Nucleic acids encoding anti-VEGF entity and complement negative modulators and their use for treatment of age-related macular degeneration

Abstract

The present invention relates to a product comprising (i) an anti-VEGF entity; and (ii) a complement negative modulator, or a nucleotide sequence encoding the same, as a combination formulation for simultaneous, separate or sequential use in therapy.

Description

Field of the Invention

[0001] The present invention relates to agents for gene therapy. In particular, the present invention relates to a combination of an anti-VEGF entity and a negative complement regulator, or a nucleotide sequence encoding the same, and their use in treating or preventing complement-mediated and complement-related disorders, including complement-mediated eye diseases, such as age-related macular degeneration (AMD). Background of the Invention

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

[0004] Age-related macular degeneration (AMD) is the most common cause of functional blindness in developed countries over 50 years old (Seddon, JM, Epidemiology of age-related macular degeneration. In Ogden, TE et al. editors, Ryan SJ, editor's Retina Vol. 2, 3rd edition, St.Louis, Mo:Mosby; 2001:1039-1050). Wet AMD is related to the neovascularization that originates from the choroidal vascular system and extends to the subretinal space. In addition, the feature of AMD is that retina, retinal pigment epithelium (RPE) and bottom choroid (underlying choroid, the highly vascularized tissue below RPE, between retina and sclera) degenerate gradually.

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

[0006] The clinical progression of AMD is characterized by multiple stages, based on changes in the macula. Early AMD is characterized by the appearance of drusen, which are accumulations of extracellular debris under the retina that appear as yellow spots on the retina during clinical examination and on fundus photographs. Drusen can be classified by size as small (<63 microns), medium (63-124 microns), and large (>124 microns). They are also considered hard or soft, based on the appearance of their edges during fundus examination. Hard drusen have clearly defined edges, while soft drusen have less defined, fluid edges. The Age-Related Eye Disease Study (AREDS) Fundus Photography Severity Scale is one of the main classification systems used for this condition.

[0007] AMD is divided into two forms of "dry" and "wet" (exudative or neovascular). The typical feature of dry AMD is the progressive apoptosis of the cells of the RPE layer, the overlying photoreceptor cells, and the underlying cells of the choroid capillaris layer that are often also present. The dense area of ​​RPE cell death is accompanied by the atrophy of the overlying photoreceptors, which is called geographic atrophy, and this represents the late stage of dry AMD. Patients suffering from this form of AMD, in which visual acuity can appear slow and progressive deterioration.

[0008] Wet AMD is characterized by abnormal blood vessels that grow from the choroidal vessels beneath the RPE (choriocapillaris layer) into the subretinal space of the macula, with bleeding and / or fluid leakage, which may be the cause of sudden vision loss. It is estimated that most of the vision loss in patients is due to this choroidal neovascularization (CNV) and its secondary complications. A subtype of neovascular AMD is called retinal angiomatous proliferation (RAP). Here, the angiomatous growth originates in the retina and extends posteriorly into the subretinal space, eventually communicating with the choroidal new vessels in some cases. Polypoidal choroidal vasculopathy (PCV) is also known to occur.

[0009] Current treatment options for wet AMD are primarily targeted at the vascular endothelial growth factor (VEGF) pathway. Examples of such VEGF-targeted therapies include the aptamer pegaptanib (N Engl J Med (2004) 351:2805-2816) and antibodies such as ranibizumab (N Engl J Med (2006) 355:1432-1444), aflibercept (BMJ (2014) 98:i17-i21), and bevacizumab (BMJ (2010) 340:c2459). However, not all patients respond to anti-VEGF antibody therapy, either failing to recover vision or progressing to registered blindness. There are also reports that the visual acuity (VA) gains achieved with monthly treatment decrease incrementally (Singer et al.; Ophthalmology; 2012; 119(6); 1175-83).

[0010] Furthermore, there is evidence supporting an association between anti-VEGF therapy and progression of geographic atrophy (Gemenetza et al.; Eye; 2017; 31; 1-9, Enslow et al.; Ophthalmology and Eye Diseases; 2016; 8; 21-32 & Sadda et al.; Ophthalmology; 2020; 127(5); 648-659).

[0011] There is also a need for a treatment strategy that reduces the patient's medication burden and addresses issues of adherence and / or underuse.

[0012] Therefore, there is a significant need in the art for new methods of treating eye diseases such as AMD. SUMMARY OF THE INVENTION

[0014] Without wishing to be bound by theory, the present invention is based on such insight, at least in part, that while targeting is generally associated with the potential mechanism of "wet" AMD, targeting is generally associated with the mechanism of "dry" AMD may be advantageous. In addition, the present invention is based at least in part on considering that treatment for a form of AMD may affect another.

[0015] In a first aspect, the invention provides a product comprising (i) an anti-VEGF entity; and (ii) a negative complement regulator, or a nucleotide sequence encoding the same, as a combined preparation for simultaneous, separate or sequential use in therapy.

[0016] In one embodiment, the product may be one or more polynucleotides encoding, among others, (i) an anti-VEGF entity; and (ii) a negative complement regulator.

[0017] Thus, in a preferred embodiment, polynucleotides encoding an anti-VEGF entity and a negative complement regulator are provided.

[0018] Preferably, the polynucleotide is a bicistronic polynucleotide encoding an anti-VEGF entity and a negative complement regulator.

[0019] Specifically, applicants have provided vectors that can be used to deliver both anti-VEGF entities and complement negative regulators to patients. Specifically, applicants have successfully designed functional AAV vectors that can be produced in good titers and contain nucleotide sequences encoding both anti-VEGF entities and complement negative regulators.

[0020] The anti-VEGF entity can be selected from Ig fusion proteins, antibodies, aptamers, polypeptides, peptides, polynucleotides (e.g., antisense oligonucleotides targeting VEGF gene sequences or sequences of its mRNA, siRNA, shRNA, CRISPRi guide strands, whose sequences are directed to VEGF gene sequences) and non-antibody scaffolds. Suitably, the anti-VEGF entity is selected from aflibercept, ranibizumab, bevacizumab, brolucizumab and pegaptanib or fragments or variants thereof.

[0021] The negative complement regulator may be selected from complement factor I (CFI), complement factor H-like protein 1 (FHL1), complement factor H (CFH), complement receptor type 1 (CR1), membrane cofactor protein (MCP), complement decay accelerating factor (DAF) and MAC inhibitory protein (MAC-IP), C1 inhibitor, anaphylatoxin inhibitor, C4b binding protein (C4BP), clusterin, vitronectin or a fragment or variant thereof.

[0022] In some embodiments, the polynucleotide further comprises a nucleotide sequence encoding a CMV promoter or a chicken β-actin promoter. Preferably, the CMV or CAG promoter is located upstream of the nucleotide sequence encoding the anti-VEGF entity and the complement negative regulator. The chicken β-actin promoter can be provided in combination with the CMV early enhancer (i.e., as a CAG promoter).

[0023] In some embodiments, the polynucleotide further comprises a nucleotide sequence encoding a WPRE regulatory element.Preferably, the WPRE regulatory element is located downstream of the nucleotide sequence encoding the anti-VEGF entity and the complement negative regulator.

[0024] In some embodiments, the polynucleotide further comprises a nucleotide sequence encoding a poly-A signal. Preferably, the poly-A signal is downstream of the nucleotide sequence encoding the anti-VEGF entity and the complement negative regulator.

[0025] In some embodiments, the polynucleotide further comprises a nucleotide sequence encoding a bovine growth hormone poly-A signal. Preferably, the bovine growth hormone poly-A signal is downstream of the nucleotide sequence encoding the anti-VEGF entity and the complement negative regulator.

[0026] In some embodiments, the polynucleotide further comprises a nucleotide sequence encoding:

[0027] (a) a CMV promoter or a CAG promoter, wherein the CMV promoter or the CAG promoter is located upstream of the nucleotide sequence encoding the anti-VEGF entity and the complement negative regulator; and

[0028] (b) a bovine growth hormone poly-A signal, wherein the bovine growth hormone poly-A signal is downstream of a nucleotide sequence encoding an anti-VEGF entity and a complement negative regulator.

[0029] In other embodiments, the polynucleotide further comprises a nucleotide sequence encoding:

[0030] (a) CMV promoter or CAG promoter, wherein the CMV promoter or CAG promoter is located upstream of the nucleotide sequence encoding the anti-VEGF entity and the complement negative regulator;

[0031] (b) a WPRE regulatory element, wherein the WPRE regulatory element is located downstream of the nucleotide sequence encoding the anti-VEGF entity and the complement negative regulator; and

[0032] (c) a bovine growth hormone poly-A signal, wherein the bovine growth hormone poly-A signal is downstream of the nucleotide sequence encoding the anti-VEGF entity and the complement negative regulator.

[0033] The WPRE regulatory element may be a WPRE3 regulatory element.

[0034] The nucleotide sequence encoding the anti-VEGF entity may be upstream of the nucleotide sequence encoding the negative regulator of complement.

[0035] In some embodiments, the nucleotide sequence encoding the anti-VEGF entity is located upstream of the nucleotide sequence encoding the negative regulator of complement, wherein the anti-VEGF entity is selected from aflibercept, ranibizumab, bevacizumab, brocuzumab and pegaptanib or a fragment or variant thereof. In some embodiments, the nucleotide sequence encoding the anti-VEGF entity is upstream of the nucleotide sequence encoding the negative regulator of complement, wherein the anti-VEGF entity is aflibercept or a fragment or variant thereof. In some embodiments, the nucleotide sequence encoding the anti-VEGF entity is upstream of the nucleotide sequence encoding the negative regulator of complement, wherein the negative regulator of complement is CFI or a variant or fragment thereof. In some embodiments, the nucleotide sequence encoding the anti-VEGF entity is upstream of the nucleotide sequence encoding the negative regulator of complement, wherein the negative regulator of complement is FHL1 or a variant or fragment thereof.

[0036] In some embodiments, the nucleotide sequence encoding the anti-VEGF entity is located upstream of the nucleotide sequence encoding the negative complement regulator, wherein the anti-VEGF entity is aflibercept or a fragment or variant thereof and the negative complement regulator is CFI or a variant or fragment thereof.

[0037] In some embodiments, the nucleotide sequence encoding the anti-VEGF entity is located upstream of the nucleotide sequence encoding the negative complement regulator, wherein the anti-VEGF entity is aflibercept or a fragment or variant thereof and the negative complement regulator is FHL1 or a variant or fragment thereof.

[0038] The nucleotide sequence encoding the negative regulator of complement may be located upstream of the nucleotide sequence encoding the anti-VEGF entity.

[0039] In some embodiments, the nucleotide sequence encoding the negative complement regulator is located upstream of the nucleotide sequence encoding the anti-VEGF entity, wherein the anti-VEGF entity is selected from aflibercept, ranibizumab, bevacizumab, brocuzumab and pegaptanib or a fragment or variant thereof. In some embodiments, the nucleotide sequence encoding the negative complement regulator is located upstream of the nucleotide sequence encoding the anti-VEGF entity, wherein the anti-VEGF entity is aflibercept or a fragment or variant thereof. In some embodiments, the nucleotide sequence encoding the negative complement regulator is upstream of the nucleotide sequence encoding the anti-VEGF entity, wherein the negative complement regulator is CFI or a variant or fragment thereof. In some embodiments, the nucleotide sequence encoding the negative complement regulator is upstream of the nucleotide sequence encoding the anti-vascular endothelial cell entity, wherein the negative complement regulator is FHL1 or a variant or fragment thereof.

[0040] In some embodiments, the nucleotide sequence encoding the complement negative regulator is located upstream of the nucleotide sequence encoding the anti-VEGF entity, wherein the anti-VEGF entity is aflibercept or a fragment or variant thereof and the complement negative regulator is CFI or a variant or fragment thereof.

[0041] In some embodiments, the nucleotide sequence encoding the negative complement regulator is located upstream of the nucleotide sequence encoding the anti-VEGF entity, wherein the anti-VEGF entity is aflibercept or a fragment or variant thereof and the negative complement regulator is FHL1 or a variant or fragment thereof.

[0042] In some embodiments, the nucleotide sequence encoding the anti-VEGF entity and the complement negative regulator are operably linked via a linker. In some embodiments, the linker is a Furin, GSG, 11aa1D and F2A linker. In preferred embodiments, the linker comprises a self-cleaving 2A peptide sequence, such as P2A or a sequence comprising or defined by a Furin cleavage site, GSG, 11a1D and F2A sequence.

[0043] In some embodiments, the polynucleotide further comprises one or more adeno-associated virus (AAV) inverted terminal repeats (ITRs). In a preferred embodiment, the polynucleotide further comprises two AAV ITRs.

[0044] In some embodiments, the polynucleotide comprises one AAV ITR at its 5' end and one AAV ITR at its 3' end.

[0045] In some embodiments, the polynucleotide comprises:

[0046] (a) 5' AAV ITR.

[0047] (b) CMV promoter or CAG promoter.

[0048] (c) Nucleotide sequences encoding anti-VEGF entities.

[0049] (d) a linker, optionally wherein the linker comprises a Furin cleavage site, GSG, 11a1D and a F2A sequence.

[0050] (e) A nucleotide sequence encoding a negative regulator of complement.

[0051] (f) a poly-A signal, preferably a bovine growth hormone poly-A signal; and

[0052] (g) 3' AAV ITR.

[0053] In some embodiments, the polynucleotide comprises:

[0054] (a) 5' AAV ITR.

[0055] (b) CMV promoter or CAG promoter.

[0056] (c) Nucleotide sequences encoding anti-VEGF entities.

[0057] (d) a linker, optionally wherein the linker comprises a Furin cleavage site, GSG, 11a1D and a F2A sequence.

[0058] (e) A nucleotide sequence encoding a negative regulator of complement.

[0059] (f) a WPRE regulatory element, optionally wherein the WPRE regulatory element is a WPRE3 regulatory element.

[0060] (g) a poly-A signal, preferably a bovine growth hormone poly-A signal; and

[0061] (h) 3' AAV ITR.

[0062] Suitably, the polynucleotides defined in (c) and (e) may be at reciprocal positions in the polynucleotide.

[0063] Specifically, in some embodiments, the polynucleotide comprises:

[0064] (a) 5' AAV ITR.

[0065] (b) CMV promoter or CAG promoter.

[0066] (c) A nucleotide sequence encoding a negative regulator of complement.

[0067] (d) a linker, optionally wherein the linker comprises a Furin cleavage site, GSG, 11a1D and a F2A sequence.

[0068] (e) Nucleotide sequences encoding anti-VEGF entities.

[0069] (f) a poly-A signal, preferably a bovine growth hormone poly-A signal; and

[0070] (g) 3' AAV ITR.

[0071] In some embodiments, the polynucleotide comprises:

[0072] (a) 5' AAV ITR.

[0073] (b) CMV promoter or CAG promoter.

[0074] (c) A nucleotide sequence encoding a negative regulator of complement.

[0075] (d) a linker, optionally wherein the linker comprises a Furin cleavage site, GSG, 11a1D and a F2A sequence.

[0076] (e) Nucleotide sequences encoding anti-VEGF entities.

[0077] (f) a WPRE regulatory element, optionally wherein the WPRE regulatory element is a WPRE3 regulatory element.

[0078] (g) a poly-A signal, preferably a bovine growth hormone poly-A signal; and

[0079] (h) 3' AAV ITR.

[0080] Suitably, in embodiments wherein (b) comprises a CAG promoter; (f) may be a WPRE3 regulatory element.

[0081] The polynucleotide may consist of (a)-(g) or (a)-(h) in sequence from 5' to 3'.

[0082] In some embodiments, the AAV ITR is an AAV2 or AAV8 ITR. In preferred embodiments, the AAV ITR is an AAV2 ITR.

[0083] In some embodiments, the nucleotide sequence encoding the anti-VEGF entity and / or the complement negative regulator is codon optimized. In some embodiments, the nucleotide sequence encoding the anti-VEGF entity is codon optimized. In some embodiments, the nucleotide sequence encoding the complement negative regulator is codon optimized. In preferred embodiments, the nucleotide sequence encoding aflibercept and CFI, FHL1 or CFH is codon optimized.

[0084] In some embodiments, the nucleotide sequence encoding aflibercept is at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:11.

[0085] In a preferred embodiment, the nucleotide sequence encoding aflibercept is SEQ ID NO:11.

[0086] In some embodiments, the nucleotide sequence encoding CFI is at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:35 or 36.

[0087] In a preferred embodiment, the nucleotide sequence encoding CFI is SEQ ID NO:35 or 36.

[0088] In some embodiments, the nucleotide sequence encoding aflibercept is SEQ ID NO: 11 and the nucleotide sequence encoding CFI is SEQ ID NO: 35 or 36.

[0089] In some embodiments, the nucleotide sequence encoding FHL1 is at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:41.

[0090] In a preferred embodiment, the nucleotide sequence encoding FHL1 is SEQ ID NO:41.

[0091] In some embodiments, the nucleotide sequence encoding aflibercept is SEQ ID NO:11 and the nucleotide sequence encoding FHL1 is SEQ ID NO:41.

[0092] In some embodiments, the polynucleotide comprises the nucleotide sequence of SEQ ID NO:45, or a nucleotide sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto.

[0093] In some embodiments, the polynucleotide comprises the nucleotide sequence of SEQ ID NO:46, or a nucleotide sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto.

[0094] In some embodiments, the polynucleotide comprises the nucleotide sequence of SEQ ID NO:47, or a nucleotide sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto.

[0095] In some embodiments, the polynucleotide comprises the nucleotide sequence of SEQ ID NO:48, or a nucleotide sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto.

[0096] In some embodiments, the polynucleotide comprises the nucleotide sequence of SEQ ID NO:49, or a nucleotide sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto.

[0097] In some embodiments, the polynucleotide comprises the nucleotide sequence of SEQ ID NO:50, or a nucleotide sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto.

[0098] In some embodiments, the polynucleotide comprises the nucleotide sequence of SEQ ID NO:51, or a nucleotide sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto.

[0099] In some embodiments, the polynucleotide comprises the nucleotide sequence of SEQ ID NO:52, or a nucleotide sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto.

[0100] In some embodiments, the polynucleotide comprises the nucleotide sequence of SEQ ID NO:53, or a nucleotide sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto.

[0101] In some embodiments, the polynucleotide comprises the nucleotide sequence of SEQ ID NO:54, or a nucleotide sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto.

[0102] In some embodiments, the polynucleotide is less than or equal to 5.2, 5.1, 5.0, 4.9, 4.8 or 4.7 kb. In a preferred embodiment, the polynucleotide is less than or equal to 4.7 kb.

[0103] In another aspect, the present invention provides a vector comprising a polynucleotide of the present invention. Thus according to one aspect, the present invention provides a vector comprising (i) a polynucleotide encoding an anti-VEGF entity and (ii) a polynucleotide encoding a negative complement regulator. Preferably, the anti-VEGF entity and the negative complement regulator are provided on the same polynucleotide, which is preferably a bicistronic polynucleotide.

[0104] Suitably, a bicistronic polynucleotide as used herein may refer to a polynucleotide comprising at least two protein coding regions, each coding region being operably linked to the same promoter region. Suitably, a bi-region polypeptide may comprise two protein coding regions, each coding region being operably linked to the same promoter region.

[0105] Suitably, the present invention also provides more than one (eg two) vectors comprising between them (i) a polynucleotide encoding an anti-VEGF entity and (ii) a polynucleotide encoding a negative complement regulator.

[0106] In some embodiments, the vector is an adeno-associated virus (AAV), retrovirus, lentivirus, or adenovirus vector.

[0107] In a preferred embodiment, the vector is an AAV vector.

[0108] In some embodiments, the vector is in the form of a viral vector particle.

