Codon-Optimized Complement Factor I

Abstract

To provide a pharmaceutical composition for treating eye diseases such as AMD.SOLUTION: Disclosed herein are: an isolated polynucleotide comprising a nucleotide sequence encoding a codon-optimized Complement Factor I (CFI) which enables substantially increased expression of the CFI and FHL1 proteins compared to the wild type sequences; a vector comprising the polynucleotide; and use of them in the treatment or prevention of complement-mediated and complement-associated disorders, including eye diseases such as age-related macular degeneration (AMD).SELECTED DRAWING: None

Description

Technical Field

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

Background Art

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

[0003] Age-related macular degeneration (AMD) is the most common cause of functional blindness in people over 50 years old in developed countries (Seddon, J.M., Epidemiology of age-related macular degeneration. In: Ogden, T.E et al., eds., Ryan S.J., ed-in-chief. Retina Vol II. 3rd ed., St. Louis, Mo.: Mosby; 2001: 1039-1050). AMD is associated with angiogenesis that originates from the choroidal vasculature and extends into the subretinal space. Furthermore, AMD is characterized by the progressive degeneration of the retina, retinal pigment epithelium (RPE), and the underlying choroid (a vascular-rich tissue beneath the retina and sclera, under the RPE).

[0004] Various factors, including oxidative stress, inflammation with potential autoimmune components, genetic background (such as mutations), and environmental or behavioral factors such as smoking and diet, can be involved in the etiology of AMD.

[0005] The clinical progression of AMD is characterized by stages corresponding to changes in the macula. A feature of early AMD is the appearance of drusen, which are accumulations of extracellular debris beneath the retina and appear as yellow spots on the retina during clinical examination and on fundus photographs. Drusens are classified by size into small (<63 μm), medium (63–124 μm), and large (>124 μm). They are also considered hard or soft depending on the appearance of their edges on ophthalmic examination. Hard drusen have clearly defined edges, while soft drusen have less defined, fluid edges. The Age-Related Eye Disease Study (AREDS) fundus photographic severity scale is one of the primary classification systems used for this condition.

[0006] AMD is classified into "dry" and "wet" (exudative or neovascular) forms. Dry AMD is more common than wet AMD, but the dry form can progress to the wet form, and the two occur simultaneously in a significant number of cases. Dry AMD is typically characterized by progressive apoptosis of cells in the RPE layer, the photoreceptor cells above them, and often the underlying cells of the choroidal capillary layer. Confluent areas of RPE cell death with overlapping photoreceptor atrophy are called geographic atrophy. Patients with this form of AMD experience a slow, progressive deterioration of central vision.

[0007] Wet AMD is characterized by bleeding and / or leakage of fluid from abnormal blood vessels growing from the choroidal vessels (choroidal capillary plates) beneath the retina and macula, which can cause sudden vision loss. Much of the vision loss experienced by patients is presumed to be due to such choroidal neovascularization (CNV) and its secondary complications. A subtype of neovascular AMD is called retinal angiomatous proliferation (RAP), where the angiomatous proliferation begins in the retina, spreads posteriorly into the subretinal space, and eventually connects with new blood vessels in the choroid, if possible.

[0008] The complement system (CS) is involved in the early pathogenesis of AMD, based on the identification of CS components in drusen from the eyes of AMD patients. In AMD, at least 129 drusen-depositing proteins have been identified, including various apolipoprotein types (E, B, or A-I), several amyloid peptides (P, Aβ, or SA-1), TIMP-3, serum albumin, and specific proteins related to cellular function (e.g., ATP synthase β subunit, scavenger receptor B2, and retinol dehydrogenase). AMD-derived drusen also contain nearly all complement proteins, including regulatory proteins (CFH, complement receptor 1 (CR1), vitronectin, and clathelin), products of CS activation and degradation (C1q, C3, C3a, C3b, and C5a), and members of the terminal CS pathway, including MAC components in isolated complex forms (i.e., 5, 6, 8 (α, β, and γ) and 9). The accumulating drusen can activate CS, triggering local production of inflammatory mediators, attracting leukocytes, which in turn can exacerbate the local inflammatory state present in AMD.

[0009] Current treatment options for AMD include photodynamic therapy with benzoporphyrins (Arch Ophthalmol (1999) 117:1329-1345) and several therapies targeting the vascular endothelial growth factor (VEGF) pathway. Examples of such VEGF-targeted therapies include antibodies such as aptamer-pegaptanib (N Engl J Med (2004) 351:2805-2816), ranibizumab (N Engl J Med (2006) 355:1432-1444), and bevacizumab (BMJ (2010) 340:c2459). However, not all patients respond to treatment with anti-VEGF antibodies, and vision may not recover or may progress to certified blindness.

[0010] A therapy for treating geographic atrophy has been developed and used in a Phase III clinical trial. Lampalizumab is a humanized monoclonal inhibitory antibody against complement factor D, administered by intravitreal injection, which halts the progression of geographic atrophy. However, in a Phase III randomized clinical trial involving 906 participants, lampalizumab failed to reduce GA expansion compared to a 48-week sham.

[0011] Therefore, there is a critical need for new approaches in this field to treat eye diseases such as AMD.

[0012] Due to the ubiquity of the complement system, overactive or improperly functioning complement systems are associated with many chronic inflammatory conditions for which there are no treatment options or which require years of regular intervention to manage symptoms. Therefore, there is a general need to develop gene therapies that offer novel or alternative treatments for complement-mediated and complement-related disorders, particularly those associated with chronic inflammatory conditions, and especially those related to overactivity of the complement C3b feedback cycle (Figure 1). [Overview of the Initiative] [Problems that the invention aims to solve]

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

[0014] The improved CFI and FHL1 encoding sequences developed by the applicant enable higher doses of each protein to be delivered to the patient without increasing the amount of vector administered. The present invention therefore brings improvements in terms of production output (i.e., protein delivery can be achieved with a smaller amount of vector produced), drug efficacy, and safety. In particular, since higher doses of encoding proteins can be achieved with the delivery of 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, if the vector is delivered to the eye by subretinal injection, the risk of retinal damage or retinal detachment caused by the injection of a large amount of drug is reduced. Furthermore, the risk of off-target effects resulting from the diffusion of a large amount of drug into adjacent tissues is reduced. In addition, the use of the claimed nucleotide sequences in gene therapy has the potential to deliver therapy with a single dose, enabling long-term and stable expression of the protein and avoiding the need for monthly or regular injections. The nucleotide sequences and compositions of the present invention have additional advantages and therefore have the potential to provide a one-time or "single-dose" therapy that avoids repeated or regular surgical interventions.

[0015] In one embodiment, 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 with SEQ ID NO: 10.

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

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

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

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

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

[0021] In some embodiments, the AAV ITR is an AAV2 or AAV8 ITR. In a preferred embodiment, the AAV ITR is an AAV2 ITR.

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

[0023] In some embodiments, the vector is an adeno-associated virus (AAV), retrovirus, lentivirus, or adenovirus vector.

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

[0025] In some embodiments, the vector is in the form of virus vector particles.

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

[0027] In some embodiments, the AAV vector particles comprise an AAV2 or AAV8 capsid protein.

[0028] In some embodiments, the AAV vector particles comprise an AAV2 genome and an AAV2 capsid protein (AAV2 / 2). In other embodiments, the AAV vector particles comprise an AAV2 genome and an AAV-8 capsid protein (AAV2 / 8). In other embodiments, the AAV vector particles comprise an AAV8 genome and an AAV8 capsid protein (AAV8 / 8).

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

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

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

[0032] In a preferred embodiment, the nucleotide sequence encoding FHL1 is operably coupled to the CMV promoter, a WPRE regulatory element (preferably a WPRE3 regulatory element), and a bovine growth hormone poly(A) signaling pathway.

[0033] In another aspect, the present invention provides cells containing the polynucleotide of the present invention.

[0034] In another aspect, the present invention provides cells transduced with the vector of the present invention.

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

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

[0037] In certain embodiments, the pharmaceutical composition is suitable for topical administration (e.g., intra-arachnoid administration).

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

[0039] In another aspect, the present invention provides polynucleotides, vectors, or cells for use in therapeutic applications.

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

[0041] In preferred embodiments, the polynucleotides, vectors, or cells of the present invention are used to treat disorders associated with hyperactivity of the complement C3b feedback cycle.

[0042] In another aspect, the present invention provides polynucleotides, vectors, or cells for use in treating or preventing eye disorders.

[0043] In another aspect, the present invention provides polynucleotides, vectors, or cells for use in treating or preventing complement-mediated ocular disorders.

[0044] In another aspect, the present invention provides a method for treating or preventing complement-mediated disorders of the eye, comprising administering the polynucleotides, vectors, or cells of the present invention to a subject in need thereof.

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

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

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

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

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

[0050] In some embodiments, the subject is diagnosed with AMD or is at risk of developing AMD.

[0051] In some embodiments, the use is for treating or preventing disorders in the following subjects: (a) Having lower-than-normal complement factor I activity or concentration in the eye and / or serum, preferably with serum concentrations of 0-30, 0-20, or 0-10 μg / mL or equivalent activity; and / or (b) Age-related macular degeneration (AMD)-associated SNPs, preferably heterozygous or homozygous for rare complement factor I variants.

[0052] In some embodiments, the use is for treating or preventing disorders in the following subjects: (a) Having normal levels of complement factor I activity or concentration in the eyes and / or serum, preferably at least 30 μg / mL, for example, 30-40 μg / mL in serum; and / or (b) Does not carry a rare complement factor I mutation allele.

[0053] In another embodiment, the present invention provides polynucleotides, vectors, or cells for use in treating or preventing age-related macular degeneration (AMD). In a preferred embodiment, the AMD is dry AMD.

[0054] In another aspect, the present invention provides polynucleotides, vectors, or cells for use in treating or preventing diabetic retinopathy.

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

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

[0057] In some embodiments, the increase in geographic atrophy area is reduced by at least 10% over 12 months after administration to the treated eye of the subject, compared to an untreated eye for the same period. In other embodiments, the increase in geographic atrophy area is reduced by at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% over 12 months after administration to the treated eye of the subject, compared to an untreated eye for the same period.

[0058] In some embodiments, administration of polynucleotides, vectors, or cells increases the level of C3b inactivation and iC3b degradation activity in a subject or in the eye, such as in the retinal pigment epithelium (RPE) of the subject, to a level optionally exceeding normal levels in the subject or its eye or RPE.

