and codon optimized sequences are detailed below:

[0377] GT005:CFI wild-type sequence:

[0378]

[0379] RC001: FHL-1 wild-type sequence:

[0380]

[0381] RC128: CFI GeneArt-Basic:

[0382]

[0383] RC129: CFI GeneArt-Manual Optimization:

[0384]

[0385] RC130: CFI GenScript – The Basics:

[0386]

[0387] RC131: CFI Genscript – Manual Optimization:

[0388]

[0389] RC132: CFI IDT-Basics:

[0390]

[0391] RC133: CFI IDT – Manual Optimization:

[0392]

[0393] RC134: CFI JCat – Basics:

[0394]

[0395] RC135: CFI JCat – Manual Optimization:

[0396]

[0397] RC136: CFI COOL - Base: See SEQ ID NO: 10 above. RC137: CFI COOL - Manually Optimized:

[0398]

[0399] RC138: FHL-1GeneArt-Base:

[0400]

[0401] RC139: FHL-1GeneArt – Manual Optimization:

[0402]

[0403] RC140: FHL-1GenScript – Basics:

[0404]

[0405] RC141: FHL-1Genscript – Manual Optimization:

[0406]

[0407]

[0408] RC142: FHL-1IDT-Basic:

[0409]

[0410] RC143: FHL-1IDT - Manual Optimization:

[0411]

[0412] RC144: FHL-1JCat-Basic

[0413]

[0414] RC145: FHL-1JCat – Manual Optimization

[0415]

[0416] RC146: FHL-1COOL-basic: See SEQ ID NO: 12 above. RC147: FHL-1COOL-manually optimized

[0417]

[0418]

[0419] Plasmid generation

[0420] All 10 codon optimized sequences were synthesized and cloned into the AAV vector backbone. The vector also contained the AAV-2 left and right inverted terminal repeats (ITRs) flanking a modified CBA / CAG promoter (chicken β-actin with a CMV enhancer; "CBA"). The promoter drove the expression of codon optimized FHL1 or CFI. In addition, downstream of the transgene was a modified woodchuck hepatitis virus posttranscriptional regulatory element (WPRE) sequence and a bovine growth hormone poly-A (bGHpolyA) sequence provided to the 3' end of the cDNA.

[0421] Transfection

[0422] Transfect all 20 plasmids into ARPE19 cells using the following procedure:

[0423] Day 1: Isolate and count ARPE19 cells using ViCell. Cells were plated at 6x10 in 500 μL DMEM, 10% FBS / well. 4 Cells / well were seeded in 48-well plates.

[0424] Day 2: Check confluency and find it to be between 70-80%. Then transfect cells with 0.25 μg of plasmid DNA using PEI at a 1:3 DNA:PEI ratio, repeat twice:

[0425] 1. Dilute 2x 0.25μg DNA in 2x 5μL PBS.

[0426] 2. Dilute 2x 0.75 μL PEI in 2x 5 μL PBS.

[0427] 3. Add the PEI mixture dropwise to the DNA mixture, mix, and then incubate at room temperature for 20 minutes.

[0428] 4. Add 2 x 250 μL DMEM / Glutamax / 10% FBS to the mixture.

[0429] 5. Remove the culture medium from the cells and replace with 250 μL of DNA / PEI complex per well.

[0430] Day 3: Remove medium and replace with 125 μL serum-free DMEM / Glutamax.

[0431] Day 5: Harvest the culture medium by centrifugation at 14,000 rpm for 10 min at 4 °C, and transfer the supernatant to a new tube.

[0432] Western blotting

[0433] Supernatants from transfections were analyzed by Western blotting (primary antibodies to CFI and FHL-1 were used: goat antiserum CFI 1:3000; Quidel A312 1:3000; and secondary antibody rabbit anti-goat HRP 1:5000).

[0434] Western blot analysis Figure 2 shown.

[0435] CFI ELISA

[0436] Supernatants from transfections were analyzed for CFI by ELISA using the following procedure:

[0437] Day 1: ELISA plates were coated with 50 μL per well of sheep anti-CFI polyclonal antibody diluted 1:4000 in 1x coating buffer. Plates were stored at 4°C overnight.

[0438] Day 2: Wash the plates 3 times with 200 μL of PBS-Tween (0.05%) per well and blot dry with paper towels. Apply 200 μL of 1% BSA fifth component in PBS-Tween (0.05%) to each well and block for 2 hours at room temperature.

[0439] Samples and standard curves were prepared during the blocking incubation. Standard curves were prepared using 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.

[0440] After blocking for 2 hours, the plates were washed three times as described above, and 50 μL of samples or standards were loaded into each well and incubated for 1 hour at room temperature.

[0441] After 1 hour, the plates were washed as above, then anti-CFI (0x21) antibody was diluted 1:2000 in DMEM 5% FBS and 50 μL was applied to each well and incubated for 1 hour at room temperature.

[0442] After 1 hour, the plates were washed as above, then donkey anti-mouse-HRP antibody was diluted 1:5000 in DMEM 5% FBS and 50 μL was applied to each well and incubated for 1 hour at room temperature.

[0443] After 1 hour, the plates were washed as above and 100 μL of TMB reagent was applied to each well and incubated in the dark at room temperature for approximately 15 minutes. Once a sufficient blue color was obtained, 100 μL of 1 M sulfuric acid was added to each well to stop the reaction.

[0444] A450 was then recorded, and the data were processed and transferred to Microsoft Excel for analysis.

[0445] CFI ELISA results in Figure 3 Shown in.

[0446] FHL1 ELISA

[0447] Supernatants from transfections were analyzed for FHL1 by ELISA using the following procedure:

[0448] Day 1: ELISA plates were coated with 50 μL per 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.