[0109] In some embodiments, the AAV vector particle comprises an AAV2 or AAV8 genome, preferably an AAV2 genome.

[0110] In some embodiments, the AAV vector particle comprises an AAV2 or AAV8 capsid protein.

[0111] In some embodiments, the AAV vector particles comprise an AAV2 genome and an AAV2 capsid protein (AAV2 / 2). In a preferred embodiment, the AAV vector particles comprise an AAV2 genome and an AAV8 capsid protein (AAV2 / 8).

[0112] In another aspect, the invention provides a cell comprising a polynucleotide of the invention.

[0113] In another aspect, the invention provides cells transduced with the vector of the invention.

[0114] In another aspect, the present invention provides a pharmaceutical composition comprising a polynucleotide, vector or cell of the present invention in combination with a pharmaceutically acceptable carrier, diluent or excipient.

[0115] In a preferred embodiment, the pharmaceutical composition is for intraocular administration.

[0116] In another aspect, the present invention provides a product, polynucleotide, vector, cell or pharmaceutical composition of the present invention for use in therapy.

[0117] In another aspect, the present invention provides a product, polynucleotide, vector, cell or pharmaceutical composition of the present invention for use in treating or preventing an eye disease.

[0118] In another aspect, the present invention provides a product, polynucleotide, vector, cell or pharmaceutical composition of the present invention for use in treating or preventing a complement-mediated disease of the eye.

[0119] In another aspect, the invention provides a method of treating or preventing a complement-mediated disease of the eye, comprising administering a product, polynucleotide, vector, cell or pharmaceutical composition of the invention to a subject in need thereof.

[0120] In another aspect, the invention provides a method of providing an anti-VEGF entity and a negative complement regulator to a subject, comprising delivering a product, polynucleotide, vector or cell of the invention to an eye of the subject.

[0121] In specific embodiments, the products, polynucleotides, vectors, cells or pharmaceutical compositions of the invention are used to treat complement-mediated disorders, particularly chronic inflammation, and even more particularly those diseases associated with excessive activity of the complement C3b feedback loop.

[0122] In some embodiments, the disorder is associated with an overactive complement C3b feedback loop and / or an underactive C3b lytic loop (see Figure 5 ).

[0123] In some embodiments, the condition is age-related macular degeneration (AMD) or diabetic retinopathy. In other embodiments, the condition is glaucoma, Stargardt disease, central serous choroidopathy, retinitis pigmentosa, polypoidal choroidal vasculopathy, diabetic macular edema, branch retinal vein occlusion, or uveitis.

[0124] In preferred embodiments, the disorder is AMD.

[0125] In some embodiments, the condition is wet AMD and / or dry AMD.In some embodiments, the product, polynucleotide, vector, cell or pharmaceutical composition is used to treat and / or prevent both wet AMD and dry AMD.

[0126] In some embodiments, AMD is wet AMD, and uses according to the present invention further prevent and / or treat dry AMD from developing in the subject. In some embodiments, AMD is dry AMD, and uses according to the present invention further prevent and / or treat wet AMD from developing in the subject.

[0127] In some embodiments, the subject has been diagnosed with or is at risk of acquiring AMD.

[0128] In some embodiments, the use is for treating or preventing a disease in a subject:

[0129] (a) having a lower than normal complement factor I activity or concentration in the eye and / or serum, preferably having a concentration in serum of 0-30, 0-20 or 0-10 μg / mL or an activity equivalent thereto; and / or

[0130] (b) being heterozygous or homozygous for a SNP associated with age-related macular degeneration (AMD), preferably a rare complement factor I and / or complement factor H variant.

[0131] In some embodiments, the use is for treating or preventing a disorder in a subject:

[0132] (a) having normal levels of complement factor I activity or concentration in the eye and / or serum, preferably at least 30 μg / mL in serum, such as 30-40 μg / mL; and / or

[0133] (b) does not carry rare complement factor I variant alleles.

[0134] In another aspect, the present invention provides a product, polynucleotide, vector, cell or pharmaceutical composition of the present invention for use in treating or preventing diabetic retinopathy.

[0135] In some embodiments, the development of geographic atrophy is prevented or reduced, and / or the total amount of geographic atrophy is reduced.

[0136] In some embodiments, the progression of geographic atrophy is slowed.

[0137] In some embodiments, the increase in the area of ​​geographic atrophy is at least 10% reduced within 12 months after administration to the treated eye of the subject relative to the untreated eye during the same period. In other embodiments, the increase in the area of ​​geographic atrophy is at least 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90% reduced within 12 months after administration to the treated eye of the subject relative to the untreated eye during the same period.

[0138] In some embodiments, administration of the product, polynucleotide, vector, cell or pharmaceutical composition increases the level of C3b inactivation and iC3b degradation activity in a subject or in the eye of a subject (such as in the retinal pigment epithelial (RPE) aqueous fluid and / or vitreous humor), optionally above normal levels in the subject or in their eye or RPE.

[0139] In another aspect, the invention provides products, polynucleotides, vectors, cells or pharmaceutical compositions of the invention for use in improving or restoring vision or visual acuity, e.g., in a subject suffering from an ocular disorder such as an ocular disorder disclosed herein. In another aspect, the invention provides products, polynucleotides, vectors or cells of the invention for use in reducing loss of vision or visual acuity, e.g., loss of vision or visual acuity associated with an ocular disorder such as an ocular disorder disclosed herein.

[0140] In another aspect, the invention provides a product, polynucleotide, vector, cell or pharmaceutical composition of the invention for use in improving or restoring reading speed in a subject, e.g., in a subject suffering from an ocular disorder such as an ocular disorder disclosed herein. In another aspect, the invention provides a product, polynucleotide, vector or cell of the invention for use in alleviating a decrease in reading speed in a subject, e.g., loss of vision or visual acuity associated with an ocular disorder such as an ocular disorder disclosed herein.

[0141] In another aspect, the present invention provides a product, polynucleotide, vector, cell or pharmaceutical composition of the present invention for reducing or preventing the loss of photoreceptors and / or retinal pigment epithelium (RPE), e.g., the loss of photoreceptors and / or RPE associated with an ocular disorder such as an ocular disorder disclosed herein.

[0142] In another aspect, the present invention provides a product, polynucleotide, vector, cell or pharmaceutical composition of the present invention for use in reducing or preventing neovascularization, vascular leakage or retinal edema in an ocular disorder such as an ocular disorder disclosed herein.

[0143] In some embodiments, the product, polynucleotide, vector, cell or pharmaceutical composition is administered intraocularly.

[0144] In some embodiments, the product, polynucleotide, vector, cell, or pharmaceutical composition is administered to the eye of the subject by subretinal, direct retinal, suprachoroidal, or intravitreal injection.

[0145] In some embodiments, the product, polynucleotide, vector, cell, or pharmaceutical composition is administered to the eye of the subject by subretinal injection.

[0146] In some embodiments, the product, polynucleotide, vector, cell or pharmaceutical composition of the invention is not administered systemically. In other embodiments, the product, polynucleotide, vector, cell or pharmaceutical composition of the invention is not administered intravenously. BRIEF DESCRIPTION OF THE DRAWINGS

[0147] Figure 1- Schematic depicting the design of an illustrative monocistronic aflibercept vector and the size of the AAV genome (ITR-ITR).

[0148] Figure 2 - Comparison of aflibercept expression between different codon-optimized AAV2-vectors. Data show two independent experiments. Figure 3 - Representative images of Western blots performed under (A) non-reducing and (B) reducing conditions illustrating expression of aflibercept protein in culture supernatants 72 hours after induction. (C) Densitometric analysis of aflibercept expression under non-reducing and reducing conditions from two independent experiments.

[0149] Figure 4 - Schematic diagram illustrating the design of an illustrative bicistronic vector expressing a negative complement regulator and aflibercept.

[0150] Figure 5 - Cycle of C3b feedback (amplification) and cleavage (downregulation) of the vertebrate alternative complement pathway ("I" = complement factor I; "H" = complement factor H; "B" = complement factor B; and "D" = complement factor D).

[0151] Figure 6 - Administration of monocistronic vectors expressing CFI (GT005) or FHL1 (RC001) in a mouse CNV model.

[0152] Figure 7 - Comparison of bicistronic vector expression in HEK293 cells. Representative expression levels of: (A) AAV8 bicistronic expressing aflibercept and CFI; (B) AAV2 bicistronic expressing aflibercept (left-hand bar) and CFI (right-hand bar) at an MOI of 5e3 (C) AAV8 bicistronic expressing aflibercept and FHL-1; and (D) AAV2 bicistronic expressing aflibercept (left-hand bar) and FHL-1 (right-hand bar) at an MOI of 5e3; (E) AAV8 bicistronic expressing aflibercept and CFI; (F) AAV8 bicistronic expressing aflibercept and FHL-1.

[0153] Figure 8 - Protein expression from the bicistronic vector was demonstrated by Western blot analysis under non-reducing conditions.

[0154] Fig. 9 - Binding affinity of aflibercept. Affinity of aflibercept expressed from monocistronic and bicistronic vectors for VEGFA as determined by using a binding assay measuring unbound VEGFA after incubation of a fixed concentration of human VEGFA with varying concentrations of vector-derived aflibercept. Kd curves derived IC50 value = 2.3 nM

[0155] Fig.10 - Inhibitory properties of aflibercept. In the presence of a fixed concentration of VEGFA, vector-derived aflibercept was used at different concentrations to inhibit VEGFA-induced proliferation of HUVEC cells.

[0156] Fig.11 -C3b cleavage experiments with CFI and FHL1 showed that CFI and FHL1 expressed by the bicistronic vector were functional and could promote the cleavage of C3b into iC3b.

[0157] Fig.12 - Expression of Aflibercept mRNA - Monocistronic Aflibercept (mouse). AAV8 (monocistronic) vector expressing anti-VEGF (aflibercept) was administered subretinal to the right eye of 9-week-old male mice. mRNA produced by the transgene was quantified by reverse transcription and qRT-PCR.

[0158] Fig.13 - Expression of Aflibercept Protein - Monocistronic Aflibercept (mouse). AAV8 (monocistronic) vector expressing anti-VEGF (aflibercept) was administered subretinal to the right eye of 9-week-old male mice. Unbound aflibercept in intraocular fluid was measured using a quantitative sandwich ELISA.

[0159] Fig.14 - Expression of Aflibercept mRNA - Bicistronic Candidates (Mouse) Bicistronic AAV vectors co-expressing anti-VEGF (aflibercept) and complement modulators (hCFI or FHL-1) were subretinally administered into the right eye (OD) of 10-week-old male mice. mRNA produced by the transgene was quantified by reverse transcription and qRT-PCR.

[0160] Fig.15 - Expression of Aflibercept, CFI and FHL-1 proteins (bicistronic vectors). Bicistronic AAV vectors co-expressing anti-VEGF (aflibercept) and complement modulators (hCFI or FHL-1) were administered subretinal into the right eye (OD) of 10-week-old male mice. Unbound aflibercept in ocular fluid was measured using a quantitative sandwich ELISA. Human CFI and FHL-1 were quantified in ocular fluid using electrochemiluminescence detection technology from MSD.

[0161] Fig.16- In vivo efficacy in a mouse laser-induced choroidal neovascularization model. Five groups of mice received unilateral (right eye) subretinal injections of different AAV vectors. Four weeks after vector administration, CNV was induced by laser photocoagulation. Mice were imaged on the fourth and seventh days after laser irradiation and then sacrificed. (A) CNV leakage area and (B) CNV lesion size were quantified. DETAILED DESCRIPTION OF THE INVENTION

[0163] As used herein, the terms "comprising", "comprise" and "comprised of" are synonymous with "including" or "include"; or "containing" or "contain", and are inclusive or open-ended and do not exclude additional, unrecited members, elements or steps. The terms "comprising", "comprise" and "comprised of" also encompass the term "consisting of".

[0164] VEGF

[0165] Vascular endothelial growth factor (VEGF) is a potent angiogenic protein produced by cells that stimulates the formation of blood vessels. VEGF is a subfamily of growth factors, the platelet-derived growth factor family of cystine knot growth factors. They are involved in both vasculogenesis (the de novo formation of the embryonic circulatory system) and angiogenesis (the growth of blood vessels from pre-existing vasculature).

[0166] In mammals, the VEGF family consists of five members: VEGF-A, placental growth factor (PGF), VEGF-B, VEGF-C, and VEGF-D.

[0167] Suitably, the VEGF referred to herein may be VEGF-A.

[0168] There are multiple isoforms of VEGF-A, which are formed by alternative splicing of the mRNA of the single 8-exon VEGFA gene. These are divided into two groups, based on the splicing site of their terminal exon (exon 8): the proximal splice site (denoted as VEGF xxx ) or distal splice site (VEGF xxx b) In addition, alternative splicing of exons 6 and 7 changes its heparin binding affinity and amino acid number (in humans: VEGF 121 , VEGF 121 b. VEGF 145, VEGF 165 , VEGF 165 b. VEGF 189 , VEGF 206 ; the rodent homologous sequences of these proteins contain one less amino acid). These domains have important functional implications for VEGF splice variants because the terminal (exon 8) splice site determines whether these proteins are pro-angiogenic (the proximal splice site, expressed during angiogenesis, VEGF xxx ) or anti-angiogenic (distal splice site, expressed in normal tissues, VEGF xxx b). In addition, inclusion or exclusion of exons 6 and 7 mediates interaction with heparan sulfate proteoglycans (HSPGs) and schwannocyte protein co-receptors on the cell surface, enhancing their ability to bind and activate VEGF receptors (VEGFRs).

[0169] VEGF interacts with three families of receptor tyrosine kinases. VEGFR1 (VEGF receptor 1), VEGFR2, and VEGFR3. VEGFA and VEGFB bind to VEGFR1, VEGFA binds to VEGFR2, and VEGFC and VEGFD bind to both VEGFR2 and VEGFR3. Placental growth factor (PGF) interacts primarily with VEGFR1. VEGFRs are found on a variety of cell types. VEGFR1, also called Flt-1 (fms-like tyrosine kinase 1), is found in vascular endothelial cells, hematopoietic stem cells, monocytes, and macrophages. VEGFR2, also called KDR (kinase insert domain) or Flk-1 (fetal liver kinase 1), is expressed on vascular and lymphatic endothelial cells; VEGFR3 (also called Flt-4) is restricted to lymphatic endothelial cells. Upon ligand binding, VEGFRs transduce intracellular signals through a variety of mediators. VEGFR2 is the best characterized and includes phosphatidylinositol-3 kinase (PI3K) / Akt, mitogen-activated kinase, non-receptor tyrosine kinase Src, and PLCγ (phospholipase Cγ) / PKC (protein kinase C), which promote angiogenesis, lymphangiogenesis, vascular permeability, and vascular homeostasis.

[0170] VEGF-A is important in the etiology of diabetic retinopathy (DR). Microcirculatory problems in the retina of patients with diabetes can lead to retinal ischemia, which results in the release of VEGF-A and a shift in the balance of pro-angiogenic VEGF isoforms to the normally expressed anti-angiogenic VEGF isoforms. Pro-angiogenic VEGF may lead to the creation of new blood vessels in the retina and elsewhere in the eye, heralding changes that may affect vision.

[0171] VEGF-A is also important in the disease pathology of wet age-related macular degeneration (AMD), the leading cause of blindness in older adults in the industrialized world. The vascular pathology of AMD shares certain similarities with diabetic retinopathy, although the etiology and typical sources of neovascularization differ between the two diseases.

[0172] As used herein, an anti-VEGF entity may refer to any entity that is capable of reducing the expression and / or activity of VEGF.

[0173] It will be apparent that an anti-VEGF entity will be able to reduce the expression and / or activity of one or more pro-angiogenic isoforms of VEGF, in particular VEGF-A.

[0174] Suitably, the VEGF may be VEGFA, VEGFB and / or PGF.

[0175] Suitably, the VEGF may be VEGFA, VEGFB and PGF.

[0176] The anti-VEGF entity may be selected from one or more of the following: an antibody, an Ig fusion protein, a polypeptide, a peptide, a polynucleotide (such as an antisense oligonucleotide, siRNA, shRNA), a small molecule, a non-antibody scaffold, an aptamer, or a combination thereof.

[0177] Antibodies used herein are intended to include complete antibodies and antibody fragments thereof. Suitable antibodies include, but are not limited to, monoclonal and polyclonal antibodies, engineered antibodies, including chimeric antibodies, CDR grafted antibodies and humanized antibodies, single-chain antibodies, antibody fragments, and artificially selected antibodies produced using phage display or alternative technologies.

[0178] Suitable antibody fragments capable of binding to a selected target include Fv, ScFv, F(ab') and F(ab') 2. In addition, alternatives to classical antibodies can also be used in the present invention, such as "avibody", "avimer", "anticalin", "nanobody" and "DARPin".

[0179] The "scFv" or "single chain variable fragment" mentioned herein comprises a molecule in which the variable heavy chain (VH) and the variable light chain (VL) of an antibody are connected via a flexible oligopeptide. Thus, scFv is a fusion between at least one variable heavy chain and at least one variable light chain.

[0180] It will be apparent that in embodiments where the anti-VEGF entity and the negative complement regulator are provided as separate polynucleotides, the anti-VEGF entity will be an entity that can be encoded by the polynucleotide. For example, the anti-VEGF entity can be an Ig fusion protein, an antibody or antigen-binding fragment thereof, a polypeptide, a peptide, a polynucleotide (e.g., an antisense oligonucleotide, siRNA, shRNA, a CRISPRi guide chain whose sequence is directed to a VEGF gene sequence or a sequence of its mRNA), a non-antibody scaffold, or an aptamer.

[0181] Anti-VEGF entities may act by binding to one or more VEGFs and / or one or more VEGF receptors.

[0182] Anti-VEGF entities may be able to reduce the pro-proliferative and / or pro-angiogenic activities of VEGF.

[0183] For example, anti-VEGF entities may be able to reduce the proliferation of VEGF-dependent cell lines. Suitable cell lines include, but are not limited to, endothelial cells, such as human umbilical vein endothelial cells (HUVEC) and the Ba / F3-VEGFR2 cell line described by Wentink et al. (Br J Cancer; 2016; 115(8); 940-948).

[0184] The anti-VEGF entity may reduce the proliferation of a VEGF-dependent cell line by at least 1 fold (suitably at least 1.5 fold, at least 2 fold, at least 2.5 fold, at least 3 fold, at least 4 fold or at least 5 fold) relative to the proliferation in the absence of the anti-VEGF entity. The anti-VEGF entity may reduce the proliferation of a VEGF-dependent cell line by at least 10% (suitably at least 20%, at least 30%, at least 40%, at least 50%, at least 100%, at least 150% or at least 200%) relative to the proliferation in the absence of the anti-VEGF entity.

[0185] The anti-VEGF entity may be selected from aflibercept, ranibizumab, bevacizumab and pegaptanib or a fragment or variant thereof.

[0186] The anti-VEGF entity may be selected from aflibercept, ranibizumab and pegaptanib or a fragment or variant thereof.

[0187] The anti-VEGF entity may be selected from aflibercept and ranibizumab or a fragment or variant thereof.

[0188] The anti-VEGF entity may be aflibercept or a fragment or variant thereof.

[0189] The anti-VEGF entity may be ranibizumab or a fragment or variant thereof.

[0190] The anti-VEGF entity may be bevacizumab or a fragment or variant thereof.

[0191] The anti-VEGF entity may be pegaptanib or a fragment or variant thereof.