[0059] In another aspect, the present invention provides polynucleotides, vectors, or cells for use in improving or restoring vision or visual acuity in subjects suffering from eye disorders, such as the eye disorders disclosed herein. In another aspect, the present invention provides polynucleotides, vectors, or cells for use in mitigating vision or visual acuity loss associated with eye disorders, such as the eye disorders disclosed herein.

[0060] In another aspect, the present invention provides polynucleotides, vectors, or cells for use in improving or restoring reading speed in subjects suffering from eye disorders, such as the eye disorders disclosed herein. In another aspect, the present invention provides polynucleotides, vectors, or cells for use in mitigating a decrease in reading speed in subjects, such as a decrease in reading speed associated with an eye disorder, such as the eye disorders disclosed herein.

[0061] In another aspect, the present invention provides polynucleotides, vectors, or cells for use in reducing or preventing photoreceptor loss and / or retinal pigment epithelium (RPE), such as RPE associated with eye disorders, including photoreceptor loss and / or eye disorders as disclosed herein.

[0062] In some embodiments, polynucleotides, vectors, or cells are administered intraocularly.

[0063] In some embodiments, polynucleotides, vectors, or cells are administered to the target eye by subretinal, direct retinal, choroidal, or intravitreal injection.

[0064] In some embodiments, polynucleotides, vectors, or cells are administered to the target eye by subretinal injection.

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

[0066] In some embodiments, the vectors of the present invention do not contain the AAV2 genome and AAV8 capsid protein; that is, the vectors of the present invention are not AAV2 / 8 vectors.

[0067] In some embodiments, the polynucleotides, vectors, or cells of the present invention are not administered systemically. In other embodiments, the polynucleotides, vectors, or cells of the present invention are not administered intravenously. [Brief explanation of the drawing]

[0068] [Figure 1] The C3b feedback (amplification) and degradation (downregulation) cycle of the alternative complement pathway in vertebrates ("I" = complement factor I; "H" = complement factor H; "B" = complement factor B; and "D" = complement factor D). [Figure 2] Western blot analysis of supernatants from codon-optimized CFI and FHL1 plasmid transfections of ARPE19 cells. [Figure 3] ELISA analysis of supernatant from codon-optimized CFI plasmid transfection of ARPE19 cells. [Figure 4] ELISA analysis of supernatant from codon-optimized FHL1 plasmid transfection of ARPE19 cells. [Figure 5] ELISA analysis of the supernatant from transduction of ARPE19 cells with codon-optimized CFI AAV vectors. [Figure 6] ELISA analysis of the supernatant from transduction of ARPE19 cells with a codon-optimized FHL1 AAV vector. [Modes for carrying out the invention]

[0069] As used herein, the terms “comprising,” “comprises,” and “comprised of” are synonymous with “including” or “includes,” or “containing” or “contains,” and are comprehensive or open-ended, not excluding additional, uncited members, elements, or steps. The terms “comprising,” “comprises,” and “comprised of” also include the term “consisting of.” complement system

[0070] The complement system is an essential part of the humoral immune system and is involved in tissue inflammation, cellular opsonization, and cytolysis. It provides protection against microorganisms and mediates the clearance of exogenous and endogenous cellular debris from host tissues.

[0071] The complement system cascade consists of four activation pathways. All pathways ultimately terminate in the central cleavage of the C3 factor and the generation of its active fragments C3a and C3b. C3a is an anaphylatoxin that elicits various chemotactic and pro-inflammatory responses, such as the recruitment of inflammatory cells and increased permeability of the microvascular system, while C3b is responsible for the opsonization of foreign surfaces covalently bound to C3b. Opsonization by the activated C3 fragments (C3b and iC3b) performs three main functions: (i) removal of cellular debris by phagocytic cells (e.g., macrophages or microglia) and stimulation of the adaptive immune system (B cells and T cells), (ii) amplification of complement activation via the formation of surface-bound C3 convertases, and (iii) assembly of C5 convertases.

[0072] The assembly of C5 convertases is responsible for C5 cleavage, which leads to the formation of cell-lysic membrane invasion complexes (MACs) that can create perforations in the cell membrane, thereby promoting cell lysis and the elimination of unwanted cells. Through all of these activities, the innate complement cascade supports and promotes the function of downstream immune system mechanisms that protect the integrity of host tissues. As a whole, activation of the complement pathway results in a pro-inflammatory response that includes the generation of MACs that mediate cell lysis, the release of chemokines to attract inflammatory cells to the site of injury, and increased capillary permeability to promote extravasation of infiltrating leukocytes. Under physiological conditions, complement activation is effectively regulated by the coordinated action of soluble and membrane-bound complement regulatory molecules (CRMs). Soluble complement regulatory factors such as C1 inhibitors, anaphylatoxin inhibitors, C4b-binding protein (C4BP), complement factor H (CFH), complement factor I (CFI), clathelin, and vitronectin limit the action of complement in human tissues at multiple sites in the cascade reaction. Furthermore, each individual cell is protected from homologous complement attack by surface proteins such as complement receptor 1 (CR1, CD35), membrane cofactor protein (CD46), and glycosylphosphatidylinositol-anchored proteins such as the catalytic complement (CD55) or CD59 molecule. Notably, host cells and tissues that are inadequately protected from complement attack may undergo bystander cell lysis.

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

[0074] In some embodiments, prior to administration of the polynucleotide, vector, cell, or pharmaceutical composition of the present invention, the subject has low levels (e.g., lower than normal) of complement factor I activity, e.g., low levels of complement factor I activity in the eyes and / or low levels of complement factor I activity in serum. Lower-than-normal levels of complement factor I activity may result from lower-than-normal expression of normally functioning complement factor I, or at least partial (e.g., heterozygous) expression (normal or lower-than-normal levels) of a non-functional or subfunctional variant of complement factor I (such a subject may harbor one or more copies of an AMD-associated SNP, e.g., the subject may be homozygous or heterozygous for one of the rare complement factor I variants further described below). Thus, the subject may have low concentrations (e.g., lower than normal) of complement factor I in the eyes and / or serum. In human subjects, normal levels of complement factor I activity (activation of C3b inactivation and iC3b degradation) may be equivalent to those provided by 30–40 μg / mL of complement factor I in the subject's serum. Therefore, in subjects with low complement factor I activity, serum complement factor I activity may correspond to complement factor I levels below 30 μg / mL and above 0 μg / mL (e.g., 0–20 or 0–10 μg / mL) (these are ranges of serum complement factor I concentrations, which may include subjects with low concentrations of complement factor I).

[0075] Therefore, subjects treated by the present invention may suffer from or be at risk of developing complement-mediated ocular disorders such as AMD, more specifically dry AMD (e.g., characterized by geographic atrophy). For example, subjects may be homozygous or heterozygous and sensitive to one or more SNPs associated with complement-mediated disorders.

[0076] In some embodiments, subjects are at risk of developing AMD. For example, subjects may be homozygous or heterozygous for one or more SNPs associated with AMD, such as rare mutations in complement factor I that are associated with progressive AMD and generally result in decreased serum complement factor I levels (Kavanagh et al., (2015) Hum Mol Genet 24:3861-3870). In particular, subjects may possess one or two copies of one or more of the following rare complement factor I variants: rs144082872 (encodes P50A); 4:110687847 (encodes P64L); rs141853578 (encodes G119R); 4:110685721 (encodes V152M); 4:110682846 (encodes G162D); 4:110682801 (encodes N177I); rs146444258 (encodes A240G); rs182078921 (encodes G287R); rs41278047 (encodes K441R); and rs121964913 (encodes R474).

[0077] The present invention may further include determining whether a subject is at risk of developing a complement-mediated disorder (e.g., AMD) by determining whether the subject is homozygous or heterozygous susceptible to one or more SNPs associated with complement-mediated disorders (e.g., by determining whether the subject is homozygous or heterozygous susceptible to one or more rare complement factor I variants associated with the above-mentioned AMD).

[0078] Alternatively, the subjects may have normal levels of endogenous complement factor I activity or concentration, for example, in the eyes and / or serum, and / or may not possess rare variant complement factor I alleles.

[0079] In some embodiments, administration of the polynucleotides, vectors, cells, or pharmaceutical compositions of the present invention thereby increases the level of activity that inactivates C3b and degrades iC3b in the eye of a subject. In other embodiments, administration of the polynucleotides, vectors, cells, or pharmaceutical compositions of the present invention thereby increases the level of activity that inactivates C3b and degrades iC3b in the eye of a subject to a level above the normal level in the eye. More specifically, the level of activity that inactivates C3b and degrades iC3b increases in the RPE of the eye.

[0080] The activity of C3b inactivation and iC3b degradation in a subject after expression of complement factor I from a polynucleotide or vector according to the present invention may include the activity of C3b inactivation and iC3b degradation from the subject's endogenous complement factor I (i.e., complement factor I of the subject not produced by expression from a polynucleotide or vector), as well as the activity of C3b inactivation and iC3b degradation produced by expression from a polynucleotide or vector according to the present invention, thereby exceeding the normal level of the total level of C3b inactivation and iC3b degradation activity in the subject.

[0081] In some embodiments, the level of activity for inactivating C3b and degrading iC3b in a subject, for example in the eye, is increased to a level at least 5%, 10%, 15%, 20%, or 25% higher than the normal level.

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

[0083] For example, the level of activity for inactivating C3b and degrading iC3b in a subject, such as the eye, can be increased to levels of 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%.

[0084] In some embodiments, administration of the polynucleotides, vectors, cells, or pharmaceutical compositions of the present invention does not detectably increase the level of activity that inactivates C3b and degrades iC3b in the plasma / serum of the subject. In other embodiments, administration of the polynucleotides, vectors, cells, or pharmaceutical compositions of the present invention does not detectably increase the level of activity that inactivates C3b and degrades iC3b in the plasma / serum of the subject to levels higher than normal.