[0449] Day 2: Wash the plates 3 times with 200 μL of PBS-Tween (0.05%) per well and blot dry with paper towels. Apply 200 μL of 1% BSA fifth component in PBS-Tween (0.05%) to each well and block for 2 hours at room temperature.

[0450] Samples and standard curves were prepared during the blocking incubation. Standard curves were prepared using FHL1-His protein diluted in DMEM + 2% FBS. Samples were diluted 1:5, 1:10 and 1:30 in blocking solution.

[0451] After blocking for 2 hours, the plates were washed three times as described above, and 50 μL of samples or standards were loaded into each well and incubated for 1 hour at room temperature.

[0452] After 1 hour, the plates were washed as above, 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 for 1 hour at room temperature.

[0453] After 1 hour, the plates were washed as above, then donkey anti-mouse-HRP antibody was diluted 1:5000 in DMEM 5% FBS and 50 μL was applied to each well and incubated for 1 hour at room temperature.

[0454] After 1 hour, the plates were washed as above and 100 μL of TMB reagent was applied to each well and incubated in the dark at room temperature for approximately 15 minutes. Once a sufficient blue color was obtained, 100 μL of 1 M sulfuric acid was added to each well to stop the reaction.

[0455] A450 was then recorded, and the data were processed and transferred to Microsoft Excel for analysis.

[0456] The FHL1 ELISA results are shown in Figure 4 middle.

[0457] Generation of AAV2 vectors

[0458] The best four (CFI) and five (FHL1) sequences were used for studies using AAV.

[0459] HEK293 cells were transfected with the selected codon-optimized plasmids along with pRepCap and pHelper following a typical triple transfection protocol, specifically:

[0460] Day 1: Dissociate and count HEK293 cells using ViCell. Plate cells at 6x10 in 10 mL DMEM 10% FBS / dish. 5 Cells / cm 2 Inoculate in a 10 cm culture dish.

[0461] Day 2: Confluence was checked and found to be between 70-80%.

[0462] The culture medium was replaced with 10 mL of DMEM / Glutamax containing 5% FBS.

[0463] After 4 hours, cells were transfected with 5 μg of plasmid using PEI at a 1:3 DNA:PEI ratio.

[0464] Day 3: Add 15 mM butyrate to 11 mL of medium per plate.

[0465] Day 5: The supernatant was harvested and centrifuged at 1000 rpm for 10 minutes to remove cell debris.

[0466] The supernatant was transferred to a new tube, and 1 / 5 volume of AAVanced (AAV110A-1, Cambridge Bioscience) reagent (2.75 mL / 11 mL) was added.

[0467] The mixture was then stored at 4°C.

[0468] Day 8: The supernatant / AAVanced mixture was centrifuged at 1000 rpm for 30 minutes at 4°C.

[0469] The supernatant was discarded and the pellet was resuspended in 500 μL PBS. This was then transferred to a 1.5 mL tube and centrifuged at 1500 g for 3 minutes.

[0470] The supernatant was discarded and the remaining pellet was resuspended in 1 / 100 the original volume (ie, 100 μL / 11 mL supernatant).

[0471] The vector was stored at -80°C.

[0472] Transduction of ARPE19 cells

[0473] ARPE19 cells were transduced with vectors containing codon-optimized transgenes.

[0474] Day 1: ARPE19 cells were dissociated and counted on ViCell, then cultured at 1x 10 per well in 200 μL DMEM / Glutamax + 10% FBS. 5 Cell inoculation.

[0475] Day 2: Add vector to cells.

[0476] Day 3: Replace the culture medium with serum-free medium.

[0477] Day 4: Harvest the supernatant, centrifuge at 14,000 rpm for 10 min at 4 °C, and transfer to a fresh tube.

[0478] Total protein concentration was estimated by Bradford assay.

[0479] CFI ELISA

[0480] Supernatants from transductions were analyzed for CFI by ELISA according to the above protocol.

[0481] CFI ELISA results in Figure 5 Shown in.

[0482] FHL-1 ELISA

[0483] Supernatants from transductions were analyzed for FHL1 by ELISA according to the above protocol.

[0484] The FHL1 ELISA results are shown in Figure 6 middle.

[0485] in conclusion

[0486] Both RC136 (CFI; SEQ ID NO: 10) and RC146 (FHL-1; SEQ ID NO: 12) provided higher transgene expression than the wild-type sequence and other codon-optimized sequences tested.

[0487] All publications mentioned in the above description are incorporated herein by reference. Without departing from the scope and spirit of the present invention, various modifications and variations of the disclosed agents, compositions, purposes and methods of the present invention will be apparent to those skilled in the art. Although the present invention has been disclosed in conjunction with specific preferred embodiments, it should be understood that the claimed invention should not be overly limited to these specific embodiments. In fact, various modifications disclosed for carrying out the mode of the present invention that are apparent to those skilled in the art are intended to fall 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. The isolated polynucleotide of claim 1, wherein the nucleotide sequence is SEQ ID NO:

10.

3. 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 with SEQ ID NO:

12.

4. The isolated polynucleotide of claim 3, wherein the nucleotide sequence is SEQ ID NO:

12.

5. A vector comprising the polynucleotide according to any one of the preceding claims.

6. The vector of claim 5, wherein the vector is an adeno-associated virus (AAV) vector.

7. The vector of claim 5 or 6, wherein the vector is in the form of a viral vector particle.

8. The vector of claim 7, wherein the AAV vector particle comprises an AAV2 or AAV8 genome, and an AAV2 or AAV8 capsid protein.

9. The vector of any one of claims 5 to 8, wherein the nucleotide sequence encoding CFI or FHL1 is operably linked to a CMV promoter; a WPRE regulatory element; and / or a poly-A signal.

10. A cell comprising the polynucleotide of any one of claims 1-4.

Images