[0192] The anti-VEGF entity may be aflibercept. Aflibercept binds to circulating VEGF and acts as a "VEGF trap". It thereby inhibits the activity of VEGF isoforms VEGF-A and VEGF-B, as well as PGF. Aflibercept is a recombinant protein composed of the binding domains of VEGFR1 and VEGFR2 fused to the Fc region of human immunoglobulin gamma 1 (IgG1). Structurally, aflibercept is a dimeric glycoprotein with a protein molecular weight of 96.9 kDa.

[0193] An illustrative amino acid sequence of aflibercept is shown in SEQ ID NO:1.

[0194] SEQ ID NO:1

[0195]

[0196] The amino acid sequence of aflibercept may comprise the sequence shown in SEQ ID NO: 1. The variant has at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 1. Preferably, a fragment or variant substantially retains the functional activity of the protein shown in SEQ ID NO: 1.

[0197] The amino acid sequence of Aflibercept can be encoded by the polynucleotide sequence shown in SEQ ID NO: 2-11.

[0198] SEQ ID NO:2

[0199]

[0200] In some embodiments, the nucleotide sequence encoding aflibercept (or any other anti-VEGF entity) used in the present invention is codon optimized. Different cells differ in the use of specific codons. This codon bias corresponds to a bias in the relative abundance of a particular tRNA in a cell type. By changing the codons in the sequence to match the relative abundance of the corresponding tRNA, it is possible to increase expression. By the same token, expression can be reduced by deliberately selecting codons for which the corresponding tRNA is known to be rare in a particular cell type. Thus, there is also a degree of translational control available.

[0201] Illustrative codon-optimized nucleotide sequences encoding aflibercept are shown as SEQ ID NOs: 3-11.

[0202] SEQ ID NO:3

[0203]

[0204]

[0205] SEQ ID NO: 4

[0206]

[0207]

[0208] SEQ ID NO:5

[0209]

[0210] SEQ ID NO:6

[0211]

[0212]

[0213] SEQ ID NO: 7

[0214]

[0215] SEQ ID NO:8

[0216]

[0217]

[0218] SEQ ID NO: 9

[0219]

[0220] SEQ ID NO:10

[0221]

[0222]

[0223] SEQ ID NO: 11

[0224]

[0225]

[0226] Preferably, the polynucleotide encoding the aflibercept polypeptide is SEQ ID NO: 11 or a variant thereof.

[0227] In some embodiments, the nucleotide sequence encoding aflibercept is at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to any one of SEQ ID NOs: 2-11, preferably SEQ ID NO: 11. Preferably, the protein encoded by the nucleotide sequence substantially retains the functional activity of the protein represented by SEQ ID NO: 1.

[0228] Ranibizumab is a monoclonal antibody fragment (Fab) directed against VEGFA. Suitably, the Ranibizumab polypeptide may comprise heavy and light chain variable sequences as shown in SEQ ID NOs: 12 and 13, respectively. The complementarity determining regions (CDRs) in each sequence are underlined.

[0229] SEQ ID NO:12

[0230]

[0231] SEQ ID NO:13

[0232]

[0233] The variable sequences of the heavy and light chains of ranibizumab may comprise the polypeptides set forth in SEQ ID NO: 12 or 13. These variants may have at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 12 or 13. Preferably, the fragments or variants substantially retain the functional activity of the heavy and light chain variable sequences set forth in SEQ ID NO: 12 and 13.

[0234] The ranibizumab heavy and light chain polypeptides can be encoded by any suitable polynucleotide sequence. The polynucleotide sequences encoding the heavy and light chains can be operably linked in a separate polynucleotide. For example, the polynucleotide sequences encoding the heavy and light chains can be operably linked to the same promoter and separated from each other by an internal ribosome entry site (IRES) or a polynucleotide sequence encoding a self-cleaving polypeptide.

[0235] Examples of polynucleotide sequences encoding the ranibizumab heavy and light chain polypeptides are shown in SEQ ID NOs: 14 and 15, respectively.

[0236] SEQ ID NO: 14

[0237]

[0238]

[0239] SEQ ID NO:15

[0240]

[0241] For example, WO2019 / 164854 provides examples of isolated polynucleotides and AAV vectors encoding ranibizumab.

[0242] Bevacizumab is a monoclonal antibody directed against VEGFA. Suitably, the bevacizumab polypeptide sequence may comprise the heavy and light chain sequences shown in SEQ ID NOs: 16 and 17, respectively. The complementarity determining regions (CDRs) in each sequence are underlined.

[0243] SEQ ID NO:16

[0244]

[0245] SEQ ID NO:17

[0246]

[0247] The bevacizumab heavy and light chain sequences may comprise the polypeptides set forth in SEQ ID NO: 16 or 17. These variants have at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 16 or 17. Preferably, the fragments or variants substantially retain the functional activity of the heavy and light chain sequences set forth in SEQ ID NO: 16 and 17.

[0248] Examples of polynucleotide sequences encoding bevacizumab heavy and light chain polypeptides are shown in SEQ ID NOs: 18 and 19, respectively.

[0249] SEQ ID NO: 18

[0250]

[0251] SEQ ID NO:19

[0252]

[0253] Bevacizumab heavy chain and light chain polypeptides can be encoded by any suitable polynucleotide sequence. The polynucleotide sequence encoding the heavy chain and light chain can be operably connected in a separated polynucleotide. For example, the polynucleotide sequence encoding the heavy chain and light chain can be operably connected to the same promoter and separated from each other by an internal ribosome entry site (IRES) or a polynucleotide sequence encoding a self-cleaving polypeptide.

[0254] Pegaptanib is a VEGF- 165 ) subtype-specific RNA aptamer. The RNA sequence of pegatanib is shown as SEQ ID NO: 20.

[0255] SEQ ID NO: 20

[0256]

[0257] The pegaptanib polynucleotide sequence may comprise the sequence shown in SEQ ID NO: 20. The variant may have at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 20. Preferably, the fragment or variant substantially retains the functional activity of the polynucleotide shown as SEQ ID NO: 20.

[0258] An example of a polynucleotide sequence encoding pegaptanib is shown as SEQ ID NO:21.

[0259] SEQ ID NO: 21

[0260]

[0261] Signal peptide

[0262] The polynucleotide sequence encoding the anti-VEGF entity may further comprise a polynucleotide sequence encoding a signal peptide to allow the anti-VEGF entity to be secreted from the cell.

[0263] A signal peptide is a short peptide, usually 5-30 amino acids long, that is usually present at the N-terminus of most newly synthesized proteins destined to enter the secretory pathway. These proteins include those localized in certain organelles (such as the endoplasmic reticulum, Golgi apparatus, or endosomes), proteins that are secreted from the cell, and transmembrane proteins.

[0264] Signal peptides usually contain a core sequence, which is a long stretch of hydrophobic amino acids that tends to form a single alpha helix. The signal peptide may begin with a short stretch of positively charged amino acids, which helps to enforce the correct topology of the polypeptide during transport. At the end of the signal peptide, there is usually a stretch of amino acids that is recognized and cleaved by a signal peptidase. The signal peptidase can cleave during the translocation process or after the translocation is completed, resulting in a free signal peptide and a mature protein. The free signal peptide is then digested by a specific protease.

[0265] The signal peptide is usually located at the amino terminus of the molecule, although some carboxyl-terminal signal peptides are also known.

[0266] The signal sequence usually has a tripartite structure consisting of a hydrophobic core region (h-region) flanked by n- and c-regions. The latter contains a common cleavage site for signal peptidase (SPase). Typically, the signal sequence is cleaved off in a co-translational manner, and the resulting cleaved signal sequence is called a signal peptide.

[0267] Signal sequences can be detected or predicted using software techniques (see, for example, http: / / www.predisi.de / ). A large number of signal sequences are known and can be found in databases. For example, http: / / www.signalpeptide.de lists 2109 confirmed mammalian signal peptides in its database.

[0268] Suitably, the signal peptide may be a CFH signal peptide.

[0269] Illustrative polynucleotide sequences encoding CFH signal peptides are shown as SEQ ID NOs: 22-31.

[0270]

[0271]

[0272] Complement system

[0273] The complement system is a component of the humoral immune system and is involved in tissue inflammation, cell opsonization, and cytolysis. It provides protection against microorganisms and mediates the clearance of exogenous and endogenous cellular debris from host tissues.

[0274] The complement system cascade consists of three activation pathways. All pathways culminate in the central cleavage of factor C3 and the generation of its active fragments, C3a and C3b. C3a is an anaphylatoxin that triggers a series of chemotactic and proinflammatory responses, such as the recruitment of inflammatory cells and increased microvascular permeability, while C3b is responsible for the opsonization of foreign surfaces to which C3b is covalently linked. Three major functions are achieved through opsonization by activated C3 fragments (C3b and iC3b): (i) elimination of cell debris by phagocytes (such as macrophages or microglia) and stimulation of the adaptive immune system (B and T cells); (ii) amplification of complement activation by the formation of surface-bound C3 convertases; and (iii) assembly of C5 convertases.

[0275] The assembly of C5 convertase is responsible for the cleavage of C5, thereby forming a cytolytic membrane attack complex (MAC) that is capable of creating pores in the cell membrane, thereby promoting cell lysis and elimination of unwanted cells. Through all of these activities, the innate complement cascade supports and promotes the function of downstream mechanisms of the immune system to protect the integrity of host tissues. In general, activation of complement system pathways results in a pro-inflammatory response, including the production of MAC, which mediates cell lysis, the release of chemokines to attract inflammatory cells to the site of injury, and the enhancement of capillary permeability to promote the extravasation of infiltrating leukocytes. Under physiological conditions, complement activation is effectively controlled by the coordinated actions of soluble and membrane-associated complement regulatory molecules (CRMs). Soluble complement regulators, such as C1-inhibitor, anaphylatoxin inhibitor, C4b binding protein (C4BP), complement factor H (CFH), complement factor I (CFI), clusterin, and vitronectin, limit the action of the complement cascade at multiple sites in human tissues. In addition, every cell is protected by surface proteins such as complement receptor 1 (CR1, CD35), membrane accessory proteins (MCP, CD46), and glycosylphosphatidylinositol-anchored proteins such as decay-accelerating factor (CD55) or CD59 molecules against attack by homologous complement. Of note, host cells and tissues that are insufficiently protected against complement attack may be subject to bystander cell lysis.

[0276] In some embodiments, the invention relates to treating or preventing complement-mediated ocular disorders. For example, complement-mediated disorders may be disorders associated with defects in alternative pathway regulation, particularly with overactivity of the complement C3b feedback loop and / or underactivity of the C3b cleavage loop.

[0277] The present invention encompasses the use of negative complement regulators. A negative complement regulator may refer to an entity that limits complement action at one or more sites in the cascade. For example, a negative complement regulator may be a C1-inhibitor, an anaphylatoxin inhibitor, a C4b binding protein (C4BP), complement factor H (CFH), factor H-like protein 1 (FHL1), complement factor I (CFI), aggregin, or vitronectin. A negative complement regulator may also be a surface protein that protects cells from attack by homologous complement; for example, complement receptor 1 (CR1, CD35), membrane cofactor (CD46), and glycosylphosphatidylinositol anchored proteins, such as decay accelerating factor (CD55) or CD59 molecules.

[0278] Suitably, a negative complement regulator may be an inhibitor of a component of the complement activation cascade, for example an inhibitor of the complement C3b feedback loop.

[0279] The negative complement regulator may be selected from CFI, FHL1, CFH, CR1 and MCP, or variants or fragments thereof.

[0280] The negative complement regulator may be selected from CFI, FHL1 and CFH or variants or fragments thereof.

[0281] The negative complement regulator may be CFI or a variant or fragment thereof.

[0282] The negative complement regulator may be FHL1 or a variant or fragment thereof.

[0283] The negative complement regulator may be CFH or a variant or fragment thereof.

[0284] The negative complement regulator may be CR1 or a variant or fragment thereof.

[0285] The negative complement regulator may be MCP or a variant or fragment thereof.

[0286] Suitably, the product may comprise one or more negative complement regulators.

[0287] Suitably, the polynucleotide may encode one or more negative regulators of complement.

[0288] In some embodiments, prior to administration of the product, polynucleotide, vector, cell or pharmaceutical composition of the invention, the subject has a low level (e.g., below normal) of complement factor I activity, e.g., a low level of complement factor I activity in the eye and / or a low serum level of complement factor I activity. Complement factor I activity below normal levels may be due to expression of normally functional complement factor I below normal levels, or at least partial (e.g., heterozygous) expression (normal or below normal levels) of a non- or subfunctional variant of complement factor I (such a subject may carry a copy of one or more AMD-associated SNPs, e.g., the subject may be a homozygous or heterozygous for one of the rare complement factor I variants discussed further below). Thus, the concentration of complement factor I in the subject's eye and / or serum may be low (e.g., below normal concentration). For human subjects, a normal level of complement factor I activity (C3b inactivation and iC3b degradation activity) may be equivalent to that provided by 30-40 μg / mL of complement factor I in the subject's serum. Thus, in a subject with low complement factor I activity, the complement factor I activity in the serum may be equivalent to less than 30 μg / mL and greater than 0 μg / mL of complement factor I, such as 0-20 or 0-10 μg / mL (these are ranges of complement factor I serum concentrations that may include subjects with low complement factor I concentrations).

[0289] Therefore, the subject to be treated by the present invention may suffer from complement-mediated eye diseases, such as AMD, or may be at risk of such diseases.AMD can be wet AMD and / or dry AMD.For example, the subject may have a homotypic or heterotypic susceptibility to one or more SNPs relevant to complement-mediated diseases.

[0290] In some embodiments, the subject has the risk of AMD.For example, the subject may be susceptible to the same type or heterotype of one or more SNPs associated with AMD, such as the rare mutation of the complement factor I associated with late AMD, usually resulting in serum complement factor I levels reduced (Kavanagh et al. (2015) Hum Mol Genet 24:3861-3870). In particular, subjects may carry one or two copies of one or more of the following rare complement factor I variants: rs144082872 (encoding P50A); 4:110687847 (encoding P64L); rs141853578 (encoding G119R); 4:110685721 (encoding V152M); 4:110682846 (encoding G162D); 4:110682801 (encoding N177I); rs146444258 (encoding A240G); rs182078921 (encoding G287R); rs41278047 (encoding K441R); and rs121964913 (encoding R474).

[0291] The invention may further comprise determining whether a subject is at risk for a complement-mediated disease (e.g., AMD), for example, by determining whether the subject is susceptible to the homotype or heterotype of one or more SNPs associated with a complement-mediated disease (e.g., by determining whether the subject is homotype or heterotype of one or more rare complement factor I variants associated with AMD as described above).

[0292] Alternatively, the subject may have normal levels of endogenous complement factor I activity or concentration, for example in the eye and / or serum and / or may not carry a rare variant complement factor I allele.

[0293] In some embodiments, the product, polynucleotide, vector, cell or pharmaceutical composition of the invention is administered to increase the level of C3b inactivation and iC3b degradation activity in the eye of a subject. In other embodiments, the product, polynucleotide, vector, cell or pharmaceutical composition of the invention is administered to increase the level of C3b inactivation and iC3b degradation activity in the eye of a subject to a level that exceeds the normal level in the eye. More specifically, the level of C3b inactivation and iC3b degradation activity is increased in the RPE of the eye.

[0294] It is understood that after providing the product of the present invention and / or expressing complement factor I and complement factor H-like protein 1 by the polynucleotide or vector of the present invention, the C3b inactivation and iC3b degradation activity in the subject may include C3b inactivation and iC3b degradation activity from the subject's endogenous complement factor I (i.e., the subject's complement factor I is not provided by the product of the present invention or produced by the expression of the polynucleotide or vector of the present invention), as well as C3b inactivation and iC3b degradation activity provided by the product of the present invention or produced by the expression of the polynucleotide or vector of the present invention, so that the total level of C3b inactivation and iC3b degradation activity in the subject exceeds the normal level.

[0295] In some embodiments, the level of C3b inactivation and iC3b degradation activity in a subject, eg, in the eye, is increased by at least 5%, 10%, 15%, 20%, or 25% above normal levels.

[0296] In other embodiments, the level of C3b inactivation and iC3b degradation activity in a subject, eg, in the eye, is increased to twice normal levels, or to greater than 80%, 60%, 40%, or 20% of normal levels.

[0297] For example, the level of C3b inactivation and iC3b degradation activity in a subject, such as in the eye, can be increased to 5-100%, 5-80%, 5-60%, 5-40%, 5-20%, 10-100%, 10-80%, 10-60%, 10-40%, 10-20%, 15-100%, 15-80%, 15-60%, 15-40%, 15-20%, 20-100%, 20-80%, 20-60%, 20-40%, 25-100%, 25-80%, 25-60% or 25-40% above normal levels.

[0298] In some embodiments, administration of the products, polynucleotides, vectors, cells or pharmaceutical compositions of the invention does not detectably increase the level of C3b inactivation and iC3b degradation activity in the plasma / serum of a subject. In other embodiments, administration of the products, polynucleotides, vectors, cells or pharmaceutical compositions of the invention does not detectably increase the level of C3b inactivation and iC3b degradation activity in the plasma / serum of a subject to greater than normal levels.

[0299] In the foregoing sections, except where clearly inapplicable, references to complement factor I and C3b inactivation and iC3b degradation activities may be replaced with complement factor H or complement factor H-like protein 1, and the ability to act as a cofactor for complement factor I-mediated C3b cleavage and increase the dissociation rate of C3 convertase and C5 convertase, respectively. In some embodiments, prior to administration of the product, polynucleotide, vector, cell or pharmaceutical composition of the invention, the subject has a low level of complement factor H (e.g., lower than normal), such as a low level of complement factor H in the eye and / or a low level of complement factor H in serum. For human subjects, a normal level of complement factor H may be about 200-500 μg / mL in the serum of the subject. Thus, in a subject with a low level of complement factor H, the level in serum may be less than 200 μg / mL, greater than 0 μg / mL, such as 0-100 μg / mL. In addition, the subject may have a normal level of endogenous complement factor H, such as in the eye and / or serum.

[0300] Complement Factor I (CFI)

[0301] Complement factor I (CFI), also known as C3b / C4b inactivator, is a protein encoded by the CFI gene in humans.

[0302] Complement factor I is a serine protease that circulates in a zymogen-like state (Roversi et al. (2011) PNAS 108:12839-12844) at a concentration of approximately 35 μg / mL (Nilsson et al. (2011) Mol Immunol 48:1611-1620). The complement factor I protein is a heavily N-glycosylated heterodimer consisting of two polypeptide chains linked by a disulfide bond. The heavy chain (50 kDa) contains an N-terminal region; an FI membrane attack complex (FIMAC) domain; a CD5-like domain or scavenger receptor with cysteine-rich domain (SRCR); two low-density lipoprotein receptor (LDLr) domains; and a C-terminal region of unknown function that is the site of sequence variation between species (Roversi et al. (2011) PNAS 108:12839-12844). The light chain (38 kDa) contains a serine protease (SP) domain and conserved catalytic residues (Goldberger et al. (1987) J Biol Chem 262: 10065-10071).

[0303] Complement factor I inactivates C3b by cleaving it into iC3b, C3d and C3dg, and cleaves C4b into C4c and C4d in a similar manner. In order to function properly, complement factor I requires the presence of auxiliary factor proteins, such as C4b binding protein (C4BP), complement factor H (CFH), complement receptor 1 (CR1 / CD35) and membrane accessory protein (MCP / CD46) (Degn et al. (2011) Am J Hum Genet 88:689-705).

[0304] iC3b is unable to bind to factor B and therefore cannot perpetuate amplification of the complement cascade or activation via the alternative pathway. Therefore, once C3b is cleaved to iC3b, initiation of the alternative pathway and activation of the terminal complement cascade cannot occur.