[0085] In the previous section, unless clearly inapplicable, references to the activity of inactivating complement factor I and C3b and degrading iC3b may be replaced with the ability of complement factor H or complement factor H-like protein 1, and complement factor I, to act as cofactors for C3b cleavage mediated by complement factor I and to increase the dissociation rates of C3 convertase and C5 convertase, respectively. In some embodiments, prior to administration of the polynucleotide, vector, cell, or pharmaceutical composition of the present invention, the subject has low levels (e.g., lower than normal levels) of complement factor H, for example, low levels of complement factor H in the eye and / or low serum levels of complement factor H. In human subjects, normal levels of complement factor H may be about 200-500 μg / mL in the subject's serum. Therefore, in subjects with low levels of complement factor H, serum levels may be less than 200 μg / mL and greater than 0 μg / mL, for example, 0-100 μg / mL. Alternatively, the subject may have normal levels of endogenous complement factor H, for example, in the eyes and / or serum. Complement Factor I (CFI)

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

[0087] Complement factor I is a serine protease that circulates in a zymogen-like state at a concentration of approximately 35 μg / mL (Roversi et al., (2011) PNAS 108:12839-12844) (Nilsson et al., (2011) Mol Immunol 48:1611-1620). The complement factor I protein is a highly N-glycosylated heterodimer consisting of two polypeptide chains linked by a single disulfide bond. The heavy chain (50 kDa) includes an N-terminal region; a FI membrane invasion complex (FIMAC) domain; a CD5-like domain or a scavenger receptor cysteine-rich (SRCR) domain; two low-density lipoprotein receptor (LDLr) domains; and a C-terminal region of unknown function that is a site of interspecies sequence diversity (Roversi et al., (2011) PNAS 108:12839-12844). The light chain (38 kDa) contains a serine protease (SP) domain with a conserved catalytic residue (Goldberger et al., (1987) J Biol Chem 262:10065-10071).

[0088] Complement factor I inactivates C3b by cleaving it into iC3b, C3d, and C3d,g, and similarly inactivates C4b by cleaving it into C4c and C4d. To properly perform its function, complement factor I requires the presence of cofactor proteins such as C4b-binding protein (C4BP), complement factor H (CFH), complement receptor 1 (CR1 / CD35), and membrane cofactor protein (MCP / CD46) (Degn et al., (2011) Am J Hum Genet 88:689-705).

[0089] iC3b cannot bind to factor B, and therefore cannot sustain amplification of the complement cascade or activation via an alternative pathway. Consequently, when C3b is cleaved by iC3b, neither the initiation of an alternative pathway nor activation of the terminal complement cascade occurs.

[0090] iC3b can exert pro-inflammatory effects by binding to and activating complement receptor 3 (CR3) (CD11b / CD18) on mononuclear phagocytic cells such as polymorphonuclear leukocytes (mainly neutrophils), NK cells, and macrophages.

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

[0092] The unique ability of complement factor I to promote the cleavage of C3b to iC3b and accelerate the degradation of iC3b, combined with its relatively low concentration in human serum, which influences the amount that needs to be delivered for therapeutic effect, makes it a particularly advantageous target.

[0093] In some embodiments, complement factor I polypeptides can cleave C3b into inactive degradation products. For example, complement factor I polypeptides can cleave C3b into iC3b.

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

[0095] In a preferred embodiment, the complement factor I polypeptide can cleave C3b to iC3b and process iC3b to C3d,g.

[0096] Preferably, a fragment or derivative of complement factor I may inactivate at least 50%, 60%, 70%, 80%, 90%, 95%, or 100% of the C3b of native complement factor I and retain iC3b degradation activity.

[0097] The activity of complement factor I, or its fragments or derivatives, inactivating C3b and degrading iC3b can be determined using any suitable method known to those skilled in the art. For example, the measurement of the proteolytic activity of complement factor I is described by Hsiung et al. (Biochem. J. (1982) 203:293-298). Both hemolytic and agglutinative assays of CFI activity are described by Lachmann PJ & Hobart MJ (1978) “Complement Technology” (Handbook of Experimental Immunology, 3rd edition, DM Weir Blackwells Scientific Publications Chapter 5A p17). A more detailed description, including proteolytic assays, is provided by Harrison RA (1996) in “Weir's Handbook of Experimental Immunology” (5th edition, edited by Herzenberg Leonore A'Weir DM, Herzenberg Leonard A & Blackwell C Blackwells Scientific Publications Chapter 75 36-37). Agglutination assays are highly sensitive and can be used to detect both initial (double) clips that convert immobilized C3b to iC3b and acquire reactivity with conglutinin, and to detect the final clip to C3dg by starting with immobilized iC3b and looking for loss of reactivity with conglutinin. Hemolysis assays are used for C3b to iC3b conversion, and proteolysis assays detect all clips.

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

[0099] An example of a human complement factor I protein is the human complement factor I protein with UniProtKB accession number P05156. This exemplary sequence is 583 amino acids long (disclosed as SEQ ID NO: 1), and amino acids 1-18 form a signal sequence.

[0100] In some embodiments, the amino acid sequence of complement factor I is sequence number 1. In other embodiments, the amino acid sequence of complement factor I is the sequence disclosed as positions 19 to 583 of sequence number 1. [ka] (Sequence ID 1)

[0101] In some embodiments, the amino acid sequence of complement factor I is sequence number 9, which corresponds to NCBI accession number NP_000195. In other embodiments, the amino acid sequence of complement factor I is the sequence disclosed as positions 19-583 of sequence number 9. [ka] (Sequence ID 9)

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

[0103] The nucleotide sequence of complement factor I used in this invention is preferably codon-optimized. Different cells use their particular codons differently. This codon bias corresponds to the bias in the relative abundance of a particular tRNA in a given cell type. Expression can be increased by modifying codons in the sequence so that they are adjusted to match the relative abundance of the corresponding tRNA. Similarly, expression can be decreased by deliberately selecting codons for which the corresponding tRNA is known to be rare in a given cell type. Thus, additional translational control is available.

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

[0105] In some embodiments, the nucleotide sequence encoding complement factor I has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homology to SEQ ID NO: 10. Preferably, the protein encoded by the nucleotide sequence substantially retains the functional activity of the protein represented by SEQ ID NO: 1 or 9.

[0106] In some embodiments, the nucleotide sequence encoding complement factor I is sequence number 10.

[0107] In other embodiments, the nucleotide sequence encoding complement factor I has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with positions 55-1752 of SEQ ID NO: 10. Preferably, the protein encoded by the nucleotide sequence substantially retains the functional activity of the protein represented by SEQ ID NO: 1 or 9.

[0108] In another embodiment, the nucleotide sequence encoding complement factor I is located at positions 55-1752 of sequence number 10.

[0109] A further exemplary codon-optimized nucleotide sequence encoding complement factor I is sequence number 8. [ka] (Sequence 8)

[0110] An advantage of the present invention is that it is particularly difficult to prepare complement factor I in the form of a purified protein. Therefore, the inventors have devised a method to modulate the complement system to enable the treatment of, for example, age-related macular degeneration (AMD) by administering complement factor I in the form of an AAV vector containing a nucleotide sequence encoding complement factor I. The AAV vector can be administered to a target site, for example, the eye, to enable in situ translation of the complement factor I polypeptide. Complement factor H (CFH)

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

[0112] Complement factor H is a large (155 kDa) soluble glycoprotein that is typically present in human plasma at concentrations of 200–300 μg / mL (Hakobyan et al., (2008) 49(5):1983-90). The primary function of complement factor H is to regulate alternative pathways in the complement system.

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

[0114] Complement factor H is composed of 20 complement regulatory protein (CCP) modules (also called short consensus repeats or sci domains), interconnected by short linkers (3-8 amino acid residues) and arranged in an extended head-to-tail configuration. Each CCP module consists of approximately 60 amino acids with four cysteine ​​disulfide residues linked in a 1-3 2-4 arrangement, and a hydrophobic core built around a nearly constant tryptophan residue. The CCP modules are numbered 1-20 (from the N-terminus of the protein). CCP1-4 and CCP19-20 bind to C3b, while CCP7 and CCP19-20 bind to GAG and sialic acid (Schmidt et al., (2008) Journal of Immunology 181:2610-2619).

[0115] Gene therapy using complement factor H has been shown to improve induced AMD-like symptoms in mice (Cashman et al., (2015) J. Gene Med. 17:229-243). The following were co-injected subretinally into mice: (i) an adenovirus vector expressing complement component C3, which has been previously shown to replicate many pathological features of human AMD; and (ii) an adenovirus vector expressing complement factor H. Compared to control animals receiving GFP instead of complement factor H, mice transduced with complement factor H showed a 91% reduction in endothelial cell proliferation and a 69% reduction in RPE atrophy. Electroretinography showed improved retinal function in mice administered with complement factor H, and immunocytochemistry of rhodopsin and RPE65 was consistent with photoreceptor and RPE rescue in such animals.

[0116] In some embodiments, complement factor H polypeptides or fragments or derivatives thereof can act as cofactors for complement factor I-mediated C3b cleavage. In some embodiments, complement factor H polypeptides or fragments or derivatives thereof can increase the dissociation rates of C3 and C5 convertases.

[0117] In preferred embodiments, complement factor H polypeptides or fragments or derivatives thereof can act as cofactors for complement factor I-mediated C3b cleavage and can increase the dissociation rates of C3 convertases and C5 convertases.

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

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

[0120] In some embodiments, the amino acid sequence of complement factor H is that of SEQ ID NO: 3. In other embodiments, the amino acid sequence of complement factor H is that of positions 19-1231 of SEQ ID NO: 3. [ka] (Sequence ID 3)

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

[0122] In some embodiments, the nucleotide sequence encoding complement factor H is sequence number 4. [ka] TIFF0007833489000008.tif112161 (Sequence ID 4)

[0123] In some embodiments, the nucleotide sequence encoding complement factor H has at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 4. Preferably, the protein encoded by the nucleotide sequence substantially retains the functional activity of the protein represented by SEQ ID NO: 3.

[0124] In another embodiment, the nucleotide sequence encoding complement factor H is sequence number 4.

[0125] In other embodiments, the nucleotide sequence encoding complement factor H has at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with positions 55-3696 of SEQ ID NO: 4. Preferably, the protein encoded by the nucleotide sequence substantially retains the functional activity of the protein represented by SEQ ID NO: 3.

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

[0127] In other embodiments, the nucleotide sequence encoding complement factor H encodes an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 3. Preferably, the amino acid sequence substantially retains the functional activity of the protein represented by SEQ ID NO: 3.

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

[0129] In other embodiments, the nucleotide sequence encoding complement factor H encodes an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, and 99% identity with positions 19-1231 of SEQ ID NO: 3. Preferably, the amino acid sequence substantially retains the functional activity of the protein represented by SEQ ID NO: 3.