[0305] iC3b can provide pro-inflammatory effects by binding to and activating complement receptor 3 (CR3) (CD11b / CD18) on polymorphonuclear leukocytes (mainly neutrophils), NK cells, and mononuclear phagocytes (such as macrophages).

[0306] Complement factor I is able to process iC3b into C3dg through a protease activity that requires the cofactor CR1. C3d,g cannot bind to CR3. Since the reaction of iC3b with the complement receptor CR3 is the main mechanism of inflammation caused by complement activation, the breakdown of iC3b into C3dg is crucial for reducing complement-induced inflammation (Lachmann (2009) Adv. Immunol. 104: 115-149).

[0307] The unique ability of complement factor I to both promote the cleavage of C3b to iC3b and accelerate the breakdown of iC3b—combined with its relatively low concentration in human serum, which has implications for the amount required to be delivered for therapeutic effect—makes it a particularly advantageous target.

[0308] In some embodiments, the complement factor I polypeptide is capable of cleaving C3b into inactive degradation products. For example, the complement factor I polypeptide can cleave C3b into iC3b.

[0309] In some embodiments, the complement factor I polypeptide is capable of processing iC3b into inactive degradation products. For example, the complement factor I polypeptide can process iC3b into C3dg.

[0310] In preferred embodiments, the complement factor I polypeptide is capable of cleaving C3b into iC3b and processing iC3b into C3dg.

[0311] Suitably, the fragment or variant of complement factor I may retain at least 50%, 60%, 70%, 80%, 90%, 95% or 100% of the C3b inactivation and iC3b degradation activity of native complement factor I.

[0312] The C3b inactivation and iC3b degradation activity of complement factor I or its fragments or derivatives can be determined by any suitable method known to the skilled person. For example, the measurement of the proteolytic activity of complement factor I is described in Hsiung et al. (Biochem. J. (1982) 203: 293-298). Lachmann PJ & Hobart MJ (1978) "Complement Technology" in Handbook of Experimental Immunology 3rd edition Ed DM Weir Blackwells Scientific Publications Chapter 5A p17 describes the hemolytic and agglutination assays of CFI activity. Harrison RA (1996) in "Weir's Handbook of Experimental Immunology" Fifth Edition Eds; Herzenberg Leonore A'Weir DM, Herzenberg Leonard A & Blackwell C Blackwells Scientific Publications Chapter 75 36-37 gives a more detailed description, also including a proteolytic assay. The conglutinating assay is highly sensitive and can be used to detect the first (dual) segment that converts fixed C3b to iC3b and acquires collagen reactivity, as well as the final segment starting from fixed iC3b to C3dg and looking for loss of reactivity with collagen. Hemolytic assays are used to detect conversion of C3b to iC3b, while proteolytic assays detect all segments.

[0313] In some embodiments, the complement factor I is human complement factor I.

[0314] An example of a human complement factor I protein is the human complement factor I protein with UniProtKB accession number P05156. The sequence of this example is 583 amino acids in length (disclosed as SEQ ID NO: 32), of which 1 to 18 amino acids form a signal sequence.

[0315] In some embodiments, the amino acid sequence of complement factor I is SEQ ID NO: 32. In other embodiments, the amino acid sequence of complement factor I is the sequence disclosed as positions 19 to 583 of SEQ ID NO: 32. Preferably, the variant or fragment substantially retains the functional activity of the protein represented by SEQ ID NO: 32.

[0316]

[0317] In some embodiments, the amino acid sequence of complement factor I is SEQ ID NO: 33, which corresponds to NCBI Accession No. NP_000195. In other embodiments, the amino acid sequence of complement factor I is the sequence disclosed as positions 19 to 583 of SEQ ID NO: 33.

[0318]

[0319] The wild-type nucleotide sequence encoding complement factor I is the nucleotide sequence having NCBI Accession No. NM_000204, disclosed herein as SEQ ID NO:34.

[0320]

[0321]

[0322] In some embodiments, the nucleotide sequence of complement factor I used in the present invention is codon-optimized.

[0323] A preferred nucleotide sequence encoding complement factor I is the nucleotide sequence shown in SEQ ID NO:35.

[0324]

[0325]

[0326] Another example of a codon-optimized nucleotide sequence encoding complement factor I is SEQ ID NO:36.

[0327]

[0328]

[0329] In some embodiments, the nucleotide sequence encoding complement factor I is at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 34, 35 or 36, preferably SEQ ID NO: 35. Preferably, the protein encoded by the nucleotide sequence substantially retains the functional activity of the protein represented by SEQ ID NO: 34, 35 or 36.

[0330] In other embodiments, the nucleotide sequence encoding complement factor I is SEQ ID NO:34, 35 or 36, preferably SEQ ID NO:35.

[0331] In other embodiments, the nucleotide sequence encoding complement factor I is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 34, 35 or 36, preferably SEQ ID NO: 35 from position 55 to 1752. Preferably, the protein encoded by the nucleotide sequence substantially retains the functional activity of the protein represented by SEQ ID NO: 32 or 33.

[0332] In other embodiments, the nucleotide sequence encoding complement factor I is SEQ ID NO:34, 35 or 36, preferably positions 55 to 1752 of SEQ ID NO:35.

[0333] In other embodiments, the amino acid sequence encoded by the nucleotide sequence encoding complement factor I is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 32 or 33. Preferably, the amino acid sequence substantially retains the functional activity of the protein represented by SEQ ID NO: 32 or 33.

[0334] In other embodiments, the nucleotide sequence encoding complement factor I encodes the amino acid sequence of SEQ ID NO:32 or 33.

[0335] In another embodiment, the amino acid sequence encoded by the nucleotide sequence encoding complement factor I is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identical to positions 19 to 583 of SEQ ID NO: 32 or 33. Preferably, the amino acid sequence substantially retains the functional activity of the protein represented by SEQ ID NO: 32 or 33.

[0336] In other embodiments, the nucleotide sequence encoding complement factor I encodes the amino acid sequence at positions 19 to 583 of SEQ ID NO:32 or 33.

[0337] One advantage of the present invention is that complement factor I is particularly difficult to prepare as a purified protein. Therefore, the inventors have designed a method of modulating the complement system, for example, by administering complement factor I in the form of an AAV vector comprising a nucleotide sequence encoding complement factor I to achieve treatment of age-related macular degeneration (AMD). The AAV vector can be administered to a target site, such as the eye, to achieve in situ translation of complement factor I polypeptides.

[0338] Complement Factor H (CFH)

[0339] Complement factor H (CFH) is a complement control protein.

[0340] Complement factor H is a large (155 kDa), soluble glycoprotein with a typical concentration of 200-300 μg / mL in human plasma (Hakobyan et al. (2008) 49(5). 1983-90). The main function of complement factor H is to regulate the alternative pathway of the complement system.

[0341] Complement factor H provides cofactor activity for the cleavage of C3b mediated by complement factor I. Complement factor H also increases the dissociation rate of the C3bBb complex (C3 convertase) and the (C3b)NBB complex (C5 convertase), thereby reducing the activity of the alternative complement pathway.

[0342] Complement factor H is composed of 20 complement control protein (CCP) modules (also known as short common repeats or sushi domains), which are interconnected by short linkers (between 3 and 8 amino acid residues) and arranged in a head-to-tail manner. Each CCP module consists of approximately 60 amino acids, four cysteine ​​residues are disulfide bonded in a 1-3 2-4 arrangement, and there is a hydrophobic core around a nearly invariant tryptophan residue. CCP modules are numbered from 1-20 (starting from the N-terminus of the protein). CCP 1-4 and CCP 19-20 engage C3b, while CCP 7 and CCP 19-20 bind to GAG and sialic acid (Schmidt et al. (2008) Journal of Immunology 181: 2610-2619).

[0343] Studies have shown that gene therapy with complement factor H can improve induced AMD-like lesions in mice (Cashman et al. (2015) J. Gene Med. 17: 229-243). Mice were co-injected subretina with: (i) an adenoviral vector expressing complement component C3, which has previously been shown to reproduce many pathological features of human AMD; and (ii) an adenoviral vector expressing complement factor H. Relative to control animals that received 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. Electroretinograms showed that retinal function was improved in mice that received complement factor H, and immunocytochemical reactions for rhodopsin and RPE65 were consistent with the rescue of photoreceptors and RPE in such animals.

[0344] In some embodiments, the complement factor H polypeptide or fragment or variant thereof is capable of acting as a co-factor for complement factor I-mediated cleavage of C3b. In some embodiments, the complement factor H polypeptide or fragment or variant thereof is capable of increasing the dissociation rate of C3 convertase and C5 convertase.

[0345] In a preferred embodiment, the complement factor H polypeptide or fragment or variant thereof is capable of acting as a cofactor for complement factor I-mediated cleavage of C3b and increasing the dissociation rate of C3 convertase and C5 convertase.

[0346] The cofactor activity and dissociation acceleration activity of complement factor H or its fragment or derivative can be determined by any suitable method known to the skilled person. For example, the determination of the cofactor activity of complement factor H is described in Sánchez-Corral, P. et al., 2002. The American Journal of Human Genetics, 71 (6), pp. 1285-1295 and the measurement of the dissociation acceleration activity of complement factor H are described in Wong, EK et al., 2014, Journal of the American Society of Nephrology, 25 (11), pp. 2425-2433.

[0347] In some embodiments, the complement factor H is human complement factor H.

[0348] An example of a human complement factor H protein is the human complement factor H protein with UniProtKB accession number P08603. The sequence of this example is 1231 amino acids in length (disclosed as SEQ ID NO: 37), of which 1 to 18 amino acids form a signal sequence.

[0349] In some embodiments, the amino acid sequence of complement factor H is SEQ ID NO: 37. In other embodiments, the amino acid sequence of complement factor H is positions 19 to 1231 of SEQ ID NO: 37. Preferably, the variant or fragment substantially retains the functional activity of the protein represented by SEQ ID NO: 37.

[0350]

[0351] An example of a nucleotide sequence encoding complement factor H is the nucleotide sequence having NCBI Accession No. NM_000186.

[0352] In some embodiments, the nucleotide sequence encoding complement factor H is SEQ ID NO:38.

[0353]

[0354]

[0355]

[0356] In some embodiments, the nucleotide sequence encoding complement factor H is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 38. Preferably, the protein encoded by the nucleotide sequence substantially retains the functional activity of the protein represented by SEQ ID NO: 37.

[0357] In other embodiments, the nucleotide sequence encoding complement factor H is SEQ ID NO:38.

[0358] In other embodiments, the nucleotide sequence encoding complement factor H is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to positions 55 to 3696 of SEQ ID NO: 38. Preferably, the protein encoded by the nucleotide sequence substantially retains the functional activity of the protein represented by SEQ ID NO: 37.

[0359] In other embodiments, the nucleotide sequence encoding complement factor H is positions 55 to 3696 of SEQ ID NO:38.

[0360] In other embodiments, the amino acid sequence encoded by the nucleotide sequence encoding complement factor H is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 37. Preferably, the amino acid sequence substantially retains the functional activity of the protein represented by SEQ ID NO: 37.

[0361] In other embodiments, the nucleotide sequence encoding complement factor H encodes the amino acid sequence of SEQ ID NO:37.

[0362] In another embodiment, the amino acid sequence encoded by the nucleotide sequence encoding complement factor H is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identical to positions 19 to 1231 of SEQ ID NO: 37. Preferably, the amino acid sequence substantially retains the functional activity of the protein represented by SEQ ID NO: 37.

[0363] In other embodiments, the nucleotide sequence encoding complement factor H encodes the amino acid sequence of positions 19 to 1231 of SEQ ID NO:37.

[0364] Complement factor H-like protein 1 (FHL1)

[0365] Complement factor H-like protein 1 (FHL1) is a splice variant of complement factor H, containing the first 7 CCPs of complement factor H, followed by a 4-amino acid carboxyl terminus (Clark, SJ et al. (2015) J Clin Med 4:18-31). The regulatory activity of FHL1 has been shown to be comparable to that of complement factor H (Mannes, M., et al. 2020. Frontiers in Immunology, 11; 596415).

[0366] In some embodiments, the FHL1 polypeptide or fragment or variant thereof can function as a cofactor for C3b cleavage mediated by complement factor I. In some embodiments, the FHL1 polypeptide or fragment or variant thereof can increase the dissociation rate of C3 convertase and C5 convertase.

[0367] In a preferred embodiment, the FHL1 polypeptide or a fragment or variant thereof is capable of acting as a cofactor for complement factor I-mediated cleavage of C3b, increasing the dissociation rate of C3 convertase and C5 convertase.

[0368] The cofactor activity and dissociation accelerating activity of the FHL1 polypeptide or its fragment or derivative can be determined using any suitable method known to the skilled person, for example, those described above for complement factor H detection methods or any detection method described in Mannes, M. et al., 2020. Frontiers in Immunology, 11; 596415.

[0369] In some embodiments, FHL1 is human FHL1.

[0370] In some embodiments, the amino acid sequence of FHL1 is SEQ ID NO: 39. Preferably, the variant or fragment substantially retains the functional activity of the protein represented by SEQ ID NO: 39. Suitably, the fragment or variant of FHL1 may retain at least 50%, 60%, 70%, 80%, 90%, 95% or 100% of the cofactor activity and / or dissociation accelerating activity of the protein represented by SEQ ID NO: 39.

[0371]

[0372] An example of a nucleotide sequence encoding FHL1 is.

[0373]

[0374]

[0375] The nucleotide sequence of FHL1 used in the present invention is preferably codon-optimized.

[0376] A preferred nucleotide sequence encoding FHL1 is SEQ ID NO:41.

[0377]

[0378] In some embodiments, the nucleotide sequence encoding FHL1 has at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 40 or 41, preferably SEQ ID NO: 41. Preferably, the protein encoded by the nucleotide sequence substantially retains the functional activity of the protein represented by SEQ ID NO: 39.

[0379] In other embodiments, the nucleotide sequence encoding FHL1 is SEQ ID NO:40 or 41, preferably SEQ ID NO:41.

[0380] Complement receptor 1 (CR1)

[0381] Complement receptor 1 (CR1) mediates cellular binding to particles and immune complexes with activated complement.

[0382] In some embodiments, CR1 is human CR1.

[0383] In some embodiments, the amino acid sequence of CR1 is SEQ ID NO: 42. Preferably, the variant or fragment substantially retains the functional activity of the protein represented by SEQ ID NO:42.

[0384]

[0385] Membrane cofactor protein (MCP)

[0386] MCP acts as a cofactor for CFI.

[0387] In some embodiments, the MCP is human MCP.

[0388] In some embodiments, the amino acid sequence of MCP is SEQ ID NO: 43 or a variant or fragment thereof. Preferably, the variant or fragment substantially retains the functional activity of the protein represented by SEQ ID NO: 43.

[0389]

[0390] Connectors

[0391] In preferred embodiments, the nucleotide sequence encoding the anti-VEGF entity is upstream of the nucleotide sequence encoding the negative regulator of complement. In other embodiments, the nucleotide sequence encoding the negative regulator of complement is upstream of the nucleotide sequence encoding the anti-VEGF entity.

[0392] Here, "upstream" and "downstream" both refer to the relative position of the DNA or RNA. Each strand of the DNA or RNA has a 5' end and a 3' end, and by convention, "upstream" and "downstream" are related to the 5' to 3' direction in which RNA transcription occurs. For example, when considering double-stranded DNA, "upstream" is toward the 5' end of the coding strand and downstream is toward the 3' end of the coding strand.

[0393] In some embodiments, the nucleotide sequence encoding the anti-VEGF entity and the complement negative regulator are operably linked via a linker. In some embodiments, the linker comprises a self-cleaving 2A peptide sequence, such as a sequence comprising or defined by a Furin cleavage site, GSG, 11a1D, and F2A sequence.

[0394] In some embodiments, the linker is SEQ ID NO:44.

[0395]

[0396] In other embodiments, the linker is at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:44, and preferably, the linker substantially retains the functional activity of SEQ ID NO:44.

[0397] By "operably connected", it should be understood that the various components are connected together in a manner that enables them to perform their functions substantially unhindered.

[0398] product

[0399] For example, the product of the invention can be a composition (e.g., a pharmaceutical composition) comprising (i) an anti-VEGF entity; and (ii) a complement negative regulator, or a nucleotide sequence encoding thereof, mixed together. Alternatively, the product can be a kit comprising a formulation of (i) an anti-VEGF entity; and (ii) a complement negative regulator, or a nucleotide sequence encoding thereof, and, optionally, instructions for administering the formulation simultaneously, sequentially, or separately to a subject in need thereof.

[0400] Protein transduction

[0401] As an alternative to delivering polynucleotides to cells, the products and formulations of the invention may be delivered to cells by protein transduction.

[0402] Protein transduction can be delivered via vectors (Cai, Y. et al. (2014) Elife 3:e01911; Maetzig, T. et al. (2012) Curr. Gene Ther. 12:389-409). Vector delivery involves engineering of viral particles (such as lentiviral particles) to contain the protein to be delivered to the cell. Thus, when the engineered viral particles enter the cell as part of their natural life cycle, the protein contained in the particles is brought into the cell.

[0403] Protein transduction may be by protein delivery (Gaj, T. et al. (2012) Nat. Methods 9:805-7). For example, protein delivery can be achieved by using carriers such as liposomes or even by directly administering the protein itself into the cell.

[0404] Polynucleotide

[0405] The polynucleotides of the present invention may comprise DNA or RNA, preferably DNA. They may be single-stranded or double-stranded. It will be appreciated by the skilled artisan that, due to the degeneracy of the genetic code, many different polynucleotides may encode the same polypeptide. In addition, it will be appreciated that the skilled artisan may utilize conventional techniques to perform nucleotide substitutions that do not affect the polypeptide sequence encoded by the polynucleotides of the present invention to reflect the codon usage of any particular host organism in which the polypeptides of the present invention are to be expressed.

[0406] The nucleotide sequences of the invention disclosed herein may contain or lack a stop codon at their 3' end, for example depending on their position in a bicistronic vector. Thus, the present disclosure encompasses the SEQ ID NOs disclosed herein with or without a stop codon.

[0407] The polynucleotides may be modified by any method known in the art. Such modifications may be performed in order to increase the in vivo activity or lifespan of the polynucleotides of the present invention.

[0408] Polynucleotides such as DNA polynucleotides can be produced by recombinant, synthetic or any means available to those skilled in the art. They can also be cloned by standard techniques.

[0409] Longer polynucleotides are usually produced by recombinant methods, such as using polymerase chain reaction (PCR) cloning technology. This involves making a pair of primers (e.g., about 15 to 30 nucleotides), contacting the primers with mRNA or cDNA obtained from animal or human cells on both sides of the target sequence you want to clone, performing a polymerase chain reaction under conditions that amplify the desired region, separating the amplified fragments (e.g., by purifying the reaction mixture on an agarose gel) and recovering the amplified DNA. The primers can be designed to contain appropriate restriction endonuclease recognition sites so that the amplified DNA can be cloned into a suitable vector.

[0410] Polynucleotides can be delivered to cells via plasmid transfection or electroporation; for example as non-viral vectors such as naked DNA plasmids, bacterial artificial chromosomes, minicircle technology. These entities can also package larger transgenes than AAV vectors, but may be slightly inferior in terms of stability, bioavailability and target specificity. For example, Eyevensys uses a naked DNA electroporation platform to deliver therapeutic transgenes to muscles / eyes, etc.

[0411] In some embodiments, the polynucleotide comprises, or consists of, the nucleotide sequence of SEQ ID NO:45, or a nucleotide sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto.

[0412]

[0413]

[0414]

[0415] In some embodiments, the polynucleotide comprises or consists of the nucleotide sequence of SEQ ID NO:46, or a nucleotide sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto.