[0130] In another embodiment, the nucleotide sequence encoding complement factor H encodes the amino acid sequence from positions 19 to 1231 of SEQ ID NO: 3. Complement factor H-like protein 1 (FHL1)

[0131] Complement factor H-like protein 1 (FHL1) is a splice variant of complement factor H that contains the first seven CCPs of complement factor H followed by a carboxyl-terminal tail of four amino acids (Clark, SJ et al., (2015) J Clin Med 4:18-31).

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

[0133] In some embodiments, the amino acid sequence of FHL1 is sequence number 11. [ka] (Sequence ID 11)

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

[0135] The preferred nucleotide sequence encoding FHL1 is sequence number 12. [ka] (Sequence ID 12)

[0136] In some embodiments, the nucleotide sequence encoding FHL1 has at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 12. Preferably, the protein encoded by the nucleotide sequence substantially retains the functional activity of the protein represented by SEQ ID NO: 11.

[0137] In another embodiment, the nucleotide sequence encoding FHL1 is sequence number 12. Polynucleotides

[0138] The polynucleotides of the present invention may include DNA or RNA, preferably DNA. They may be single-stranded or double-stranded. Those skilled in the art will understand that, as a result of the degeneracy of the genetic code, many different polynucleotides may encode the same polypeptide. Furthermore, those skilled in the art will understand that, using conventional techniques, nucleotide substitutions that do not affect the polypeptide sequence encoded by the polynucleotides of the present invention may be made to reflect the codon usage frequency of any particular host organism in which the polypeptide of the present invention is expressed.

[0139] Polynucleotides can be modified by any method available in the art. Such modifications may be carried out to enhance the in vivo activity or extend the lifespan of the polynucleotides of the present invention.

[0140] Polynucleotides, such as DNA polynucleotides, can be produced by recombination, synthesis, or any means available to those skilled in the art. They may also be cloned by standard methods.

[0141] Longer polynucleotides will generally be produced using recombination methods, for example, polymerase chain reaction (PCR) cloning techniques. This involves creating a pair of primers (e.g., about 15–30 nucleotides) adjacent to the target sequence to be cloned, contacting the primers with mRNA or cDNA obtained from animal or human cells, performing a polymerase chain reaction under conditions that result in amplification of the desired region, isolating the amplified fragment (e.g., by purifying the reaction mixture on an agarose gel), and recovering the amplified DNA. The primers may be designed to contain suitable restriction enzyme recognition sites so that the amplified DNA can be cloned into a suitable vector. Structure of the eye

[0142] The agents disclosed herein may be delivered to the eyes of mammals, preferably humans, in connection with the treatment or prevention of eye diseases such as age-related macular degeneration (AMD).

[0143] Those skilled in the art of treating eye diseases will have a detailed and complete understanding of the structure of the eye. However, the following structure is particularly relevant to the present invention. retina

[0144] The retina is a multilayered structure that lines the inside of the posterior chamber of the eye and senses the image of the visual world transmitted to the brain via the optic nerve. From the inside to the outside of the eye, the retina consists of layers of the neurosensory retina and retinal pigment epithelium, while the choroid is located outside the retinal pigment epithelium. Neurosensory retina and photoreceptor cells

[0145] The neurosensory retina contains photoreceptor cells that directly sense light. The neurosensory retina consists of the following layers: internal limiting membrane (ILM); nerve fiber layer; ganglion cell layer; internal plexiform layer; internal granular layer; external plexiform layer; external granular layer (nuclei of photoreceptors); external limiting membrane (ELM); and photoreceptors (medial and lateral segments of rods and cones).

[0146] Those skilled in the art will have a detailed understanding of photoreceptor cells. In short, photoreceptor cells are specialized neurons located within the retina that convert light into biological signals. Photoreceptor cells include rod cells and cone cells, and are distributed differently throughout the retina.

[0147] Rod cells are primarily distributed on the outer retina. These cells are highly sensitive and provide vision at low light levels. A normal human retina contains an average of about 125 million rod cells.

[0148] Cone cells are found throughout the retina, but are particularly concentrated in the fovea, the depression of the neurosensory retina responsible for central high-resolution vision. Cone cells are less sensitive than rod cells. A normal human retina contains an average of 6 to 7 million cone cells. retinal pigment epithelium

[0149] The retinal pigment epithelium (RPE) is a layer of pigment cells located just outside the neurosensory retina. The RPE performs multiple functions, including the transport of nutrients and other substances to photoreceptor cells and the absorption of scattered light to improve vision. choroid

[0150] The choroid is a vascular layer located between the retina pleoplasm (RPE) and the outer sclera of the eye. The choroidal vascular system enables the supply of oxygen and nutrients to the retina. Age-related macular degeneration (AMD)

[0151] The clinical progression of age-related macular degeneration (AMD) is characterized by stages corresponding to changes in the macula. A feature of early AMD is the appearance of drusen, which are accumulations of extracellular debris beneath the retina and appear as yellow spots on the retina during clinical examination and on fundus photographs. Drusens are classified by size into small (<63 μm), medium (63–124 μm), and large (>124 μm). They are also considered hard or soft depending on the appearance of their edges on ophthalmic examination. Hard drusen have clearly defined edges, while soft drusen have less defined, fluid edges. The Age-Related Eye Disease Study (AREDS) fundus photographic severity scale is one of the primary classification systems used for this condition.

[0152] AMD is classified into "dry" and "wet" (exudative or neovascular) forms. Dry AMD is more common than wet AMD, but the dry form can progress to the wet form, and the two occur simultaneously in a significant number of cases. Dry AMD is typically characterized by progressive apoptosis of cells in the RPE layer, the photoreceptor cells above them, and often the underlying cells of the choroidal capillary layer. Confluent areas of RPE cell death with overlapping photoreceptor atrophy are called geographic atrophy (GA). Patients with this form of AMD experience a slow, progressive deterioration of central vision.

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

[0154] The treatments or preventive measures for AMD described herein may reduce or prevent the occurrence of the above-described AMD phenotype. Preferably, the treatment of AMD allows for the maintenance or improvement of visual function.

[0155] In some embodiments, treatment or prevention of AMD results in prevention or reduction of geographic atrophy formation. In other embodiments, treatment or prevention of AMD slows the progression of geographic atrophy. For example, it results in a reduction of at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the increase in GA area over 12 months after administration to the treated eye of the subject, compared to an untreated eye for the same period. In other embodiments, treatment or prevention of AMD results in treatment of geographic atrophy, e.g., a reduction in the amount of geographic atrophy.

[0156] In some embodiments, treatment or prevention of AMD results in the prevention or reduction of drusen formation. In other embodiments, treatment or prevention of AMD results in a reduction of existing drusen, e.g., a reduction in the size and / or number of existing drusen.

[0157] In some embodiments, treatment or prevention of AMD results in prevention or reduction of complement deposition. In other embodiments, treatment or prevention of AMD results in a reduction of existing complement deposition.

[0158] In some embodiments, the treatment or prevention of AMD results in an improvement or restoration of vision or visual acuity. In other embodiments, the treatment or prevention of AMD reduces vision or visual acuity loss.

[0159] In some embodiments, treating or preventing AMD results in an improvement or recovery of the target's read speed. In other embodiments, treating or preventing AMD results in a reduction of the target's read speed degradation.

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

[0161] Diabetic retinopathy is a condition characterized by damage to the blood vessels in the retina caused by high blood sugar levels associated with diabetes. If left untreated, diabetic retinopathy can lead to blindness.

[0162] While patients with mild diabetic retinopathy may have good vision, two types of diabetic retinopathy, namely diabetic macular edema (DMO) and proliferative diabetic retinopathy (PDR), can threaten their visual acuity.

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

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

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

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

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

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

[0169] AAV vectors may contain the AAV genome or fragments or derivatives thereof.

[0170] AAV is known to be able to package genomes up to 5.2kb in size (Dong, J.-Y. et al., (1996) Human Gene Therapy 7:2101-2112).

[0171] The AAV genome is a polynucleotide sequence that can encode functions necessary for the generation of AAV particles. These functions include those manipulated in the AAV replication and packaging cycle in host cells, including the capsid formation of the AAV genome onto the AAV particle. Spontaneously occurring AAVs are replication-deficient and rely on the provision of helper functions in trans to complete the replication and packaging cycle. Therefore, the AAV genome of the AAV vector of the present invention is typically replication-deficient.

[0172] AAV genomes can be either positive-sense or negative-sense, single-stranded, or double-stranded. The use of double-stranded genomes allows for bypassing the DNA replication step in target cells, thereby promoting transgene expression.

[0173] The AAV genome can originate from any naturally occurring serotype, isolate, or clade of AAV. Therefore, the AAV genome can be the entire genome of a naturally occurring AAV. As is well known to those skilled in the art, naturally occurring AAVs can be classified according to various biological systems.

[0174] Generally, AAVs are referred to in terms of their serotypes. Serotypes correspond to variant subtypes of AAV, and they have a unique reactivity that can be used to distinguish them from other variant subtypes based on the expression profile of their capsid surface antigens. Typically, viruses with a particular AAV serotype do not efficiently cross-react with neutralizing antibodies specific to any other AAV serotype.

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

[0176] In some embodiments, the AAV vector particles are AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, Rec2, or Rec3AAV vector particles.

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

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

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

[0180] The capsid protein may be a mutant capsid protein, such as those disclosed in International Publication No. 2008 / 124724, which is incorporated herein by reference.

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

[0182] Reviews of AAV serotypes can be found in Choi et al. (2005) Curr. Gene Ther. 5:299-310 and Wu et al. (2006) Molecular Therapy 14:316-27. The sequences of the AAV genome for use in this invention, or the sequences of elements of the AAV genome such as ITR sequences, rep genes, or cap genes, can be derived from the following accession numbers for the whole AAV genome sequence: adeno-associated virus 1 NC_002077, AF063497; adeno-associated virus 2 NC_001401; adeno-associated virus 3 NC_001729; adeno-associated virus 3B NC_001863; adeno-associated virus 4 NC_001829; adeno-associated virus 5 Y18065, AF085716; adeno-associated virus 6 NC_001862; avian AAV ATCC VR-865 AY186198, AY629583, NC_004828; avian AAV strain DA-1 NC_006263, AY629583; bovine AAV NC_005889, AY388617.

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

[0184] Those skilled in the art can, based on general knowledge, select an appropriate serotype, clade, clone, or isolate of AAV for use in the present invention. For example, the AAV5 capsid has been shown to efficiently transduce cone photoreceptors in primates, as evidenced by its successful correction of hereditary color blindness (Mancuso et al., (2009) Nature 461:784-7).

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

[0186] Typically, the AAV genome of a naturally occurring serotype, isolate, or clade of AAV contains at least one reverse-ended repeat sequence (ITR). The ITR sequence acts cis-in to provide a functional origin for replication, enabling vector incorporation and excision from the cell's genome. In preferred embodiments, one or more ITR sequences are adjacent to nucleotide sequences encoding complement factor I or FHL1. The AAV genome also typically contains packaging genes, such as rep genes and / or cap genes, which encode the packaging function of AAV particles. Rep genes encode one or more of the proteins Rep78, Rep68, Rep52, and Rep40 or their variants. Cap genes encode one or more capsid proteins, such as VP1, VP2, and VP3 or their variants. These proteins constitute the capsid of the AAV particle. Capsid variants are described below.

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

[0188] As described above, the AAV genome used in the AAV vector of the present invention can therefore be a complete genome of naturally occurring AAV. For example, an AAV vector or vector particle may be prepared in vitro using a vector containing a complete AAV genome. However, although such a vector can, in principle, be administered to a patient, this will rarely be done in practice. Preferably, the AAV genome will be derivatized for the purpose of administration to a patient. Such derivatization is standard in the art, and the present invention encompasses the use of any known derivative of the AAV genome and derivatives that can be produced by applying techniques known in the art. Derivatization of AAV genomes and AAV capsids is outlined in Coura and Nardi (2007) Virology Journal 4:99 and Choi et al., and Wu et al. (see above).

[0189] Derivatives of the AAV genome include any truncated or modified form of the AAV genome that enables the expression of a transgene from the AAV vector of the present invention in vivo. Typically, the AAV genome can be significantly truncated to contain a minimal viral sequence while still retaining the above-described functions. This is preferable for safety reasons, as it reduces the risk of vector recombination with wild-type virus and avoids the induction of a cellular immune response due to the presence of viral gene proteins in target cells.

[0190] Typically, derivatives will contain at least one reverse-terminal repeat (ITR), preferably two or more ITRs, for example, two or more ITRs. One or more of the ITRs may originate from AAV genomes with different serotypes, or they may be chimeric or mutant ITRs. Preferred mutant ITRs are those with deletions of trs (terminal degradation sites). This deletion allows for continued replication of the genome, generating a single-stranded genome containing both coding and complementary sequences, i.e., a self-complementary AAV genome. This allows for bypassing DNA replication in target cells, thus promoting the expression of the transgene.

[0191] Preferably, one or more ITRs will be adjacent to a nucleotide sequence encoding complement factor I or FHL1 at one of their ends. The inclusion of one or more ITRs is preferable, for example, to facilitate concatemer formation of the vector of the present invention in the nucleus of the host cell after conversion of the single-stranded vector DNA to double-stranded DNA by the action of host cell DNA polymerase. The formation of such an episomal concatemer protects the vector construct for the lifetime of the host cell, thereby enabling long-term expression of the transgene in vivo.

[0192] In preferred embodiments, the ITR element is the only sequence retained from the native AAV genome in the derivative. Therefore, the derivative preferably does not contain the rep gene and / or cap gene of the native genome, nor any other sequences of the native genome. This is preferred for the reasons mentioned above, and also to reduce the likelihood of the vector being integrated into the host cell genome. Furthermore, reducing the size of the AAV genome allows for greater flexibility in incorporating other sequence elements (such as regulatory elements) into the vector, in addition to the transgene.

[0193] Therefore, the following portions may be removed in the derivatives of the present invention: one reverse terminal repeat (ITR) sequence, a replication (rep) gene, and a capsid (cap) gene. However, in some embodiments, the derivatives may further include one or more rep genes and / or cap genes or other viral sequences of the AAV genome. Spontaneously occurring AAV integrates at a high frequency at specific sites on human chromosome 19, exhibiting only a very small random integration frequency; therefore, the retention of integration ability in the vector may be acceptable in a therapeutic setting.

[0194] If the derivative includes 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. In particular, the present invention encompasses providing capsid protein sequences from different serotypes, clades, clones, or isolates of AAV within the same vector (i.e., a pseudotype vector).

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

[0196] Chimeric capsid proteins include those generated by recombination between two or more capsid coding sequences of naturally occurring AAV serotypes. This may be carried out, for example, by a marker-rescue approach in which a non-infectious capsid sequence of one serotype is co-transfected with a capsid sequence of a different serotype, and a capsid sequence with the desired properties is selected using indicated selection. The capsid sequences of the different serotypes can be modified in the cell by homologous recombination to generate a novel chimeric capsid protein.

[0197] Chimeric capsid proteins also include those produced by manipulating the capsid protein sequence to transfer specific capsid protein domains, surface loops, or specific amino acid residues between two or more capsid proteins, for example, between two or more capsid proteins of different serotypes.

[0198] Shuffled or chimeric capsid proteins can also be generated by DNA shuffling or error-prone PCR. Hybrid AAV capsid genes can be created by randomly fragmenting the sequences of relevant AAV genes, for example those encoding capsid proteins of multiple different serotypes, and then reconstructing the fragments with a self-priming polymerase reaction, which can also result in crossovers in regions of sequence homology. Libraries of hybrid AAV genes thus created by shuffling several serotype capsid genes can be screened to identify viral clones with desired functionality. Similarly, error-prone PCR can be used to randomly mutate AAV capsid genes to create diverse libraries of variants, which can then be selected to match desired characteristics.

[0199] The capsid gene sequence may also be genetically modified to introduce specific deletions, substitutions, or insertions relative to the natural wild-type sequence. In particular, the capsid gene may be modified by inserting sequences of unrelated proteins or peptides within the open reading frame of the capsid coding sequence, or at the N-terminus and / or C-terminus of the capsid coding sequence.

[0200] Unrelated proteins or peptides may advantageously act as ligands for specific cell types, thereby resulting in improved binding to target cells or improving the specificity of vector targeting to specific cell populations. An example is the use of the RGD peptide, which blocks uptake in the retinal pigment epithelium and thereby promotes introduction into the surrounding retinal tissue (Cronin et al., (2008) ARVO Abstract: D1048). Unrelated proteins may also assist in the purification of viral particles as part of the production process, i.e., they may be epitopes or affinity tags. Insertion sites are typically selected so as not to interfere with other functions of viral particles (e.g., internalization, transport). Those skilled in the art can identify suitable sites for insertion based on general knowledge. Specific sites are disclosed by Choi et al., referenced above.

[0201] The present invention further encompasses providing a sequence of the AAV genome in an order and configuration different from that of the native AAV genome. The present invention also encompasses replacing one or more AAV sequences or genes with a chimeric gene composed of sequences from another virus or sequences from two or more viruses. Such a chimeric gene may consist of sequences from two or more related viral proteins of different viral species.

[0202] The AAV vector of the present invention may take the form of a nucleotide sequence comprising an AAV genome or its derivative, as well as a sequence encoding complement factor I, or an FHL1 transgene or its derivative.

[0203] The AAV particles of the present invention include a trans-capsid form in which an AAV genome or derivative having an ITR of one serotype is packaged on a capsid of a different serotype. The AAV particles of the present invention also include a mosaic form in which a mixture of unmodified capsid proteins from two or more different serotypes constitutes the viral capsid. The AAV particles also include a chemically modified form that carries a ligand adsorbed to the capsid surface. For example, such a ligand may include an antibody for targeting a specific cell surface receptor.

[0204] Therefore, for example, AAV particles of the present invention include those having the AAV2 genome and AAV2 capsid protein (AAV2 / 2), those having the AAV2 genome and AAV5 capsid protein (AAV2 / 5), those having the AAV2 genome and AAV8 capsid protein (AAV2 / 8), and those having the AAV2 genome and capsid proteins of multiple serotypes.

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

[0206] The polynucleotides or vectors of the present invention may also include elements that enable the expression of complement factor I or FHL1 transgenes in vitro or in vivo. These may also be called expression regulatory sequences. Thus, the polynucleotides or vectors typically include expression regulatory sequences (e.g., promoter sequences) operably ligated to the nucleotide sequence encoding the transgene.

[0207] Any suitable promoter can be used, and its selection is readily apparent to those skilled in the art. The promoter sequence may be constitutively active (i.e., operable in any host cell background) or active only in a specific host cell environment, thus enabling targeted expression of the transgene in a specific cell type (e.g., a tissue-specific promoter). The promoter may exhibit inducible expression in response to the presence of another factor, such as a factor present in the host cell. In either case, if the vector is administered therapeutically, it is preferable that the promoter is functional in the background of the target cell.

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

[0209] A preferred promoter that is not retinal cell-specific is 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, for example, the promoter used in the rAVE expression cassette (GeneDetect.com).

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

[0211] Examples of CMV promoter sequences are as follows: [ka] (Sequence ID 13)

[0212] In some embodiments, the polynucleotide or vector includes a promoter having a nucleotide sequence that has at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 13. Preferably, the nucleotide sequence substantially retains the functional activity of the promoter represented by SEQ ID NO: 13.

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

[0214] Further examples of promoter sequences are as follows: [ka] (Sequence ID 5)

[0215] In some embodiments, the polynucleotide or vector includes a promoter having a nucleotide sequence that has at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 5. Preferably, the nucleotide sequence substantially retains the functional activity of the promoter represented by SEQ ID NO: 5.

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

[0217] Examples of human sequence-based promoters that induce retina-specific gene expression include rod and cone rhodopsin kinases (Allocca et al., (2007) J. Virol. 81:11372-80), cone-only PR2.1 (Mancuso et al., (2009) Nature 461:784-7), and / or retinal pigment epithelium RPE65 (Bainbridge et al., (2008) N. Engl. J. Med. 358:2231-9) or VMD2 (Esumi et al., (2004) J. Biol. Chem. 279:19064-73).

[0218] The polynucleotide or vector of the present invention may also include one or more additional regulatory sequences that may act pre-transcriptionally or post-transcriptionally. The regulatory sequences may be part of a native transgene locus or heterologous regulatory sequences. The polynucleotide or vector of the present invention may include a portion of the 5'-UTR or 3'-UTR from a native transgene transcript.

[0219] A regulatory sequence is any sequence that facilitates the expression of a transgene, i.e., increases the expression of a transcript, improves the nuclear export of mRNA, or enhances its stability. Examples of such regulatory sequences include enhancer elements, post-transcriptional regulatory elements, and polyadenylation sites.

[0220] The preferred polyadenylation site is the bovine growth hormone polyA (bGH polyA) signaling pathway.