[0416]

[0417]

[0418]

[0419] In some embodiments, the polynucleotide comprises or consists of the nucleotide sequence of SEQ ID NO:47, or a nucleotide sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto.

[0420]

[0421]

[0422]

[0423] In some embodiments, the polynucleotide comprises or consists of the nucleotide sequence of SEQ ID NO:48, or a nucleotide sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto.

[0424]

[0425]

[0426]

[0427] In some embodiments, the polynucleotide comprises or consists of the nucleotide sequence of SEQ ID NO:49, or a nucleotide sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto.

[0428]

[0429]

[0430]

[0431] In some embodiments, the polynucleotide comprises or consists of the nucleotide sequence of SEQ ID NO:50, or a nucleotide sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto.

[0432]

[0433]

[0434]

[0435] In some embodiments, the polynucleotide comprises or consists of the nucleotide sequence of SEQ ID NO:51, or a nucleotide sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto.

[0436]

[0437]

[0438]

[0439] In some embodiments, the polynucleotide comprises or consists of the nucleotide sequence of SEQ ID NO:52, or a nucleotide sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto.

[0440]

[0441]

[0442] In some embodiments, the polynucleotide comprises or consists of the nucleotide sequence of SEQ ID NO:53, or a nucleotide sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto.

[0443]

[0444]

[0445] In some embodiments, the polynucleotide comprises or consists of the nucleotide sequence of SEQ ID NO:54, or a nucleotide sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto.

[0446]

[0447]

[0448]

[0449] The structure of the eye

[0450] The medicaments disclosed herein can be delivered to the eye of a mammal, preferably a human, for the treatment or prevention of an eye disease, such as age-related macular degeneration (AMD).

[0451] Those skilled in the art of eye disease treatment will have a detailed and thorough understanding of the structure of the eye. However, the following structures that are particularly relevant to the present invention will be described.

[0452] Retina

[0453] The retina is a multilayered tissue that lines the inner posterior chamber of the eye and perceives images of the visual world, which are communicated to the brain via the optic nerve. In order from the inside of the eye to the outside, the retina contains the neurosensory retina and the retinal pigment epithelium, with the choroid located distal to the retinal pigment epithelium.

[0454] Neuroreceptor retinal and photoreceptor cells

[0455] The neuroreceptor retina contains photoreceptor cells that directly sense light. It contains the following layers: inner limiting membrane (ILM); nerve fiber layer; ganglion cell layer; inner plexiform layer; inner nuclear layer; outer plexiform layer; outer nuclear layer (nuclei of the photoreceptors); external limiting membrane (ELM); and the inner and outer portions of the rods and cones.

[0456] Technicians will have a detailed understanding of photoreceptor cells. In short, photoreceptor cells are specialized neurons located in the retina that convert light into biological signals. Photoreceptor cells contain rods and cones, which are distributed throughout the retina in different patterns.

[0457] Rods are mainly found in the peripheral part of the retina. They are highly sensitive and provide vision in low light. On average, a normal person has about 125 million rods in their retina.

[0458] Cones are found throughout the retina, but are particularly highly concentrated in the fovea (a depression in the neurosensory retina responsible for central, high-resolution vision). Cones are less sensitive than rods and are responsible for color vision. The average human retina has about 6-7 million cones.

[0459] Retinal Pigment Epithelium

[0460] The retinal pigment epithelium (RPE) is a cellular pigment layer just outside the neurosensory retina, adjacent to the photoreceptor outer segments. The RPE separates the neurosensory retina from the choroidal vasculature and has functions such as recycling visual pigments from the photoreceptor outer segments. The RPE performs a number of functions, including transporting nutrients and other substances to the photoreceptor cells and absorbing scattered light to improve vision.

[0461] Choroid

[0462] The choroid is a vascular layer located between the RPE and the outer sclera of the eye. The blood vessels of the choroid supply oxygen and nutrients to the retina.

[0463] Age-related macular degeneration (AMD)

[0464] The clinical progression of age-related macular degeneration (AMD) is characterized by multiple stages based on changes in the macula. Early AMD is characterized by the appearance of drusen, which are accumulations of extracellular debris under the retina that appear as yellow spots on the retina during clinical examination and on fundus photographs. Drusen can be classified by size as small (<63 microns), medium (63-124 microns), and large (>124 microns). They are also considered hard or soft based on the appearance of their edges during fundus examination. Hard drusen have clearly defined edges, while soft drusen have less defined, fluid edges. The Age-Related Eye Disease Study (AREDS) Fundus Photography Severity Scale is one of the main classification systems used for this condition.

[0465] AMD is divided into two forms of "dry" and "wet" (exudative or neovascular). The typical feature of dry AMD is the cell of RPE layer, the overlying photoreceptor cells and the bottom cells of choriocapillaris layer that often also have to lose. The dense area of ​​RPE cell death is accompanied by the atrophy of overlying photoreceptors, and is called geographic atrophy (GA). Patients who suffer from this form of AMD, in which visual acuity can appear slow and progressive deterioration.

[0466] Wet AMD is characterized by bleeding and / or leaking of fluid from abnormal blood vessels that grow from the RPE and choroidal vessels beneath the macula (choriocapillaris layer), which may be the cause of sudden vision loss. It is estimated that most of the vision loss in patients is due to this choroidal neovascularization (CNV) and its secondary complications.

[0467] The treatment or prevention of AMD described herein can reduce or prevent the appearance of the above-mentioned AMD phenotypes.Preferably, the treatment of AMD can maintain or improve visual function.

[0468] In some embodiments, the result of treating or preventing AMD is to prevent or reduce the formation of geographic atrophy.In other embodiments, the result of treating or preventing AMD is to slow down the progress of geographic atrophy.For example, within 12 months after being applied to the treated eyes of the experimenter, relative to the untreated eyes of the same period, it causes the increase of GA area to reduce at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90%.In other embodiments, the result of treating or preventing AMD is to treat geographic atrophy, for example, reduce the quantity of geographic atrophy.

[0469] In some embodiments, treating or preventing AMD results in preventing or reducing the formation of drusen. In other embodiments, treating or preventing AMD results in reducing existing drusen, such as reducing the size and / or number of existing drusen.

[0470] In some embodiments, treating or preventing AMD results in preventing or reducing complement deposition. In other embodiments, treating or preventing AMD results in reducing existing complement deposition.

[0471] In some embodiments, treating or preventing AMD results in improved or restored vision or visual acuity. In other embodiments, treating or preventing AMD reduces the loss of vision or visual acuity.

[0472] In some embodiments, the treatment or prevention of AMD results in an improvement or restoration of the subject's reading speed. In other embodiments, the treatment or prevention of AMD reduces a decrease in the subject's reading speed.

[0473] In some embodiments, treating or preventing AMD results in reducing or preventing the loss of photoreceptors and / or retinal pigment epithelium (RPE).

[0474] Diabetic retinopathy

[0475] Diabetic retinopathy is a disease 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.

[0476] Although a subject with mild diabetic retinopathy may have good vision, certain types of diabetic retinopathy, namely diabetic macular edema (DMO) and proliferative diabetic retinopathy (PDR), may threaten a subject's vision.

[0477] Diabetic macular edema is characterized by fluid leaking from damaged blood vessels at the back of the eye. The leaking fluid accumulates in the macula, causing swelling and blurred vision. This eventually leads to poor central vision, making it difficult to read or drive. Vision on the side usually remains normal.

[0478] Proliferative diabetic retinopathy is characterized by the closure of retinal blood vessels, resulting in the growth of abnormal, fragile blood vessels on the surface of the retina. This can lead to permanent loss of vision due to bleeding into the eye, scarring, and retinal detachment. Nonproliferative retinopathy is an early stage of diabetic retinopathy and can lead to proliferative retinopathy if left untreated. Therefore, treatment is considered for all stages and types of diabetic retinopathy.

[0479] Carrier

[0480] A vector is a tool that allows or facilitates the transfer of an entity from one environment to another.

[0481] Adeno-associated virus (AAV) vector

[0482] In one aspect, the invention provides an AAV vector comprising a polynucleotide of the invention.

[0483] Preferably, the AAV vector is in the form of an AAV vector particle.

[0484] Methods for making and modifying viral vectors and viral vector particles, such as those derived from AAV, are well known in the art.

[0485] The AAV vector may comprise the AAV genome or a fragment or derivative thereof.

[0486] A polynucleotide comprising nucleotides encoding (i) an anti-VEGF entity and (ii) a negative complement regulator provided by the present invention can be suitable for packaging in AAV. AAV is known to be able to package a genome of 5.2 kb in size (Dong, J.-Y. et al. (1996) Human Gene Therapy 7:2101-2112).

[0487] Thus, the nucleotide sequences encoding the anti-VEGF entity and the complement negative regulator can be selected so that the combination is suitable for packaging in AAV. In certain embodiments, functional fragments of the anti-VEGF entity and / or the complement negative regulator (as described herein) can be used to ensure that the polynucleotide is suitable for packaging in AAV.

[0488] In some embodiments, the polynucleotide is less than or equal to 5.2, 5.1, 5.0, 4.9, 4.8 or 4.7 kb. In a preferred embodiment, the polynucleotide is less than or equal to 4.7 kb.

[0489] The AAV genome is a polynucleotide sequence that encodes the functions required for the production of AAV particles. These functions include the replication and packaging cycle of AAV in the host cell, including the encapsulation of the AAV genome into AAV particles. Naturally occurring AAV is replication-defective and relies on the provision of trans-acting auxiliary functions to complete the replication and packaging cycle. Therefore, the AAV genome of the AAV vector of the present invention is generally replication-defective.

[0490] The AAV genome can be single-stranded, either positive-sense or negative-sense, or double-stranded.

[0491] The AAV genome can be from any naturally derived AAV serotype, isolate or branch. Thus, the AAV genome can be the complete genome of a naturally occurring AAV. As known to the skilled artisan, AAV occurring in nature can be classified according to various biological systems.

[0492] AAVs are usually referred to by their serotype. A serotype is equivalent to a variant subspecies of AAV that has unique reactivity due to the expression profile of its capsid protein surface antigens, which can be used to distinguish it from other variant subspecies. In general, viruses with a specific AAV serotype will not effectively cross-react with specific neutralizing antibodies against any other AAV serotype.

[0493] 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 were recently discovered from primate brains. Any of these AAV serotypes can be used in the present invention.

[0494] In some embodiments, the AAV vector particle is an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, Rec2, Rec3, Rh10, DJ, or Anc65 AAV vector particle.

[0495] In some embodiments, the AAV vector particle may comprise an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, or AAV11 capsid protein.

[0496] In some embodiments, the AAV can be an AAV1, AAV2, AAV5, AAV7, or AAV8 serotype.

[0497] In some embodiments, the AAV can be of the AAV2 or AAV8 serotype.

[0498] In some embodiments, the AAV may be of the AAV2 serotype. In other preferred embodiments, the AAV may be of the AAV8 serotype.

[0499] The capsid protein may be a mutated capsid protein as disclosed in WO 2008 / 124724, which is incorporated herein by reference.

[0500] In some embodiments, the AAV vector comprises an AAV8 capsid protein having a Y733F mutation.

[0501] Reviews on AAV serotypes can be found in Choi et al. (2005) Curr. Gene Ther. 5:299-310 and Wu et al. (2006) Molecular Therapy 14:316-27. The sequence of the AAV genome or elements of the AAV genome (including ITR sequences, Rep or Cap genes) used in the present invention can be obtained from the accession numbers of the following AAV complete genome sequences: adeno-associated virus 1 NC_002077, AF063497; adeno-associated virus 2 NC_001401; adeno-associated virus 3 NC_001729; adeno-associated virus 3B NC_001863; adeno-associated virus 4 NC_001829; adeno-associated virus 5 Y18065, AF085716; adeno-associated virus 6 NC_001862; avian AAV ATCC VR-865 AY186198, AY629583, NC_004828; avian AAV strain DA-1 NC_006263, AY629583; bovine AAV NC_005889, AY388617.

[0502] AAVs may also be referred to as clades or clones. This refers to the phylogenetic relationships of naturally derived AAVs, usually referring to a phylogenetic group of AAVs that can be traced back to a common ancestor and contains all of its descendants. In addition, AAVs may be referred to as specific isolates, which are genetic isolates of a particular AAV found in nature. The term genetic isolate describes an AAV population that has had limited genetic mixing with other naturally occurring AAVs, thereby defining an identifiable, unique population at the genetic level.

[0503] The skilled person can select the appropriate serotype, branch, clone or isolate of AAV for use in the present invention according to their common sense. For example, AAV5 capsid has been shown to effectively transduce cone photoreceptors of primates and successfully correct hereditary color vision defects (Mancuso et al. (2009) Nature 461:784-7).

[0504] The serotype of AAV determines the tissue specificity (or tropism) of AAV infection. Therefore, preferred AAV serotypes for AAV administered to patients according to the present invention are those that have a natural tropism for target cells in the eye or have a higher infection efficiency. In some embodiments, the AAV serotypes used in the present invention are those that can transduce cells of the neurosensory retina, retinal pigment epithelium, and / or choroid.

[0505] Typically, the AAV genome of a naturally derived AAV serotype, isolate or branch contains at least one inverted terminal repeat (ITR). The function of the ITR sequence is to provide a functional replication origin in cis and allow the vector to integrate and excise from the genome of the cell. In a preferred embodiment, one or more ITR sequences flank the nucleotide sequence encoding the anti-VEGF entity and the complement negative regulator. The AAV genome also typically contains packaging genes, such as the rep and / or cap genes that encode the AAV particle packaging function. The rep gene encodes one or more proteins of Rep78, Rep68, Rep52 and Rep40 or their variants. The cap gene encodes one or more capsid proteins, such as VP1, VP2 and VP3 or their variants. These proteins constitute the capsid of the AAV particle. Variants of the capsid will be discussed below.

[0506] The promoter will be operably linked to each packaging gene. Specific examples of such promoters include p5, p19 and p40 promoters (Laughlin et al. (1979) Proc. Natl. Acad. Sci. USA 76: 5567-5571). For example, p5 and p19 promoters are generally used to express rep genes, while p40 promoters are generally used to express cap genes.

[0507] As described above, the AAV genome used in the AAV vector of the present invention can therefore be a complete genome of a naturally occurring AAV. For example, vectors containing a complete AAV genome between them can be used to prepare AAV vectors or vector particles in vitro. However, although such vectors can in principle be administered to patients, this is rarely done in practice. Preferably, the AAV genome will be derivatized for the purpose of administration to patients. Such derivatization is standard in the art, and the present invention encompasses the use of any known derivatives of the AAV genome, as well as derivatives produced by applying techniques known in the art. Derivatization of the AAV genome and AAV capsid is reviewed in Coura and Nardi (2007) Virology Journal 4:99 and in Choi et al. and Wu et al. mentioned above.

[0508] The derivative of the AAV genome includes any truncated or modified form of the AAV genome, which allows the AAV vector of the present invention to express transgenes in vivo. Generally, the AAV genome can be truncated significantly to contain minimal viral sequences, but retain the above functions. For safety reasons, this is preferred to reduce the risk of recombination between the vector and the wild-type virus, while also avoiding the presence of viral gene proteins in the target cells that trigger a cellular immune response.

[0509] Typically, a derivative will contain at least one inverted terminal repeat (ITR), preferably more than one ITR, such as two or more ITRs. One or more ITRs can be from an AAV genome with a different serotype, or a chimeric or mutant ITR. A preferred mutant ITR is an ITR with a trs (terminal resolution site) deletion. This deletion allows the genome to continue to replicate, producing a single-stranded genome containing coding and complementary sequences, i.e., a self-complementary AAV genome. This allows DNA replication in the target cell to be bypassed, thereby accelerating the expression of the transgene.

[0510] One or more ITRs preferably flank the nucleotide sequence encoding the anti-VEGF entity and the complement negative regulator. The inclusion of one or more ITRs is preferred to assist in forming concatemers in the nucleus of the host cell in order to help the vector of the present invention, for example, after the single-stranded vector DNA is converted to double-stranded DNA by the action of the host cell DNA polymerase. The formation of such episomal concatamers protects the vector construct during the life of the host cell, thereby allowing for long-term expression of the transgene in vivo.

[0511] In a preferred embodiment, the ITR element will be the only sequence from the native AAV genome retained in the derivative. Therefore, the derivative preferably does not contain the rep and / or cap genes of the native genome and any other sequence of the native genome. This is preferred for the reasons mentioned above and is to reduce the possibility of vector integration into the host cell genome. In addition, reducing the size of the AAV genome can increase the flexibility of adding other sequence elements (such as regulatory elements) in addition to the transgene in the vector.

[0512] Therefore, the following parts can be removed in the derivatives of the present invention: replication (rep) and capsid (cap) genes. However, in some embodiments, the derivatives may additionally contain one or more Rep and / or Cap genes or other viral sequences of the AAV genome. Naturally occurring AAV integrates at a high frequency at a specific site on human chromosome 19 and shows a negligible random integration frequency, so the retention of integration ability in the vector can be tolerated in a therapeutic environment.

[0513] When the derivative comprises capsid proteins, i.e., VP1, VP2 and / or VP3, the derivative may be a chimeric, shuttle or capsid-modified derivative of one or more naturally occurring AAVs. In particular, the present invention comprises providing capsid protein sequences of AAVs from different serotypes, branches, clones or isolates in the same vector (i.e., pseudotype vector).

[0514] Chimeric, shuttle-type or capsid-modified derivatives are often selected to provide one or more desired functions to AAV vectors. Therefore, these derivatives can show higher gene delivery efficiency, reduced immunogenicity (humoral or cellular), altered tropism range and / or improved targeting to specific cell types compared to AAV vectors containing naturally occurring AAV genomes, such as AAV2 or AAV8 genomes. Improved gene delivery efficiency can be achieved by improving receptor or co-receptor binding on the cell surface, improving internalization, improving intracellular and intranuclear transport, improving viral particle uncoating and improving conversion of single-stranded genomes to double-stranded forms. Improved efficiency may also be associated with altered trophic range or targeting of specific cell populations so that the dose of the vector is not diluted by administration to unwanted tissues.

[0515] Chimeric capsid proteins include those produced by recombination between two or more capsid coding sequences of naturally occurring AAV serotypes. This can be done, for example, by a marker rescue approach, where a non-infectious capsid sequence of one serotype is co-transfected with a capsid sequence of a different serotype, and directed selection is used to select for capsid sequences with desired properties. The capsid sequences of different serotypes can be altered by homologous recombination in cells to produce new chimeric capsid proteins.

[0516] Chimeric capsid proteins also include those produced by engineering the capsid protein sequence to transfer specific capsid protein domains, surface loops or specific amino acid residues between two or more capsid proteins, such as between two or more capsid proteins of different serotypes.

[0517] Shuttled or chimeric capsid proteins can also be generated by DNA shuttling or by error-prone PCR. Hybrid AAV capsid genes can be generated by randomly fragmenting the sequences of related AAV genes, such as genes encoding capsid proteins of multiple different serotypes, and then recombining these fragments in a self-excited polymerase reaction, which may also result in crossover of regions of sequence homology. A mixed AAV gene library generated by shuttling capsid protein genes of several serotypes in this way can be screened to identify viral clones with desired functions. Similarly, error-prone PCR can be used to randomly mutate the capsid genes of AAV to create a diverse library of variants that can then be selected based on desired properties.

[0518] The sequence of the capsid gene can also be genetically modified to introduce specific deletions, substitutions or additions relative to the native wild-type sequence. In particular, the capsid gene can be modified by inserting an unrelated protein or peptide sequence within the open reading frame of the capsid coding sequence, or at the N-terminus and / or C-terminus of the capsid coding sequence. For example, this can be done to confer or alter tropism for a specific cell receptor.