[0221] Examples of bovine growth hormone polyA (bGH polyA) signaling include: [ka] (Sequence ID 14)

[0222] Further examples of bovine growth hormone polyA (bGH polyA) signaling include: [ka] (Sequence ID 6)

[0223] In some embodiments, the polynucleotide or vector contains a polyadenylation signal having a nucleotide sequence that has at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 14 or 6. Preferably, the nucleotide sequence substantially retains the functional activity of the polyadenylation signal represented by SEQ ID NO: 14 or 6.

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

[0225] In the context of the polynucleotides or vectors of the present invention, such regulatory sequences are cis-acting. However, the present invention also encompasses the use of trans-acting regulatory sequences located on additional gene constructs.

[0226] A preferred post-transcriptional regulatory element for use in the AAV vector of the present invention is the woodchuck hepatitis post-transcriptional regulatory element (WPRE) or a variant thereof.

[0227] An example of WPRE is as follows: [ka] (Sequence ID 7)

[0228] WPRE is a three-part element containing gamma, alpha, and beta elements in a given order. A shortened form of WPRE containing only the minimum gamma and alpha elements (also called WPRE3; Choi, J.-H. et al., (2014) Molecular Brain 7:17) may be used in this invention.

[0229] An example of a WPRE3 array is as follows: [ka] (Sequence ID 15)

[0230] In some embodiments, the polynucleotide or vector includes 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.

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

[0232] Another regulatory sequence that may be used in the polynucleotide or vector of the present invention is a scaffold attachment region (SAR). Additional regulatory sequences can be readily selected by those skilled in the art. Method of administration

[0233] The polynucleotides or vectors of the present invention may be administered systemically (e.g., by peripheral intravenous injection) or locally (e.g., into the CNS system by intrathecal injection). In preferred embodiments, the polynucleotides or vectors are administered intraocularly.

[0234] The term "intraocular" refers to the inside of the eye, and therefore intraocular administration relates to administration into the inside of the target eye.

[0235] In some embodiments, the polynucleotide or vector is administered to the target eye by subretinal, direct retinal, choroidal, or intravitreal injection. In some embodiments, the above administration is performed by a robot.

[0236] The volume of the drug composition to be injected may be, for example, about 10 to 500 μL, for example, about 50 to 500, 100 to 500, 200 to 500, 300 to 500, 400 to 500, 50 to 250, 100 to 250, 200 to 250, or 50 to 150 μL. The volume may be, for example, about 10, 50, 100, 150, 200, 250, 300, 350, 400, 450, or 500 μL. Preferably, the volume of the drug composition to be injected is 100 μL.

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

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

[0239] In some embodiments, the polynucleotide, vector, or pharmaceutical composition is administered once or twice or less during the lifetime of the subject. subretinal injection

[0240] Subretinal injection is an injection into the subretinal space, that is, beneath the sensory retina. During subretinal injection, the injected material is directed towards the photoreceptor cells and the retinal pigment epithelium (RPE) layer, creating a space between them.

[0241] When injections are administered through a small retinal incision, retinal detachment can occur. The detached, raised layer of the retina created by the injected material is called a "bleb."

[0242] The hole created by subretinal injection must be small enough to prevent significant backflow of the injected solution into the vitreous cavity after administration. Such backflow would be particularly problematic when the drug is injected, as it directs the drug's effect away from the target area. Preferably, the injection creates a self-sealing entry point in the neurosensory retina; that is, when the needle is removed, the hole created by the needle is resealed so that little or virtually no injected material escapes from the hole.

[0243] To facilitate this process, specialized subretinal injection needles are commercially available (e.g., DORC 41G Teflon subretinal injection needle, Dutch Ophthalmic Research Center International BV, Zuidland, The Netherlands). These needles are designed specifically for performing subretinal injections.

[0244] As long as no retinal damage occurs during injection and a sufficiently small needle is used, substantially all of the injected material remains localized between the detached neurosensory retina and the retinal pulposus (RPE) at the site of retinal detachment (i.e., does not flow back into the vitreous cavity). In fact, the typical persistence of blebs over short timeframes indicates that there is usually little escape of the injected material into the vitreous. Blebs may dissipate over longer timeframes once the injected material is absorbed.

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

[0246] The volume of the drug composition to be injected may be, for example, about 10 to 500 μL, for example, about 50 to 500, 100 to 500, 200 to 500, 300 to 500, 400 to 500, 50 to 250, 100 to 250, 200 to 250, or 50 to 150 μL. The volume may be, for example, about 10, 50, 100, 150, 200, 250, 300, 350, 400, 450, or 500 μL. Preferably, the volume of the drug composition to be injected is 100 μL. Larger volumes may increase the risk of retinal elongation, while smaller volumes may result in impaired vision. Two-stage subretinal injection

[0247] The polynucleotide or vector of the present invention can be delivered with enhanced accuracy and safety by using a two-step method in which a localized retinal detachment is induced by subretinal injection of a first solution. The first solution does not contain the polynucleotide or vector. A second subretinal injection is then used to deliver the drug containing the polynucleotide or vector to the subretinal fluid of the bleb created by the first subretinal injection. Since the injection delivering the drug is not used to detach the retina, a specific volume of the solution may be injected in this second step.

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

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

[0250] The volume of the drug composition injected in step (b) may be, for example, about 10 to 500 μL, for example, about 50 to 500, 100 to 500, 200 to 500, 300 to 500, 400 to 500, 50 to 250, 100 to 250, 200 to 250, or 50 to 150 μL. The volume may be, for example, about 10, 50, 100, 150, 200, 250, 300, 350, 400, 450, or 500 μL. Preferably, the volume of the drug composition injected in step (b) is 100 μL. Larger volumes may increase the risk of retinal elongation, while smaller volumes may result in blurred vision.

[0251] A drug-free solution (i.e., the “solution” in step (a)) may be formulated in the same way as a drug-containing solution, as described below. A preferred drug-free solution is an equilibrium salt solution (BSS) or a similar buffer adapted to the pH and osmotic pressure of the subretinal space. Visualization of the retina during surgery

[0252] Under certain circumstances, such as during end-stage retinal degeneration, locating the retina is difficult because it is thin, transparent, and difficult to see against the damaged, thick pigment epithelium on which it rests. The use of blue vitreous dyes (e.g., BrilliantPeel®, Geuder; MembraneBlue-Dual®, Dorc) facilitates the identification of retinal holes created for retinal detachment procedures (i.e., procedure (a) of the two-step subretinal injection method of the present invention), and as a result, the drug can be administered through the same hole without the risk of backflow into the vitreous cavity.

[0253] The use of a blue bio-dye also helps identify any area of ​​the retina where a thick internal limiting membrane or epiretinal membrane is present, because injections through either of these structures would obstruct clear access to the subretinal space. Furthermore, contraction of either of these structures in the immediate postoperative period could lead to elongation of the retinal entrance hole, which could result in drug reflux into the vitreous cavity. suprachoroidal injection

[0254] The polynucleotides or vectors of the present invention can be delivered to the suprachoroidal space using a microcatheter-assisted ab externo approach (e.g., Peden et al. (2011) PLoS One 6(2):e17140). In this method, a limbal-periconjunctival incision is made to expose the bare sclera, followed by a scleral incision to expose the bare choroid. A microcatheter (such as iScience Interventional's iTrack250A, optionally connected to an illumination system such as an iLumin laser diode-based micro-illumination system (iScience Interventional)) is introduced into the suprachoroidal space and advanced posteriorly toward the optic disc. After manipulating the tip of the microcatheter to the desired position, injection of the polynucleotide or vector forms blebs within the retina and choroid.

[0255] Thus, in some embodiments, the polynucleotide or vector is delivered to the suprachoroid by a method that includes: (i) introducing a microcatheter into the suprachoroidal space, (ii) advancing the microcatheter within the suprachoroidal space until the tip is near the diseased area of the retina, and (iii) injecting the polynucleotide or vector from the tip of the microcatheter to form a bleb.

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

[0257] The agents of the present invention, such as polynucleotides or vectors, can be formulated into pharmaceutical compositions. These compositions can include, in addition to the agent, pharmaceutically acceptable carriers, diluents, excipients, buffers, stabilizers, or other materials well known in the art. Such materials must be non-toxic and must not interfere with the effectiveness of the active ingredient. The exact nature of the carrier or other material can be determined by one of ordinary skill in the art according to the route of administration, such as subretinal, direct retinal, suprachoroidal, or intravitreal injection.

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

[0259] For injection at the diseased site, the active ingredient can be in the form of an aqueous solution that does not contain pyrogens and has suitable pH, isotonicity and stability. One of ordinary skill in the art can adequately prepare a suitable solution using an isotonic vehicle such as, for example, sodium chloride injection, Ringer's injection, or lactated Ringer's injection. If necessary, preservatives, stabilizers, buffers, antioxidants and / or other additives may be included.

[0260] In the case of delayed release, the drug may be contained in a pharmaceutical composition formulated for sustained release, such as in microcapsules formed from biocompatible polymers by methods known in the art, or in a liposome carrier system. Treatment method

[0261] All references to treatment in this specification include curative, palliative, and preventive treatments, and in the context of the present invention, references to prevention should be understood to more generally relate to preventive treatment. Treatment may also include preventing the progression of disease severity.

[0262] Treatment of mammals, particularly humans, is preferred. However, both human and animal treatments are within the scope of the present invention. Variants, derivatives, analogues, homologs, and fragments

[0263] In addition to the specific proteins and nucleotides mentioned herein, the present invention also encompasses the use of their variants, derivatives, analogues, homologues, and fragments. In the context of the present invention, a variant of any given sequence is a sequence in which a specific sequence of residues (whether amino acids or nucleic acid residues) is modified such that the polypeptide or polynucleotide in question substantially retains its function. Variant sequences can be obtained by the addition, deletion, substitution, modification, and / or alteration of at least one residue present in a naturally occurring protein.

[0264] As used herein with respect to the proteins or polypeptides of the present invention, the term "derivative" includes any substitution, modification, alteration, substitution, deletion, and / or addition of one (or more) amino acid residues to or from the sequence of the obtained protein or polypeptide, provided that the obtained protein or polypeptide substantially retains at least one of its endogenous functions.

[0265] As used herein with respect to polypeptides or polynucleotides, the term “analog” includes any mimic, i.e., a compound possessing at least one of the endogenous functions of the polypeptide or polynucleotide being mimicked.

[0266] Typically, amino acid substitutions can involve, for example, 1, 2, or 3 to 10 or 20 substitutions, provided that the modified sequence substantially retains the desired activity or capability. Amino acid substitutions may include the use of analogues that do not occur naturally.

[0267] The proteins used in this invention may also undergo deletions, insertions, or substitutions of amino acid residues that result in silent changes and yield functionally equivalent proteins. Intentional amino acid substitutions can be made based on similarities in the polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphiphilicity of the residues, as long as the endogenous function is preserved. 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 head groups having similar hydrophilic values ​​include asparagine, glutamine, serine, threonine, and tyrosine.

[0268] Conservative substitutions may be carried out, for example, according to the following table. Amino acids in the same block in the second column, preferably in the same row in the third column, may be substituted for each other: [Table 1]

[0269] As used herein, the term “homologous” means an entity having a specific homology to wild-type amino acid sequences and wild-type nucleotide sequences. The term “homologous” may be equivalent to “identity.”

[0270] Homologous sequences may include amino acid sequences that are at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90% identical to the target sequence, preferably at least 95%, 97%, or 99% identical. Typically, homologous sequences will contain the same active sites as the target amino acid sequence. Homology can also be considered in terms of similarity (i.e., amino acid residues having similar chemical properties / functions), but in the context of the present invention, homology is preferably expressed in terms of sequence identity.

[0271] Homologous sequences may 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%, 97%, or 99% identical. Homologousity can also be considered from the viewpoint of similarity, but in the context of the present invention, homology is preferably expressed from the viewpoint of sequence identity.

[0272] Preferably, a reference to a sequence having percent identity with any one of the sequence numbers detailed herein refers to a sequence having percent identity described over the entire length of the sequence number being referenced.

[0273] Homology comparisons can be performed visually, or more commonly, with the help of readily available sequence comparison programs. These commercially available computer programs can calculate the percentage of homology or identity between two or more sequences.

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

[0275] This is a very simple and consistent method, but for example, it does not take into account that in otherwise identical sequence pairs, due to one insertion or deletion in the nucleotide sequence, the next codon can be out of alignment. For this reason, when performing a global alignment, the percent identity can be significantly reduced. As a result, most sequence comparison methods are designed to generate an optimal alignment that takes into account possible insertions and deletions without imposing an excessive penalty on the overall identity score. This is achieved by inserting "gaps" in the sequence alignment to maximize local identity.

[0276] However, these more complex methods assign a "gap penalty" to each gap that occurs in the alignment, so that for the same number of identical amino acids, a sequence alignment with as few gaps as possible, which reflects a higher relatedness between the two compared sequences, will achieve a higher score than one with many gaps. An "affine gap cost" is typically used to impose a relatively high cost for the presence of a gap and a smaller penalty for each subsequent residue in the gap. This is the most commonly used gap scoring system. A high gap penalty will of course result in an optimized alignment with fewer gaps. Most alignment programs recognize that the gap penalty can be changed. However, when using such software for sequence comparison, it is preferable to use the default values. For example, when using the GCG Wisconsin BestFit package, the default gap penalty for amino acid sequences is -12 for a gap and -4 for each extension.

[0277] Calculating maximum percentage homology therefore first requires creating an optimal alignment that takes gap penalties into account. A suitable computer program for performing such alignments is the GCG Wisconsin Bestfit package (University of Wisconsin, USA; Devereux et al., (1984) Nucleic Acids Res. 12:387). Other software capable of performing sequence comparisons include, but are not limited to, the BLAST package (Ausubel et al., (1999) ibid. - Chapter 18), FASTA (Atschul et al., (1990) J.Mol. Biol. 403-410), and the GENEWORKS suite of comparison tools. Both BLAST and FASTA are available for offline and online searches (see Ausubel et al., (1999) ibid., pp. 7-58 to 7-60). However, for some applications, it is preferable to use the GCG Bestfit program. Another tool called BLAST2 sequencing 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).

[0278] While final percent homology can be measured in terms of identity, the alignment process itself is not typically based on all-or-nothing pair comparisons. Instead, a scaled similarity score matrix is ​​commonly used, which assigns a score to each pairwise comparison based on chemical similarity or evolutionary distance. An example of such a commonly used matrix is ​​the BLOSUM62 matrix, which is the default matrix for the BLAST suite of programs. The GCG Wisconsin program typically uses either public default values ​​or custom symbol comparison tables if provided (see the user manual for details). In some applications, it is preferable to use the public default values ​​of the GCG package, or, in the case of other software, to use a default matrix such as BLOSUM62.

[0279] Once the software generates the optimal alignment, it can calculate the percentage homology, preferably the percentage sequence identity. The software typically performs this as part of a sequence comparison, generating a numerical result.

[0280] There is also a variant of "fragment" of full-length complement factor I or FHL1, and this term typically refers to a selected region of a polypeptide or polynucleotide of interest, functionally or, for example, in an assay. "Fragment" therefore refers to an amino acid or nucleic acid sequence that is part of a full-length polypeptide or polynucleotide.

[0281] Such variants can be prepared using standard recombinant DNA techniques, such as site-directed mutagenesis. If an insertion is performed, synthetic DNA encoding the insertion can be produced, along with 5' and 3' faciest regions corresponding to naturally occurring sequences on either side of the insertion site. The faciest regions include convenient restriction sites corresponding to locations in the naturally occurring sequence, and as a result, the sequence can be cleaved by an appropriate enzyme(s), with the synthetic DNA ligated to the cleavage site. The DNA is then expressed according to the present invention to produce the encoded protein. These methods are merely illustrative of numerous standard techniques known in the art for manipulating DNA sequences, and other known techniques may be used.

[0282] Those skilled in the art will understand that all features of the invention disclosed herein may be combined without departing from the scope of the disclosed invention.

[0283] Preferred features and embodiments of the present invention are described herein as non-limiting examples.

[0284] The implementation of this invention will utilize the prior art of chemistry, biochemistry, molecular biology, microbiology, and immunology, which is within the scope of the skills of those skilled in the art, unless otherwise indicated. Such art is described in the literature. For example, Sambrook, J., Fritsch, E. and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory Press; Ausubel, F. et al. (1995 and periodic supplements) Current Protocols in Molecular Biology, Chapters 9, 13 and 16, John Wiley & Sons; Roe, B., Crabtree, J. and Kahn, A. (1996) DNA Isolation and Sequencing: Essential Techniques, John Wiley & Sons; Polak, J. and McGee, J. O'D. (1990) In Situ Hybridization: Principles and Practice, Oxford University Press; Gait, M.J. (1984) Oligonucleotide Synthesis: A Practical Approach, IRL Press; and Lilley, D. and Dahlberg, J.E. (1992) Methods in See Enzymology: DNA Structures Part A: Synthesis and Physical Analysis of DNA, Academic Press. Each of these general texts is incorporated herein by reference. Examples Example 1 Codon optimization

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

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

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

[0288] For each of the above tools 1-4, a single array was generated from each tool.

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

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

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

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

[0293] The splice site was removed by altering the GT of the donor site or the AG of the acceptor site whenever possible. If this was not possible (e.g., the sequence encoding valine), the 5' adjacent base was altered.

[0294] All modified sequences were then analyzed using the www.Fruitfly.org tool to confirm that all splice sites had been removed or reduced to a threshold of less than 0.75. Removal of microRNA binding sites

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

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

[0297] Regarding FHL-1, the following miRNA binding site was identified (based on the sequences of complement factor H and CFH): hsa-mir-146a-5p.

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

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

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

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

[0302] All 20 plasmids were transfected into ARPE19 cells using the following procedure. Day 1: ARPE19 cells were isolated and counted using ViCell. Cells were placed in a 48-well plate with 500 μL of DMEM and 10% FBS per well, 6x10⁶ cells. 4 Cells were seeded in wells. Day 2: Confluence was checked and found to be 70-80%. The cells were then transfected with 0.25 μg of plasmid DNA using PEI in a 1:3 DNA:PEI ratio in a 2-chain configuration. 1.2 x 0.25 μg of DNA was diluted with 2 x 5 μL of PBS. 2.2 x 0.75 μL PEI was diluted with 2 x 5 μL PBS. 3. The PEI mix was added dropwise to the DNA mix, mixed, and then incubated at room temperature for 20 minutes. 4.2 x 250 μL DMEM / Glutamax / 10% FBS was added to the mixture. 5. The culture medium was removed from the cells and replaced with 250 μL / well of DNA / PEI complex. Day 3: Remove the culture medium and replace it with 125 μL of serum-free DMEM / Glutamax. Day 5: The culture medium was collected, centrifuged at 14,000 rpm for 10 minutes at 4°C, and the supernatant was transferred to a new tube. Western blot

[0303] The transfection supernatant was analyzed by Western blotting (primary antibodies against CFI and FHL-1: goat antiserum CFI 1:3000; Quidel A312 1:3000; and secondary antibody rabbit anti-goat HRP 1:5000 were used).

[0304] The results of the Western blot analysis are shown in Figure 2. CFI ELISA

[0305] The supernatant from transfection was analyzed by CFI ELISA using the following procedure: Day 1: ELISA plates were coated with 50 μL per well of sheep anti-CFI polyclonal antibody diluted to 1 / 4000 in 1x coating buffer. The plates were stored overnight at 4°C. Day 2: The plate was washed three times with 200 μL of PBS-Tween (0.05%) per well, and then blotted onto tissue. 1% BSA fraction V in 200 μL of PBS-Tween (0.05%) was applied to each well and blocked at room temperature for 2 hours.

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

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

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

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

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

[0311] I then recorded the A450 and transferred the data to Microsoft Excel for processing and analysis.

[0312] The results of the CFI ELISA are shown in Figure 3. FHL1 ELISA

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

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

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

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

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

[0318] Next, I recorded the data on the A450, processed it, and transferred it to Microsoft Excel for analysis.

[0319] The results of the FHL1 ELISA are shown in Figure 4. AAV2 vector creation

[0320] The best four (CFI) and five (FHL1) sequences were advanced to studies using AAV. HEK293 cells were transfected with selected codon-optimized plasmids along with pRepCap and pHelper, following a typical triple transfection protocol. Day 1: HEK293 cells were isolated and counted using ViCell. Cells were placed in 6x10 cubic meters in a 10cm dish. 5 cells / cm 2 Seeds were then sown in 10 mL of DMEM 10% FBS in a dish. Day 2: Confluence was checked and found to be 70-80%.