[0519] The unrelated protein or peptide can be a protein or peptide that advantageously acts as a ligand for a particular cell type, thereby conferring improved binding to the target cell or improving the specificity of the vector's targeting to a particular cell population. An example may include the use of an RGD peptide to block uptake by retinal pigment epithelial cells, thereby improving transduction of peripheral retinal tissue (Cronin et al. (2008) ARVO Abstract: D1048). Unrelated proteins can also be used as part of the production process to assist in the purification of viral particles, i.e., epitopes or affinity tags. The location of insertion is generally intended not to interfere with other functions of the viral particles, such as internalization and transport of the viral particles. Technicians can determine the appropriate insertion site based on their common sense. Choi et al. and Dalkara et al. (Science, Translational Medicine, 2013; 5 (189)) mentioned above disclose specific sites.

[0520] The present invention also encompasses providing sequences of the AAV genome in an order and structure different from the native AAV genome. The present invention also encompasses replacing one or more AAV sequences or genes with sequences of another virus or chimeric genes composed of sequences of more than one virus. Such chimeric genes may be composed of sequences of two or more related viral proteins from different viral species.

[0521] The AAV vector of the present invention may take the form of a nucleotide sequence comprising the AAV genome or a derivative thereof and a sequence encoding an anti-VEGF entity and a complement negative regulator transgene or a derivative thereof.

[0522] The AAV particles of the present invention include transcapsidated forms in which an AAV genome or derivative having ITRs of one serotype is packaged in a capsid of a different serotype. The AAV particles of the present invention also include mosaic forms in which a mixture of unmodified capsid proteins from two or more different serotypes constitutes the viral capsid. The AAV particles also include chemically modified forms with ligands adsorbed on the capsid surface. For example, such ligands may include antibodies for targeting specific cell surface receptors.

[0523] Thus, for example, the AAV particles of the invention include those having an AAV2 genome and AAV2 capsid proteins (AAV2 / 2), particles having an AAV2 genome and AAV5 capsid proteins (AAV2 / 5), and particles having an AAV2 genome and AAV8 capsid proteins (AAV2 / 8), as well as those having an AAV2 genome and capsid proteins of more than one serotype.

[0524] The AAV vector may contain multiple copies (eg, 2, 3, etc.) of the nucleotide sequences mentioned herein.

[0525] In some embodiments, the polynucleotide further comprises one or more AAV ITRs. In preferred embodiments, the polynucleotide further comprises two AAV ITRs. In some embodiments, the polynucleotide comprises one AAV ITR at its 5' end and one AAV ITR at its 3' end. In some embodiments, the AAV ITR is an AAV2 or AAV8 ITR. In preferred embodiments, the AAV ITR is an AAV2 ITR.

[0526] In some embodiments, the polynucleotide comprises a 5' AAV ITR having a nucleotide sequence of SEQ ID NO:55, or a nucleotide sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.

[0527]

[0528] In some embodiments, the polynucleotide further comprises a nucleotide sequence of SEQ ID NO:56, or a nucleotide sequence at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto, immediately 3' to the 5' ITR.

[0529]

[0530] In some embodiments, the polynucleotide comprises a 5' AAV ITR having a nucleotide sequence of SEQ ID NO: 65, or a nucleotide sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.

[0531]

[0532] In some embodiments, the polynucleotide comprises a 3' AAV ITR having a nucleotide sequence of SEQ ID NO:57, or a nucleotide sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.

[0533]

[0534] In some embodiments, the polynucleotide further comprises a nucleotide sequence of SEQ ID NO:58, or a nucleotide sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto, immediately 5' to the 3' ITR.

[0535]

[0536] In some embodiments, the polynucleotide comprises a 3' AAV ITR having a nucleotide sequence of SEQ ID NO:66, or a nucleotide sequence that is at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0537]

[0538] Promoter and regulatory sequences

[0539] The polynucleotides or vectors of the invention may also contain elements that allow for expression of an anti-VEGF entity and / or a negative complement regulator transgene in vitro or in vivo. These may be referred to as expression control sequences. Thus, a polynucleotide or vector typically comprises an expression control sequence (e.g., comprising a promoter sequence) operably linked to a nucleotide sequence encoding a transgene.

[0540] In some embodiments, the polynucleotide or vector comprises a nucleotide sequence encoding: (a) a CMV promoter or a CAG promoter, optionally wherein the CMV or CAG promoter is upstream of the nucleotide sequence encoding the anti-VEGF entity and / or the complement negative regulator; (b) a WPRE regulatory element, optionally wherein the WPRE regulatory element is downstream of the nucleotide sequence encoding the anti-VEGF entity and / or the complement negative regulator; and / or (c) a poly-A signal, such as a bovine growth hormone poly-A signal, optionally wherein the poly-A signal is downstream of the nucleotide sequence encoding the anti-VEGF entity and / or the complement negative regulator.

[0541] In some embodiments, the polynucleotide or vector comprises:

[0542] (a) 5' AAV ITR;

[0543] (b) a promoter, optionally a CMV promoter;

[0544] (c) a nucleotide sequence encoding a negative regulator of complement;

[0545] (d) a linker, optionally wherein the linker comprises or is defined by a Furin cleavage site, GSG, 11a1D and F2A sequence;

[0546] (e) a nucleotide sequence encoding an anti-VEGF entity, preferably aflibercept;

[0547] (f) optionally a WPRE regulatory element, optionally a WPRE3 regulatory element;

[0548] (g) a poly-A signal, optionally a bovine growth hormone poly-A signal; and

[0549] (h) 3' AAV ITR.

[0550] The negative complement regulator may be selected from CFI and FHL-1.

[0551] Elements (c) and (e) may be in reciprocal positions.

[0552] Thus, in some embodiments, the polynucleotide or vector comprises:

[0553] (a) 5' AAV ITR;

[0554] (b) a promoter, optionally a CMV promoter;

[0555] (c) a nucleotide sequence encoding an anti-VEGF entity, preferably aflibercept;

[0556] (d) a linker, optionally wherein the linker comprises or is defined by a Furin cleavage site, GSG, 11a1D and F2A sequence;

[0557] (e) a nucleotide sequence encoding a negative regulator of complement;

[0558] (f) optionally a WPRE regulatory element, optionally a WPRE3 regulatory element;

[0559] (g) a poly-A signal, optionally a bovine growth hormone poly-A signal; and

[0560] (h) 3' AAV ITR.

[0561] In some embodiments, the polynucleotide or vector comprises:

[0562] (a) 5' AAV ITR;

[0563] (b) a promoter, optionally a CAG promoter;

[0564] (c) a nucleotide sequence encoding a negative complement regulator, preferably FHL-1;

[0565] (d) a linker, optionally wherein the linker comprises or is defined by a Furin cleavage site, GSG, 11a1D and F2A sequence;

[0566] (e) a nucleotide sequence encoding an anti-VEGF entity, preferably aflibercept;

[0567] (f) optionally a WPRE regulatory element, optionally a WPRE3 regulatory element;

[0568] (g) a poly-A signal, optionally a bovine growth hormone poly-A signal; and

[0569] (h) 3' AAV ITR.

[0570] Elements (c) and (e) may be in reciprocal positions.

[0571] Thus, in some embodiments, the polynucleotide or vector comprises:

[0572] (a) 5' AAV ITR;

[0573] (b) a promoter, optionally a CAG promoter;

[0574] (c) a nucleotide sequence encoding an anti-VEGF entity, preferably aflibercept;

[0575] (d) a linker, optionally wherein the linker comprises or is defined by a Furin cleavage site, GSG, 11a1D and F2A sequence;

[0576] (e) a nucleotide sequence encoding a negative complement regulator, preferably FHL-1;

[0577] (f) optionally a WPRE regulatory element, optionally a WPRE3 regulatory element;

[0578] (g) a poly-A signal, optionally a bovine growth hormone poly-A signal; and

[0579] (h) 3' AAV ITR.

[0580] The polynucleotide may consist of (a)-(h) in order from 5' to 3'. Suitably, (a) is at the 5' end of the polynucleotide, and (h) is at the 3' end of the polynucleotide. Suitably, (b) is upstream of (c)-(g). Suitably, (f) is downstream of (b)-(e). Suitably, (g) is downstream of (b)-(f). Suitably, (c) is upstream of (d), and (e) is downstream of (d).

[0581] Any suitable promoter may be used and can be readily selected by the skilled artisan. The promoter sequence may be constitutively active (i.e., operable in any host cell background) or may be active only in a specific host cell environment, thereby allowing for 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 (e.g., a factor present in the host cell). In any case, when the vector is to be used therapeutically, it is preferred that the promoter should be functional in the target cell background.

[0582] In some embodiments, preferred promoters exhibit retinal cell-specific expression, so that the transgene is expressed only in a retinal cell population. Thus, expression of the promoter may be retinal cell-specific, for example confined to cells of the neurosensory retina and retinal pigment epithelium. The promoter may also initiate expression, particularly tissue or cell-specific expression, in ganglion cells, Mueller cells, photoreceptor cells, retinal pigment epithelial cells, and / or endothelial cells.

[0583] Preferred promoters that are not retinal cell specific include the chicken beta-actin (CBA) promoter, optionally in combination with a cytomegalovirus (CMV) enhancer element.An example for use in the present invention is the CAG promoter, such as that used in the rAVE expression cassette (GeneDetect.com).

[0584] In a preferred embodiment, the polynucleotide or vector comprises a CMV promoter.

[0585] An example of a CMV promoter sequence is.

[0586]

[0587]

[0588] In some embodiments, the polynucleotide or vector comprises a promoter, the nucleotide sequence of which is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 59. Preferably, the nucleotide sequence substantially retains the functional activity of the promoter represented by SEQ ID NO: 59.

[0589] In other embodiments, the polynucleotide or vector comprises a promoter having a nucleotide sequence of SEQ ID NO:60.

[0590] An example of a CAG promoter sequence is.

[0591]

[0592] In some embodiments, the polynucleotide or vector comprises a promoter, the nucleotide sequence of which is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 60. Preferably, the nucleotide sequence substantially retains the functional activity of the promoter represented by SEQ ID NO: 60.

[0593] In other embodiments, the polynucleotide or vector comprises a promoter having a nucleotide sequence of SEQ ID NO:60.

[0594] Examples of promoters based on human sequences that will induce retina-specific gene expression include rhodopsin kinase for rods and cones (Allocca et al. (2007) J. Virol. 81: 11372-80), PR2.1 for cones only (Mancuso et al. (2009) Nature 461: 784-7), and / or RPE65 for retinal pigment epithelial cells (Bainbridge et al. (2008) N. Engl. J. Med. 358: 2231-9), or VMD2 (Esumi et al. (2004) J. Biol. Chem. 279: 19064-73).

[0595] The polynucleotides or vectors of the present invention may also contain one or more additional regulatory sequences that may function before or after transcription. The regulatory sequences may be part of the native transgenic locus or may be heterologous regulatory sequences. The polynucleotides or vectors of the present invention may contain portions of the 5'-UTR or 3'-UTR from the native transgenic transcript.

[0596] A regulatory sequence is any sequence that promotes transgene expression, i.e., acts to increase transcript expression, improve mRNA nuclear export, or increase its stability. Such regulatory sequences include, for example, enhancer elements, post-transcriptional regulatory elements, and polyadenylation sites.

[0597] A preferred polyadenylation site is the bovine growth hormone poly-A (bGH poly-A) signal.

[0598] An example of a bovine growth hormone poly-A (bGH poly-A) signal is:

[0599]

[0600] Another example of a bovine growth hormone poly-A (bGH poly-A) signal is:

[0601]

[0602] In some embodiments, the polynucleotide or vector comprises a polyadenylation signal, the nucleotide sequence of which is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 61 or 62. Preferably, the nucleotide sequence substantially retains the functional activity of the polyadenylation signal represented by SEQ ID NO: 61 or 62.

[0603] In other embodiments, the polynucleotide or vector comprises a polyadenylation signal having a nucleotide sequence of SEQ ID NO: 61 or 62.

[0604] In the context of the polynucleotides or vectors of the invention, such regulatory sequences will be cis-acting. However, the invention also encompasses the use of trans-acting regulatory sequences located on other genetic structures.

[0605] A preferred post-transcriptional regulatory element for use in the AAV vectors of the present invention is the woodchuck hepatitis post-transcriptional regulatory element (WPRE) or a variant thereof.

[0606] An example of a WPRE is.

[0607]

[0608]

[0609] WPRE is a tripartite element containing γ, α and β elements arranged in the given order. A shortened version of WPRE containing only minimal γ and α elements (called WPRE3; Choi, J.-H. et al. (2014) Molecular Brain 7:17) can also be used in the present invention.

[0610] An example of a WPRE3 sequence is.

[0611]

[0612] In some embodiments, the polynucleotide or vector comprises a post-transcriptional regulatory element, and its nucleotide sequence is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 63 or 64. Preferably, the nucleotide sequence substantially retains the functional activity of the post-transcriptional regulatory element represented by SEQ ID NO: 63 or 64.

[0613] WPRE can be derived from a wild-type or modified woodchuck hepatitis virus posttranscriptional regulatory element (WPRE) sequence

[0614] In other embodiments, the polynucleotide or vector comprises a post-transcriptional regulatory element having a nucleotide sequence of SEQ ID NO: 63 or 64.

[0615] Another regulatory sequence that can be used in the polynucleotide or vector of the present invention is the scaffold-attachment region (SAR). A skilled person can easily select other regulatory sequences.

[0616] Dosage

[0617] The product, polynucleotide or vector of the present invention can be administered systemically (e.g., by peripheral intravenous infusion), or locally or regionally (e.g., by intrathecal injection into the central nervous system). In a preferred embodiment, the product, polynucleotide or vector is administered intraocularly.

[0618] The term "intraocular" refers to the interior of the eye, and thus intraocular administration refers to administration inside the eye of a subject.

[0619] In some embodiments, the product, polynucleotide or vector is injected into the subject's eye subretinal, directly retinal, suprachoroidal or intravitreal. In some embodiments, the administration is performed by a robot.

[0620] The volume of the injected pharmaceutical composition can be, for example, about 10-500 μL, such as about 50-500, 100-500, 200-500, 300-500, 400-500, 50-250, 100-250, 200-250 or 50-150 μL. For example, the volume can be about 10, 50, 100, 150, 200, 250, 300, 350, 400, 450 or 500 μL. Preferably, the volume of the injected pharmaceutical composition is 100 μL.

[0621] The skilled artisan will be familiar with and well-equipped to perform individual subretinal, direct retinal, suprachoroidal or intravitreal injections.

[0622] Preferably, the product, polynucleotide or vector is administered by subomental injection.

[0623] In some embodiments, the product, polynucleotide, vector, or pharmaceutical composition comprising the same is administered no more than once or no more than twice during the lifetime of the subject.

[0624] Subretinal injection

[0625] Subretinal injections are injections into the subretinal space, which is beneath the neurosensory retina. During a subretinal injection, the injected material is directed between the photoreceptor cells and the retinal pigment epithelium (RPE) layer and creates a space between the two.

[0626] When an injection is made through a small retinal incision, a retinal detachment may occur. The detached, raised layer of retina created by the injected material is called a "bleb."

[0627] The hole created by the subretinal injection must be small enough so that the injected solution does not appreciably flow back into the vitreous cavity after administration. Such backflow would be particularly problematic when injecting drugs, as the effects of the drug would be directed away from the target area. Preferably, the injection creates a self-sealing entry point in the neurosensory retina, i.e., once the injection needle is removed, the hole created by the needle reseals so that very little or substantially no injected material is released through the hole.

[0628] To facilitate this process, specialized subretinal injection needles are available on the market (e.g., DORC 41G Teflon subretinal injection needle, Dutch Ophthalmic Research Center International BV, Zuidland, The Netherlands). These are needles designed for performing subretinal injections.

[0629] Unless damage to the retina occurs during the injection procedure, and provided a sufficiently small needle is used, essentially all of the injected material will remain between the neuroreceptive retina and the RPE at the site of localized retinal detachment (i.e., will not flow back into the vitreous cavity). The fact that typical hemorrhages persist for a short period of time suggests that little of the injected material usually escapes into the vitreous. The spots may dissipate over a longer period of time as the injected material is absorbed.

[0630] Visualization of the eye, particularly the retina, such as using optical coherence tomography, can be performed prior to the procedure.

[0631] The volume of the injected pharmaceutical composition can be, for example, about 10-500 μL, such as about 50-500, 100-500, 200-500, 300-500, 400-500, 50-250, 100-250, 200-250, or 50-150 μL. For example, the volume can be about 10, 50, 100, 150, 200, 250, 300, 350, 400, 450, or 500 μL. Preferably, the volume of the injected pharmaceutical composition is 100 μL. Larger volumes may increase the risk of stretching the retina, while smaller volumes may make it difficult to see.

[0632] Two-step subretinal injection

[0633] The products, polynucleotides or vectors of the present invention can be delivered with improved accuracy and safety by using a two-step method, wherein a localized retinal detachment is formed by injecting a first solution subretina. The first solution does not contain the product, polynucleotide or vector. Then, a second subretinal injection is used to deliver the drug containing the product, polynucleotide or vector into the subretinal fluid of the blister produced by the first subretinal injection. Because the injection solution for delivering the drug is not used to detach the retina, a specific volume of solution can be injected in this second step.

[0634] In some embodiments, the subretinal injection of the vector comprises the following steps.

[0635] (a) administering to a subject by subretinal injection a solution in an amount effective to at least partially detach the retina to form a subretinal bleb, wherein the solution does not comprise a product, a polynucleotide, or a vector; and

[0636] (b) administering a pharmaceutical composition into the blister formed in step (a) by subretinal injection, wherein the drug comprises a product, a polynucleotide or a vector.

[0637] The volume of the at least partially detached retinal solution injected in step (a) can be, for example, about 10-1000 μL, such as about 50-1000, 100-1000, 250-1000, 500-1000, 10-500, 50-500, 100-500, 250-500 μL. The volume can be, for example, about 10, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900 or 1000 μL.

[0638] The volume of the pharmaceutical composition injected in step (b) can be, for example, about 10-500 μL, such as about 50-500, 100-500, 200-500, 300-500, 400-500, 50-250, 100-250, 200-250 or 50-150 μL. The volume can be, for example, about 10, 50, 100, 150, 200, 250, 300, 350, 400, 450 or 500 μL. Preferably, the volume of the pharmaceutical composition injected in step (b) is 100 μL. Larger volumes may increase the risk of stretching the retina, while smaller volumes may be difficult to see.

[0639] The solution not containing the drug (ie, the "solution" of step (a)) can be formulated similarly to the solution containing the drug, as described below. A preferred drug-free solution is balanced saline (BSS) or a similar buffered solution that matches the pH and osmotic pressure of the subretinal space.

[0640] Visualization of the retina during surgery

[0641] In some cases, such as in end-stage retinal degeneration, identification of the retina can be difficult because it is thin, transparent, and difficult to see amidst the damaged and heavily pigmented epithelium in which it resides. Geuder; Dorc) can easily identify the retinal hole opened for the retinal detachment step (i.e., step (a) in the two-step subretinal injection method of the present invention), so that the drug can be administered through the same hole without the risk of reflux into the vitreous cavity.

[0642] The use of blue vital dye also allows identification of any areas of the retina with thickened internal limiting membrane or epiretinal membrane, as injection through either of these structures will prevent clean access to the subretinal space. In addition, contraction of either of these structures in the immediate postoperative period can result in stretching of the retinal entry hole, which can lead to reflux of medication into the vitreous cavity.

[0643] Suprachoroidal injection

[0644] The products, polynucleotides or vectors of the present invention can be delivered to the suprachoroidal space by an ab externo approach using a microcatheter (e.g., see Peden et al. (2011) PLoS One 6(2): e17140). In this approach, a keratoconjunctival incision is performed to expose the bare sclera, followed by a dura mater incision to expose the bare choroid. A microcatheter (e.g., iTrack 250A from iScience Interventional, optionally connected to an illumination system, such as the iLumin laser diode-based micro-illumination system (iScience Interventional)) is introduced into the suprachoroidal space and advanced posterior to the optic disc. After manipulating the tip of the microcatheter to the desired location, the injected product, polynucleotide or vector forms a bleb within the retina and choroid.

[0645] Thus, in some embodiments, the product, polynucleotide or vector is delivered suprachoroidally by a method comprising (i) introducing a microcatheter into the suprachoroidal space; (ii) advancing the microcatheter within the space until the tip is proximate the affected area of ​​the retina; and (iii) injecting the product, polynucleotide or vector from the tip of the microcatheter to form a bleb.

[0646] In some embodiments, the above management procedures are directly executed by the robot.

[0647] Pharmaceutical composition and injection

[0648] The medicaments of the present invention, such as products, polynucleotides or vectors, can be formulated into pharmaceutical compositions. These compositions, in addition to the drug, can also contain pharmaceutically acceptable carriers, diluents, excipients, buffers, stabilizers or other materials known in the art. These materials should be non-toxic and should not interfere with the efficacy of the active ingredient. The exact nature of the carrier or other material can be determined by the technician according to the route of administration, for example, subretinal, direct retinal, suprachoroidal or intravitreal injection.

[0649] The pharmaceutical composition is usually in liquid form. Liquid pharmaceutical compositions generally include a liquid carrier, such as water, petroleum, animal or vegetable oil, mineral oil or synthetic oil. It may include physiological saline solution, magnesium chloride, glucose or other sugar solutions, or glycols such as ethylene glycol, propylene glycol or polyethylene glycol. In some cases, a surfactant may be used, such as 0.001% pluronic acid (PF68).

[0650] For injection at the site of disease, the active ingredient can be in the form of a pyrogen-free aqueous solution with appropriate pH, isotonicity and stability. The technician is well able to prepare suitable solutions using, for example, isotonic vehicles such as sodium chloride injection, Ringer's injection or lactated Ringer's injection. Preservatives, stabilizers, buffers, antioxidants and / or other additives can be added as needed.

[0651] For delayed release, the drug may be contained in a pharmaceutical composition formulated for sustained release, for example, in a microcapsule or liposome carrier system formed from biocompatible polymers according to methods known in the art.

[0652] Treatment

[0653] It should be understood that all treatments mentioned herein include curative, palliative and preventive treatments; although in the context of the present invention, references to prevention are more relevant to preventive treatments. Treatment may also include preventing the progression of the severity of the disease.

[0654] Treatment of mammals, particularly humans, is preferred. However, both human and veterinary treatments are within the scope of the present invention.

[0655] As used herein, the term "combination", or the terms "combination", "combined use" or "co-preparation" may refer to the simultaneous, sequential or separate co-administration of two or more agents.

[0656] The term "simultaneously" as used herein refers to administration at the same time, ie at the same time.

[0657] As used herein, the term "sequentially" means that the agents are administered one after the other.

[0658] The term "separately" as used herein means that the administration of the agents is independent of each other, but within a certain time interval, so that the agents show a combined, preferably synergistic effect. Therefore, "separately" administration can allow one agent to be administered within 1 minute, 5 minutes or 10 minutes after another agent.

[0659] Variants, derivatives, analogs, homologs and fragments

[0660] In addition to the specific proteins and nucleotides mentioned herein, the present invention also encompasses the use of variants, derivatives, analogs, homologs and fragments thereof.

[0661] 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 in such a way that the polypeptide or polynucleotide concerned substantially retains its function. Variant sequences can be obtained by adding, deleting, substituting, modifying, replacing and / or altering at least one residue in a naturally occurring protein.

[0662] The term "derivative" as used herein, with respect to a protein or polypeptide of the present invention, comprises any substitution, change, modification, replacement, deletion and / or addition to one (or more) amino acid residues in the sequence, as long as the resulting protein or polypeptide substantially retains at least one of its endogenous functions.

[0663] As used herein, the term "analog," with respect to a polypeptide or polynucleotide, encompasses any mimetic, ie, a chemical compound that possesses at least one endogenous function of the polypeptide or polynucleotide that it mimics.

[0664] Typically, amino acid substitutions can be made, for example from 1, 2 or 3 to 10 or 20 substitutions, as long as the modified sequence substantially retains the desired activity or ability. Amino acid substitutions can include the use of non-naturally occurring analogs.

[0665] The proteins used in the present invention may also have deletions, additions or substitutions of amino acid residues that produce silent changes and produce functionally equivalent proteins. Deliberate amino acid substitutions may be made based on similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or the amphipathic nature of the residues, as long as the endogenous function is retained. For example, negatively charged amino acids include aspartic acid and glutamic acid; positively charged amino acids include lysine and arginine; and amino acids with uncharged polar head groups of similar hydrophilicity values ​​include asparagine, glutamine, serine, threonine, and tyrosine.

[0666] Conservative substitutions can be made, 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, can be substituted for each other.

[0667]

[0668] The term "homolog" as used herein refers to an entity having a certain homology with a wild-type amino acid sequence and a wild-type nucleotide sequence. The term "homology" can be equivalent to "identity".

[0669] A homologous sequence may comprise an amino acid sequence which may be at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85% or 90% identical to the subject sequence, preferably at least 95% or 97% or 99% identical. Typically, a homologue will contain the same active sites etc. as the subject amino acid sequence. Although homology may also be considered in terms of similarity (i.e., amino acid residues having similar chemical properties / functions), in the context of the present invention, it is preferred to express homology in terms of sequence identity.

[0670] Homologous sequences may comprise a nucleotide sequence that is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85% or 90% identical to the subject sequence, and preferably at least 95%, 97% or 99% identical. Although homology may also be considered in terms of similarity, in the context of the present invention, homology is preferably expressed in terms of sequence identity.

[0671] Preferably, reference to a sequence having a percentage identity to any one of the SEQ ID NOs detailed herein refers to the sequence having that percentage identity over the entire length of the referenced SEQ ID NO.

[0672] Homology comparisons can be made by eye, or more often with the aid of readily available sequence comparison programs. These commercially available computer programs can calculate the homology or identity percentage between two or more sequences.

[0673] Percent homology can be calculated over contiguous sequences, i.e. one sequence is aligned with the other sequence and each amino acid in one sequence is directly compared with the corresponding amino acid in the other sequence, one residue at a time. This is called an "ungapped" alignment. Typically, such ungapped alignments are performed only over a relatively small number of residues.

[0674] Although this is a very simple and consistent approach, it does not take into account that, for example, within a pair of identical sequences, a single insertion or deletion in the nucleotide sequence may cause subsequent codons to not be aligned, potentially resulting in a large reduction in the homology percentage when a global alignment is performed. Therefore, most sequence comparison methods are designed to produce an optimal alignment that takes into account possible insertions and deletions without unduly penalizing the overall homology score. This is achieved by inserting "gaps" in the sequence alignment to maximize local homology.

[0675] However, these more sophisticated methods impose a "gap penalty" on each gap that occurs in the alignment, so that for the same number of identical amino acids, a sequence alignment with as few gaps as possible, reflecting a higher relatedness between the two sequences being compared, will score higher than one with many gaps. A commonly used "Affine gapcost" is one that charges a relatively high cost for the presence of a gap, and a smaller penalty for each subsequent residue in the gap. This is the most commonly used gap scoring system. A high gap penalty will of course produce an optimized alignment with few gaps. Most alignment programs allow the gap penalty to be modified. However, when using such software for sequence comparisons, it is best 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 stretch.

[0676] Therefore, calculating the maximum homology percentage first requires making an optimal alignment, taking into account the penalty for gaps. A suitable computer program for performing such an alignment is the Bestfit package of GCG Wisconsin (University of Wisconsin, USA; Devereux et al. (1984) Nucleic Acids Res. 12: 387). Other examples of software that can perform sequence comparisons include, but are not limited to, the BLAST package (see Ausubel et al. (1999) supra-Chapter 18), FASTA (Atschul et al. (1990) J. Mol. Biol. 403-410) and the GENEWORKS comparison tool suite. Both BLAST and FASTA can be used for offline and online retrieval (see Ausubel et al. (1999) supra, pages 7-58 to 7-60). However, for some applications, it is preferred to use the GCG Bestfit program. Another tool, called BLAST 2 Sequences, can also be used to compare protein and nucleotide sequences (see FEMS Microbiol. Lett. (1999) 174:247-50; FEMS Microbiol. Lett. (1999) 177:187-8).

[0677] Although the final homology percentage can be measured in terms of identity, the alignment process itself is not usually based on an all-or-nothing pairwise comparison. Instead, a scaled similarity score matrix is ​​often used, assigning scores to each pairwise comparison based on chemical similarity or evolutionary distance. An example of such a matrix commonly used is the BLOSUM62 matrix - the default matrix for the BLAST programs. GCG Wisconsin programs typically use either public defaults or a custom symbol comparison table if provided (see the user manual for further details). For some applications, it is better to use the public defaults for GCG software packages, or in the case of other software, use a default matrix such as BLOSUM62.

[0678] Once the software has produced an optimal alignment, the percent homology, preferably the percent sequence identity, can be calculated. The software usually does this as part of the sequence alignment and produces a numerical result.

[0679] "Fragment" is also a variant, and the term generally refers to a selected region of a polypeptide or polynucleotide that is functionally or, for example, useful in detection. Thus, a "fragment" refers to an amino acid or nucleic acid sequence that is a portion of a full-length polypeptide or polynucleotide.

[0680] Such variants can be prepared with standard recombinant DNA techniques, such as site-directed mutagenesis. Where insertion is desired, synthetic DNA encoding the insert can be made, as well as 5' and 3' flanking regions corresponding to the naturally occurring sequence on both sides of the insertion point. The flanking regions will contain convenient restriction sites corresponding to the sites in the naturally occurring sequence, so that the sequence can be cut with an appropriate enzyme, and the synthetic DNA can be connected to the cut. Then according to the present invention, DNA is expressed to make the encoded protein.

[0681] These methods are merely illustrative of the many standard techniques known in the art for manipulating DNA sequences, and other known techniques may also be used.

[0682] Aspects of the Invention

[0683] Various aspects of the invention are defined in the following numbered paragraphs (paragraphs).

[0684] 1. A product comprising (i) an anti-VEGF entity; and (ii) a negative complement regulator, or a nucleotide sequence encoding the same, as a combined preparation for simultaneous, separate or sequential use in therapy.

[0685] 2. The product according to paragraph 1 is for use in therapy, wherein the disease to be treated or prevented is an ocular disorder.

[0686] 3. The product according to paragraph 2 for use in treatment, wherein the ocular disorder is a complement-mediated disorder of the eye.

[0687] 4. The product according to paragraph 3 for use in treatment, wherein the complement-mediated disorder is age-related macular degeneration (AMD), diabetic retinopathy, glaucoma, Stargardt's disease, central serous choroidopathy, retinitis pigmentosa, polypoidal choroidal vasculopathy, diabetic macular edema, branch retinal vein occlusion or uveitis, preferably AMD.

[0688] 5. The product according to paragraph 4 is used for treatment, wherein the AMD is wet AMD and / or dry AMD.

[0689] 6. The product according to paragraph 4 for use in treatment, wherein the AMD is wet AMD, and said use further prevents and / or treats the onset of dry AMD in said subject.

[0690] 7. A product according to any of paragraphs 1 to 6 for use in therapy, wherein the product comprises a polynucleotide comprising a nucleotide sequence encoding (i) an anti-VEGF entity and (ii) a negative complement regulator.

[0691] 8. An isolated polynucleotide comprising a nucleotide sequence encoding (i) an anti-VEGF entity and (ii) a negative complement regulator.

[0692] 9. A product for use in therapy according to paragraph 7 or a polynucleotide isolated according to paragraph 8, wherein the anti-VEGF entity is selected from the group consisting of an Ig fusion protein, an antibody, a polypeptide, a peptide, a non-antibody scaffold, an antisense oligonucleotide, a siRNA, a shRNA, a CRISPR guide strand and an aptamer.

[0693] 10. A product for use in therapy or an isolated polynucleotide according to paragraph 9, wherein the anti-VEGF entity is selected from aflibercept, ranibizumab, bevacizumab and pegaptanib.

[0694] 11. An isolated polynucleotide according to paragraph 10, wherein the anti-VEGF entity is aflibercept, for example wherein aflibercept is encoded by a polynucleotide sequence comprising any one of SEQ ID NOs: 3 to 11.

[0695] 12. A product for use in treatment or an isolated polynucleotide according to any of the preceding paragraphs, wherein the negative complement regulator is selected from complement factor I (CFI), complement factor H-like protein 1 (FHL1), complement factor H (CFH), complement receptor type 1 (CR1), membrane cofactor protein (MCP), complement decay accelerating factor (DAF), MAC-inhibitory protein (MAC-IP), C1-inhibitor, allergic protein inhibitor, C4b binding protein (C4BP), clusterin, vitronectin or variants or fragments thereof.

[0696] 13. A product for use in therapy or an isolated polynucleotide according to paragraph 12, wherein the negative complement regulator is selected from CFI and FHL1 or variants or fragments thereof.

[0697] 14. A product according to any one of paragraphs 7 to 13 for use in therapy or an isolated polynucleotide, wherein the polynucleotide further comprises a nucleotide sequence encoding:

[0698] (a) CMV or CAG promoter, optionally wherein the CMV or CAG promoter is upstream of the nucleotide sequence encoding (i) and (ii).

[0699] (b) a WPRE regulatory element, optionally wherein the WPRE regulatory element is located downstream of the nucleotide sequence encoding (i) and (ii); and / or

[0700] (c) a poly-A signal, optionally a bovine growth hormone poly-A signal, wherein the poly-A signal is optionally downstream of the nucleotide sequence encoding (i) and (ii).

[0701] 15. A product according to any of paragraphs 7 to 14 for use in therapy or an isolated polynucleotide, wherein the polynucleotide further comprises one or more adeno-associated virus (AAV) inverted terminal repeats (ITRs).

[0702] 16. A product according to any one of paragraphs 7 to 15 for use in therapy or an isolated polynucleotide, wherein the polynucleotide comprises one AAV ITR at its 5' end and one AAV ITR at its 3' end.

[0703] 17. A product according to any one of paragraphs 7 to 16 for use in therapy or an isolated polynucleotide, wherein the polynucleotide comprises:

[0704] (a) 5' AAV ITR;

[0705] (b) CMV or CAG promoter;

[0706] (c) a nucleotide sequence encoding an anti-VEGF entity;

[0707] (d) a linker, optionally wherein the linker comprises or is defined by a Furin cleavage site, GSG, 11a1D and F2A sequence;

[0708] (e) a nucleotide sequence encoding a negative regulator of complement;

[0709] (f) optionally a WPRE regulatory element, further optionally wherein the WPRE regulatory element is a WPRE3 regulatory element;

[0710] (g) bovine growth hormone poly-A signal; and

[0711] (h) 3' AAV ITR.

[0712] 18. A product according to any one of paragraphs 7 to 17 for use in therapy or an isolated polynucleotide, wherein the AAV ITR is an AAV2 ITR.

[0713] 19. A product for use in therapy or an isolated polynucleotide according to any one of paragraphs 7 to 18, wherein the nucleotide sequence encoding the anti-VEGF entity and the complement negative regulator is codon optimized.

[0714] 20. A product for use in therapy or an isolated polynucleotide according to any one of paragraphs 7 to 19, wherein the nucleotide sequence encoding aflibercept has at least 75% sequence identity to SEQ ID NO: 11.

[0715] 21. A product for use in therapy or an isolated polynucleotide according to any one of paragraphs 7 to 20, wherein the nucleotide sequence encoding CFI has at least 75% sequence identity with SEQ ID NO: 35 or 36, or the nucleotide sequence encoding FHL-1 has at least 75% sequence identity with SEQ ID NO: 41.

[0716] 22. A product for use in therapy or an isolated polynucleotide according to any one of paragraphs 7 to 20, wherein the polynucleotide comprises a nucleotide sequence of SEQ ID NO: 45 to 54, or a nucleotide sequence having at least 75% sequence identity thereto.

[0717] 23. A product according to any one of paragraphs 7 to 22 for use in therapy or an isolated polynucleotide, wherein the polynucleotide is less than or equal to 4.7 kb.

[0718] 24. A vector comprising the polynucleotide of any one of paragraphs 8 to 23.

[0719] 25. The vector of paragraph 24, wherein the vector is an adeno-associated virus (AAV) vector.

[0720] 26. The vector of paragraph 24 or 25, wherein the vector is in the form of a viral vector particle.

[0721] 27. The vector of paragraph 26, wherein the AAV vector particle comprises an AAV2 or AAV8 genome, and an AAV2 or AAV8 capsid protein.

[0722] 28. A cell comprising the polynucleotide of any one of paragraphs 7 to 22.

[0723] 29. A cell transduced with the vector of any one of paragraphs 24 to 27.

[0724] 30. A pharmaceutical composition comprising the isolated polynucleotide, vector or cell of any one of paragraphs 8 to 29 in combination with a pharmaceutically acceptable carrier, diluent or excipient.

[0725] 31. The isolated polynucleotide, vector or cell of any one of paragraphs 8 to 29 for use in therapy.

[0726] 32. The isolated polynucleotide, vector or cell of any one of paragraphs 8 to 29 for use in treating or preventing a complement-mediated disorder of the eye.

[0727] 33. The isolated polynucleotide, vector or cell according to paragraph 32 for use in treatment or prevention, wherein the complement-mediated disorder is age-related macular degeneration (AMD), diabetic retinopathy, glaucoma, Stargardt's disease, central serous choroidopathy, retinitis pigmentosa, polypoidal choroidal vasculopathy, diabetic macular edema, branch retinal vein occlusion or uveitis, preferably AMD.

[0728] 34. The isolated polynucleotide, vector or cell according to paragraph 32 for use in treatment or prevention, wherein the AMD is wet AMD and / or dry AMD.

[0729] 35. The isolated polynucleotide, vector or cell according to paragraph 33 for use in treatment or prevention, wherein the AMD is wet AMD and the use further prevents and / or treats the onset of dry AMD in the subject.

[0730] 36. A polynucleotide, vector or cell according to any of paragraphs 32 to 35 for use in treatment or prevention, wherein the development of geographic atrophy is prevented or reduced, and / or the total amount of geographic atrophy is reduced.

[0731] 37. A polynucleotide, vector or cell according to any of paragraphs 32 to 36 for use in treatment or prevention, wherein the progression of geographic atrophy is slowed.

[0732] 38. A polynucleotide, vector or cell according to any of paragraphs 32 to 37 for use in treatment or prevention, wherein the increase in the area of ​​geographic atrophy is reduced by at least 10% over 12 months after administration to the treated eye of the subject relative to an untreated eye over the same period.

[0733] 39. A polynucleotide, vector or cell according to any of paragraphs 32-38 for use in treatment or prevention, wherein administration of the polynucleotide, vector or cell increases the level of C3b inactivation and iC3b degradation activity in a subject or in the eye of a subject (such as in retinal pigment epithelial (RPE) aqueous fluid and / or vitreous humor), optionally above normal levels in the subject or in its eye or RPE.

[0734] 40. A polynucleotide, vector or cell according to any one of paragraphs 32 to 39 for use in therapy or prevention, wherein the polynucleotide, vector or cell is administered intraocularly.

[0735] 41. A polynucleotide, vector or cell according to any one of paragraphs 32-40 for use in treatment or prevention, wherein the polynucleotide, vector or cell is administered to the eye of the subject by subretinal, direct retinal, suprachoroidal or intravitreal injection.