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

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

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

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

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

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

[0327] ARPE19 cells were transduced using a vector containing a codon-optimized transgene. Day 1: ARPE19 cells were isolated, ViCells were counted, and then 200 μL of DMEM / Glutamax was added. + 10% FBS 1x10 per middle well 5 The seeds were seeded using cells. Day 2: The vector was added to the cells. Day 3: The culture medium was replaced with serum-free medium. Day 4: The supernatant was collected, centrifuged at 14,000 rpm for 10 minutes at 4°C, and then transferred to a new tube.

[0328] Total protein concentration was evaluated using the Bradford assay. CFI ELISA

[0329] The supernatant from transduction was analyzed for CFI by ELISA according to the protocol described above.

[0330] The results of the CFI ELISA are shown in Figure 5. FHL-1 ELISA

[0331] The supernatant from transduction was analyzed for FHL1 by ELISA according to the protocol described above.

[0332] The results of the FHL1 ELISA are shown in Figure 6. conclusion

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

[0334] All publications described in the above specification are incorporated herein by reference. Various modifications and variations of the disclosed agents, compositions, uses and methods of the present invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the present invention is disclosed in relation to certain preferred embodiments, it should be understood that the claimed invention should not be excessively limited to such specific embodiments. In fact, various modifications of the disclosed embodiments for carrying out the present invention, which will be 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 98% sequence identity with SEQ ID NO:

10.

2. The isolated polynucleotide according to claim 1, wherein the nucleotide sequence encoding the CFI is sequence number 10.

3. The isolated polynucleotide according to claim 1 or 2, wherein the polynucleotide comprises an adeno-associated virus (AAV) reverse terminal repeat (ITR) at its 5' end and an AAV ITR at its 3' end.

4. The isolated polynucleotide according to claim 3, wherein the AAV ITR is AAV2 or AAV8 ITR.

5. The isolated polynucleotide according to claim 4, wherein the AAV ITR is AAV2 ITR.

6. The isolated polynucleotide according to any one of claims 1 to 5, wherein the nucleotide sequence encoding the CFI is operably linked to a promoter.

7. The isolated polynucleotide according to claim 6, wherein the promoter is a CMV promoter or a CAG promoter.

8. The isolated polynucleotide according to claim 6 or 7, wherein the promoter has the nucleotide sequence of SEQ ID NO: 5 or SEQ ID NO: 13, or a nucleotide sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 5 or SEQ ID NO:

13.

9. The isolated polynucleotide according to any one of claims 1 to 8, wherein the nucleotide sequence encoding the CFI is operably linked to a WPRE regulatory element.

10. The isolated polynucleotide according to claim 9, wherein the WPRE regulatory element is a WPRE3 regulatory element.

11. The isolated polynucleotide according to claim 9 or 10, wherein the WPRE regulatory element has the nucleotide sequence of SEQ ID NO: 7 or SEQ ID NO: 15, or a nucleotide sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 7 or SEQ ID NO:

15.

12. The isolated polynucleotide according to any one of claims 1 to 11, wherein the nucleotide sequence encoding the CFI is operably linked to a polyA signal.

13. The isolated polynucleotide according to claim 12, wherein the polyA signal is a bovine growth hormone polyA signal.

14. The isolated polynucleotide according to claim 12 or 13, wherein the polyA signal has the nucleotide sequence of SEQ ID NO: 6 or SEQ ID NO: 14, or a nucleotide sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 6 or SEQ ID NO:

14.

15. The isolated polynucleotide according to any one of claims 1 to 14, wherein the nucleotide sequence encoding the CFI is operably linked to the CMV promoter, the WPRE regulatory element, and the polyA signal.

16. A vector comprising a polynucleotide according to any one of claims 1 to 15.

17. The vector according to claim 16, wherein the vector is an adeno-associated virus (AAV) vector.

18. The vector according to claim 16 or 17, wherein the vector is in the form of a viral vector particle.

19. The vector according to claim 18, wherein the viral vector particles comprise an AAV2 or AAV8 genome and an AAV2 or AAV8 capsid protein.

20. The vector according to claim 18, wherein the viral vector particles comprise the AAV2 genome and AAV2 capsid protein (AAV2 / 2), the AAV2 genome and AAV8 capsid protein (AAV2 / 8), or the AAV8 genome and AAV8 capsid protein (AAV8 / 8).

21. A cell comprising a polynucleotide according to any one of claims 1 to 15.

22. Cells transduced with the vector according to any one of claims 16 to 20.

23. A pharmaceutical composition comprising, in combination with a pharmaceutically acceptable carrier, diluent, or excipient, a polynucleotide according to any one of claims 1 to 15, a vector according to any one of claims 16 to 20, or a cell according to claim 21 or 22.

24. The use of a polynucleotide according to any one of claims 1 to 15, a vector according to any one of claims 16 to 20, or a cell according to claim 21 or 22 in the manufacture of a pharmaceutical product.

25. Use of a polynucleotide according to any one of claims 1 to 15, a vector according to any one of claims 16 to 20, or a cell according to claim 21 or 22 in the manufacture of a pharmaceutical product for treating or preventing complement-mediated disorders of the eye.

26. The use according to claim 25, wherein the disorder is age-related macular degeneration (AMD) or diabetic retinopathy.

27. ​​The use according to claim 26, wherein the disorder is AMD.

28. The use according to claim 26 or 27, wherein the AMD is a dry AMD.

29. The use according to any one of claims 25 to 28, wherein the formation of geographic atrophy is prevented or reduced, and / or the amount of geographic atrophy is reduced.

30. The use according to any one of claims 25 to 29, wherein the progression of geographic atrophy is slowed.

31. The use according to any one of claims 25 to 30, wherein there is at least a 10% reduction in the increase in geographic atrophy areas over 12 months after administration to the treated eye of the subject, compared to an untreated eye over the same period.

32. The use according to any one of claims 25 to 31, wherein the administration of the polynucleotide, vector, or cells in the subject inactivates C3b and increases the level of activity of iC3b degradation in the eye of the subject (such as in the retinal pigment epithelium (RPE)).

33. The use according to claim 32, wherein the administration of the polynucleotide, vector or cell increases the level of activity of inactivating C3b and degrading iC3b in the subject or in the eye (such as in the RPE) of the subject to a level exceeding normal levels in the subject or in its eye or in its retinal pigment epithelium (RPE).

34. Use of a polynucleotide according to any one of claims 1 to 15, a vector according to any one of claims 16 to 20, or a cell according to claim 21 or 22 in the manufacture of a pharmaceutical for improving or restoring reading speed in a subject suffering from an eye disorder, and / or mitigating a decline in reading speed associated with an eye disorder.

35. Use of a polynucleotide according to any one of claims 1 to 15, a vector according to any one of claims 16 to 20, or a cell according to claim 21 or 22 in the manufacture of a pharmaceutical for improving or restoring reading speed in a subject suffering from AMD, and / or mitigating reading speed reduction associated with AMD.

36. Use of a polynucleotide according to any one of claims 1 to 15, a vector according to any one of claims 16 to 20, or a cell according to claim 21 or 22 in the manufacture of a pharmaceutical product for reducing or preventing the loss of photoreceptors and / or retinal pigment epithelium (RPE).

37. The use according to any one of claims 24 to 36, wherein the polynucleotide, vector, or cell is administered intraocularly.

38. The use according to any one of claims 24 to 37, wherein the polynucleotide, vector, or cell is administered to the target eye by subretinal, direct retinal, choroidal, or intravitreal injection.

39. The use according to any one of claims 24 to 38, wherein the polynucleotide, vector, or cell is administered to the target eye by subretinal injection.

40. A pharmaceutical composition for treating or preventing complement-mediated disorders of the eye, comprising a polynucleotide according to any one of claims 1 to 15, a vector according to any one of claims 16 to 20, or cells according to claim 21 or 22.

41. The pharmaceutical composition according to claim 40, wherein the disorder is age-related macular degeneration (AMD) or diabetic retinopathy.

42. The pharmaceutical composition according to claim 41, wherein the disorder is AMD.

43. The pharmaceutical composition according to claim 41 or 42, wherein the AMD is a dry AMD.

44. A pharmaceutical composition according to any one of claims 40 to 43, wherein the formation of geographic atrophy is prevented or reduced, and / or the amount of geographic atrophy is reduced.

45. The pharmaceutical composition according to any one of claims 40 to 44, wherein the progression of geographic atrophy is delayed.

46. The pharmaceutical composition according to any one of claims 40 to 45, wherein there is at least a 10% reduction in the increase in geographic atrophy areas over 12 months after administration to the treated eye of the subject, compared to an untreated eye over the same period.

47. The pharmaceutical composition according to any one of claims 40 to 46, wherein administration of the pharmaceutical composition increases the level of activity of inactivating C3b and degrading iC3b in a subject or in the eye of a subject (such as in the retinal pigment epithelium (RPE)).

48. The pharmaceutical composition according to claim 47, wherein administration of the pharmaceutical composition increases the level of activity for inactivating C3b and degrading iC3b in the subject or in the subject's eye (such as in the RPE) to a level exceeding normal levels in the subject or in its eye or retinal pigment epithelium (RPE).

49. A pharmaceutical composition for improving or restoring reading speed in a subject suffering from an eye disorder, and / or for mitigating a reading speed reduction associated with an eye disorder, comprising a polynucleotide according to any one of claims 1 to 15, a vector according to any one of claims 16 to 20, or a cell according to claim 21 or 22.

50. A pharmaceutical composition for improving or restoring read speed in a subject suffering from AMD, and / or for mitigating read speed reduction associated with AMD, comprising a polynucleotide according to any one of claims 1 to 15, a vector according to any one of claims 16 to 20, or cells according to claim 21 or 22.

51. A pharmaceutical composition for reducing or preventing the loss of photoreceptors and / or retinal pigment epithelium (RPE), comprising a polynucleotide according to any one of claims 1 to 15, a vector according to any one of claims 16 to 20, or cells according to claim 21 or 22.

52. The pharmaceutical composition according to any one of claims 40 to 51, wherein the pharmaceutical composition is administered intraocularly.

53. The pharmaceutical composition according to any one of claims 40 to 52, wherein the pharmaceutical composition is administered to the target eye by subretinal, direct retinal, choroidal, or intravitreal injection.

54. The pharmaceutical composition according to any one of claims 40 to 53, wherein the pharmaceutical composition is administered to the target eye by subretinal injection.

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