[0736] 42. The polynucleotide, vector or cell according to any one of paragraphs 32 to 41 for use in treatment or prevention, wherein the polynucleotide, vector or cell is administered to the eye of the subject by subretinal injection.

[0737] 43. A method of treating or preventing a complement-mediated disease of the eye, comprising administering the isolated polynucleotide, vector or cell of any of paragraphs 8-29 to a subject in need thereof.

[0738] 44. A method of providing (i) an anti-VEGF entity; and (ii) a negative complement regulator to a subject, comprising delivering the isolated polynucleotide, vector or cell of any of paragraphs 8-29 to an eye of the subject.

[0739] 45. An anti-VEGF entity for use in treating or preventing a disease, wherein the anti-VEGF entity is used in combination with a negative regulator of complement.

[0740] 46. ​​A complement negative regulator for use in treating or preventing a disease, wherein the complement negative regulator is used in combination with an anti-VEGF entity.

[0741] 47. An anti-VEGF entity for use in treatment or prevention according to paragraph 45 or a negative complement regulator for use in treatment or prevention according to paragraph 46, wherein the disease is a disease listed in any one of paragraphs 2 to 6.

[0742] 48. An anti-VEGF entity according to paragraph 45 or 47 for use in treatment or prevention, wherein the anti-VEGF entity is an entity as defined in any of paragraphs 9 to 11.

[0743] 49. A negative complement regulator for use in treatment or prevention according to paragraph 46 or 47, wherein the negative complement regulator is an entity as defined in paragraph 12 or 13.

[0744] The skilled person will understand that they can combine all features of the invention without departing from the scope of the invention as disclosed.

[0745] Preferred features and embodiments of the invention will now be described by way of non-limiting examples.

[0746] The practice of the present invention will employ, unless otherwise indicated, conventional techniques of chemistry, biochemistry, molecular biology, microbiology and immunology, which are within the capabilities of one of ordinary skill in the art and are explained in the literature. See, for example, Sambrook, J., Fritsch, EF, and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press; Ausubel, FM 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, JM and McGee, J.O'D. (1990) In Situ Hybridization: Principles and Practice, Oxford University Press; Gait, MJ (1984) Oligonucleotide Synthesis: A Practical Approach, IRL Press; and Lilley, DM and Dahlberg, JE (1992) Methods in Enzymology: DNA Structures Part A: Synthesis and Physical Analysis of DNA, Academic Press. These general texts are incorporated herein by reference. Example

[0747] Example 1 - Codon Optimization of Aflibercept Sequence

[0748] First, the protein sequence of Aflibercept was obtained from www.drugbank.ca (see SEQ ID NO: 1).

[0749] The protein sequence from 'Drugbank' was back-translated into DNA sequence and then aligned with human VEGF receptor and human IgG protein sequences and indicated as the 'wild-type' aflibercept sequence. The DNA sequence of wild-type aflibercept was exported and subcloned into the GT005 backbone and indicated as RC288. A native FHL-1 signal peptide was added upstream of the wild-type aflibercept sequence to allow secretion from the cells. Five online tools were used to generate the 'base' sequences (GeneArt, Genscript, Genewiz, IDT & JCat). Each base sequence was then manually optimized to remove cryptic splice sites, miRNA binding sites and tandem repeat codons. These optimized sequences were designated as 'manual'. Each codon-optimized sequence was synthesized and cloned into the GT005 backbone via Genewiz (Tables 1 and Figure 1 ).

[0750] Table 1: Table listing the changes in codon-optimized aflibercept constructs and reference links to online codon optimization tools

[0751]

[0752] In general, the monocistronic aflibercept vector consists of the following components:

[0753] Inverted terminal repeats (ITRs).

[0754] CMV early enhancer / chicken β-actin (CAG) promoter

[0755] Native or codon-optimized FHL1 secretion signal peptide

[0756] Aflibercept is composed of the second binding domain of the vascular endothelial growth factor receptor VEGFR-1 and the third binding domain of VEGFR-2 fused to the sequence of the Fc fragment of human IgG (WT = wild type; co-aflibercept = codon-optimized aflibercept sequence).

[0757] Woodchuck hepatitis virus posttranscriptional regulatory element (WPRE)

[0758] Bovine growth hormone polyadenylation signal (bGHpA)

[0759] Example 2 - Comparison of in vitro expression of vectorized aflibercept

[0760] All codon-optimized aflibercept vectors were packaged into AAV2 using triple transfection. All vectors were then titrated using qPCR and transduced into HEK293 at an MOI of 1E4. The expression level of aflibercept in the culture supernatant 72 hours after transduction was measured by ELISA. The experiment was repeated twice. Figure 2 As shown, all codon-optimized aflibercept vectors showed different expression levels.

[0761] Specifically, RC298 (Jcat basic) and RC299 (Jcat manual) showed the highest aflibercept expression levels compared to the control RC288 vector containing the non-codon-optimized wild-type sequence.

[0762] Western blot analysis was also performed to confirm the integrity of the aflibercept protein expressed by the AAV2 vectors. All vectors showed expression of the aflibercept protein at the expected molecular weight of 115 kDa (under non-reducing conditions), which is comparable to that of recombinant aflibercept protein ( Figure 3 A and B). Densitometric analysis showed that RC298 expressed the highest level of aflibercept ( Figure 3 C), and Figure 2 In particular, RC299 showed an abnormal protein banding pattern and was therefore excluded from selection.

[0763] In conclusion, RC298 was selected as the most ideal codon-optimized aflibercept sequence to be introduced into a bicistronic vector.

[0764] Example 3 - Construction of a bicistronic vector co-expressing a negative complement regulator and aflibercept

[0765] The resulting bicistronic vector contains a (CAG or CMV) promoter driving the expression of codon-optimized CFI or codon-optimized FHL1 linked to codon-optimized aflibercept (from RC298) via a furin F2A linker. Figure 4 As shown in Table 2 , the positions of complement negative regulators and aflibercept are interchanged in different configurations.

[0766] In general, the bicistronic aflibercept vector contains the following parts:

[0767] Inverted terminal repeats (ITRs)

[0768] Cytomegalovirus (CMV) promoter or CMV early enhancer / chicken β-actin (CAG) promoter

[0769] Codon-optimized complement factor I (CFI) or codon-optimized FHL1

[0770] Furin F2A cleavage peptide

[0771] Aflibercept - consists of the sequence of the second binding domain of the vascular endothelial growth factor receptor VEGFR-1 and the third binding domain of VEGFR-2 fused to the sequence of the Fc fragment of human IgG (WT = wild type; co-aflibercept = codon-optimized aflibercept sequence).

[0772] The woodchuck hepatitis virus posttranscriptional regulatory element (WPRE) or a shortened version of WPRE containing only the minimal gamma and alpha elements (WPRE3).

[0773] Bovine growth hormone polyadenylation signal (bGHpA)

[0774] Table 2: Table listing bicistronic vectors and corresponding configurations

[0775]

[0776]

[0777] The codon-optimized CFI sequence is SEQ ID NO:35.

[0778] The codon-optimized FHL1 sequence is SEQ ID NO:41.

[0779] The codon-optimized sequence of aflibercept is SEQ ID NO: 11.

[0780] The 5' ITR is SEQ ID NO:55.

[0781] The CMV promoter is SEQ ID NO: 59

[0782] The CAG promoter is SEQ ID NO:60.

[0783] The sequence of WPRE is SEQ ID NO:63.

[0784] The sequence of WPRE3 is SEQ ID NO:64.

[0785] The sequence of bGHpA is SEQ ID NO:61.

[0786] The 3' ITR is SEQ ID NO:57.

[0787] Example 4 - Functional characterization of a bicistronic vector co-expressing a negative complement regulator and aflibercept

[0788] Comparison of bicistronic vector expression by in vitro transduction was performed to compare Figure 4 Expression of aflibercept, CFI, and FHL1 from the bicistronic vectors listed in Table 1 versus their monocistronic counterparts.

[0789] Functional analysis of aflibercept in a bicistronic vector was performed to determine anti-VEGF binding and antiproliferative capabilities.

[0790] All bicistronic vectors were packaged into AAV8 or AAV2 using triple transfection. In vitro transductions were performed in HEK293 cells at different MOIs to compare the expression of the transgenes from the respective bicistronic vectors listed in Table 2. The expression levels of aflibercept, CFI, and FHL1 from each bicistronic vector were quantified using ELISA. At the same time, their expression levels were compared with their monocistronic counterparts. AAV8 and AAV2 bicistronic vectors (RC289 and RC313) showed measurable expression levels of CFI and aflibercept after transduction, and the expression levels of RC289 packaged into AAV8 and AAV2 vectors were comparable to the monocistronic counterparts ( Figure 7 A and B). In contrast, RC313 showed lower expression levels compared to RC289 and the corresponding monocistronic vectors. Similarly, AAV8 and AAV2 bicistronic vectors (RC304 and RC312) showed quantifiable expression levels of FHL-1 and aflibercept that were comparable to the corresponding monocistronic vectors (RC146 - monocistronic FHL1, RC298 - monocistronic aflibercept) ( Figure 7 C and D).

[0791] In further experiments, the bicistronic vectors RC313, RC289, RC304, and RC312 were packaged into AAV8 using a triple transfection method. The purified vectors were transduced in triplicate at 3 MOIs in HEK293. The supernatants were taken and quantified by aflibercept, CFI, and FHL-1 ELISA. For the CFI-aflibercept bicistronic vectors (RC313 and RC289), the expression of aflibercept was similar between the two vectors, with RC289 showing higher CFI expression than RC313 ( Figure 7 E). For the FHL-1-aflibercept bicistronic vector, the expression of aflibercept was also similar between the two vectors, and RC304 expressed higher FHL-1 than RC312 at an MOI of 1E4 ( Figure 7 F). Using the same cell culture supernatant samples from the expression studies, Western blot analysis was performed to assess protein expression from all bicistronic vectors. Under non-reducing conditions, all cell culture supernatants showed expression of CFI, FHL-1, and Aflibercept proteins, and all bicistronic vectors expressed the expected protein size similar to the recombinant protein control (see Figure 8 ).

[0792] These results indicate that all bicistronic vectors tested were able to achieve dual transgene expression, with some candidate vectors expressing levels similar to those of monocistronic vectors. All bicistronic vectors expressed proteins of the correct size.

[0793] VEGF binding assay

[0794] To determine the binding affinity of aflibercept expressed from a bicistronic vector for VEGFA, we performed equilibrium binding assays. HEK293 cells were transduced with a monocistronic vector expressing aflibercept (RC298) and a bicistronic vector co-expressing CFI or FHL1 and aflibercept. 72 hours after transduction, secreted aflibercept in the culture supernatant was extracted and quantified by ELISA. Secretion from the vector and recombinant aflibercept was measured by detecting free (unbound) human VEGFA in mixtures of aflibercept (over a range of concentrations) incubated with human VEGFA for a fixed period of time using a specific and sensitive ELISA. The binding affinity of Fig. 9 As shown, the binding equilibrium (kD) [IC50] of aflibercept for VEGFA from monocistronic and bicistronic vectors was found to be similar to that of resemblance.

[0795] These data indicate that the designed bicistronic vector is capable of expressing aflibercept with similar Functions of similar capabilities.

[0796] VEGF-induced proliferation assay

[0797] To determine whether AAV vector-derived aflibercept can effectively bind to VEGFA and effectively block the ability of VEGF to induce cell proliferation, a set concentration of human VEGFA and different concentrations of aflibercept (derived from the culture supernatant of HEK293 cells after AAV transduction as described above) or (Positive control) Human umbilical cord endothelial cells were challenged. 72 hours after treatment, the inhibition of VEGF-induced proliferation of HUVEC cells was measured by adding [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxypheyl)-2-(4-sulfophenyl)-2H-tetrazolium and spectrophotometric analysis at 450 / 570 nm. Fig.10 As shown, aflibercept from both monocistronic and bicistronic vectors blocked VEGF-induced proliferation in a 5-day growth assay in these cells, and the antiproliferative properties observed were consistent with those of resemblance.

[0798] These data indicate that the designed bicistronic vector is capable of expressing aflibercept with similar Functions of similar capabilities.

[0799] C3b cleavage assay

[0800] To analyze the functional activity of CFI or FHL1 secreted from transduced cells, supernatants from HEK293 cells transduced with: AAV2 expressing CFI (GT005); AAV2 expressing FHL1 (RC146); AAV2 co-expressing CFI and aflibercept (RC289 or RC313); or AAV2 co-expressing FHL1 and aflibercept (RC312 or RC304) were tested in the C3b cleavage assay ( Fig.11 The principle of this assay is based on the ability of CFI to cleave C3b into iC3b in the presence of FHL1. ELISA quantifies the total amount of iC3b, a product of C3b cleavage.

[0801] C3b cleavage experiments were performed by incubating 1 μg of C3b with recombinant FHL1 (GTP Tech) and supernatant of cells transduced with CFI expression vectors (GT005, RC289, and RC313) at a molar ratio of FHL1:CFI of 4:1. TM Reduced serum medium was used to make the reaction volume 20ul, and the reaction was incubated at 37 degrees for 20 minutes. The supernatant of cells transduced with FHL1 vector (RC146, RC312 or RC304) was mixed with recombinant CFI (Complement Technology, Inc, A138), 1ug ​​C3b (Complement Technology, Inc, A113) and Opti-MEM TM Serum-free medium was incubated at a molar ratio of FHL1:CFI of 4:1 at 37°C for 20 min in a total volume of 20 μl.

[0802] Results Samples were diluted 1:100 and 1:400 and loaded into iC3b ELISA using coating antibody: mouse anti-human activated C3 (Hycult, HM2168), detection antibody: rat anti-C3dg (Hycult, HM2199), and secondary antibody: mouse anti-rat (Southern Biotech, 3061-05). iC3b concentration (ng / mL) was calculated based on a standard curve generated using iC3b purified human protein (Complement Technology, Inc).

[0803] C3b incubated with recombinant CFI and FHL1 served as a positive control ( Fig.11 , left-hand bar). The negative control with C3b alone showed that the cleavage of C3b requires the presence of CFI and FHL1, as C3b is stable in the absence of the other components. C3b incubated with conditioned supernatant from untransfected cells (UTD) also served as a negative control. The results of the iC3b ELISA showed that CFI and FHL1 expressed by all bicistronic vectors were functional and could promote the cleavage of C3b into iC3b (see Fig.11 ).

[0804] In vivo studies

[0805] In vivo bicistronic expression and efficacy studies were performed to determine if negative complement regulators and aflibercept were expressed from bicistronic vectors in rodents. Studies were also performed to demonstrate the efficacy of the bicistronic vector in a laser CNV mouse model.

[0806] Example 5 - Use of Monocistronic Vectors Expressing CFI or FHL1 in Mouse Models of CNV

[0807] The AAV vector was first delivered subretinally into the mouse eye and left there for 4 weeks. On day 0, all pre-injected eyes received laser-induced CNV lesions by laser ablation of Bruch's membrane. Eyes with CNV lesions were also injected intravitreally as a positive control group. Retinal structure and CNV lesion area were imaged using optical coherence tomography and fluorescein angiography on days 0 and 4, respectively. On day 7 after laser ablation, all mice were culled and CNV area (determined by isolectin staining), CNV leakage area, and CNV score (grading) were measured as primary and secondary endpoints (see Figure 6 ).

[0808] Example 6—In vivo expression studies

[0809] Single cistron expression data

[0810] AAV8 (monocistronic) vector expressing anti-VEGF (aflibercept) was administered subretinal to the right eye of 9-week-old C57BL / 6JRj male mice (grouped as follows). Spectral domain optical coherence tomography (SD-OCT) was used to verify the success of subretinal administration.

[0811] Group 1: AAV8 RC298, dose 5e7 vg / eye (n=9).

[0812] Group 2: AAV8 RC298, dose 2e8 vg / eye (n=10).

[0813] Group 3: AAV8 RC298, dose 5e8 vg / eye (n=10).

[0814] Group 4: AAV8 RC298, dose 2e9 vg / eye (n=10).

[0815] Group 5: AAV8 RC298, dose 5e9 vg / eye (n=10).

[0816] Group VI: AAV8 RC298, dose 1e10 vg / eye (n=9).

[0817] The contralateral eye of each animal served as an uninjected control. Five weeks after injection, all animals were sacrificed. The injected and contralateral uninjected eyes were dissected open, and the ocular fluid was collected in 40 μl of PBS (containing protease inhibitors), placed in a screw-cap tube, and quickly frozen until protein analysis. The posterior eyecup was placed in a new tube containing 50 microliters of RNAlater solution and further dissected with dissecting scissors. The dissected eyecup was then quickly frozen in liquid nitrogen for 30 seconds, and 50uL of RLT buffer containing β-mercaptoethanol was added, followed by further homogenization with a homogenizer for 2 minutes. 200uL of RLT buffer containing β-mercaptoethanol was added to the eyecup homogenate and mixed by repeated pipetting. All eyecup homogenates were stored at -80°C for at least 24 hours before RNA extraction.

[0818] RNA extraction and quantitative RT-PCR

[0819] RNA was isolated from rat posterior eye cup tissue using the RNeasy Mini kit (Qiagen) according to the manufacturer's instructions. Frozen eye cup homogenate was thawed on ice and mechanically minced using QIAshredder. After mincing, 70% EtOH was added to the homogenate and the mixture was transferred to an RNeasy column. The column was centrifuged and washed with RW1 buffer. The column membrane was then treated with DNaseI, incubated at room temperature for 15 minutes, and then further washed with RW1 and RPE buffer. The RNA was then eluted with RNase-free water in a final volume of 50 μL. The RNA concentration of each sample was then measured using a NeoDot nanospectrometer (Generon).

[0820] Reverse transcription was performed using the SuperScript III Reverse Transcriptase Kit (Invitrogen). For mouse samples, transgene-derived mRNA was quantified by using the following primers targeting the bGHpA sequence: bGHpA FR 5′-CATCGCATTGTCTGAGTAGGT-3′ and bGHpA Rv 5′-AGCATGCCTGCTATTGTCTT-3′. All qRT-PCRs were performed using the CFX96 Touch Real-Time PCR Detection System (BioRad) and SYBR Green chemistry. As standards, linearized transgenic plasmids diluted to known concentrations were used. SYBR Green was applied as follows: initial denaturation at 95°C for 3 min, followed by 40 cycles of 95°C for 10 s and 56°C for 30 s.

[0821] Fig.12 Aflibercept mRNA expression for Groups 1 to 6 is shown.

[0822] Quantification of Aflibercept Expression

[0823] Unbound aflibercept in ocular fluid was quantitatively measured using a sandwich ELISA following the manufacturer's instructions (ImmunoGuide). Briefly, diluted aflibercept standards and ocular fluid samples were incubated in microtiter plates coated with recombinant human vascular endothelial growth factor-A (rhVEGF-A). After incubation, the wells were washed and horseradish peroxidase (HRP)-conjugated anti-human IgG monoclonal antibodies were added to bind to the Fc portion of aflibercept pre-captured by rhVEGF-A on the well surface. After incubation, the wells were washed and bound enzyme activity was detected by adding a chromogenic substrate. Color development was proportional to the amount of aflibercept in the sample or standard, and the optical density was measured at 450 nm using a photometer (OD620 nm reference value is optional).

[0824] Fig.13 Aflibercept protein expression for Groups 1 to 6 is shown.

[0825] Bicistronic expression data

[0826] Bicistronic AAV vectors co-expressing anti-VEGF (aflibercept) and complement modulators (hCFI or FHL-1) were administered subretinal injection into the right eye (OD) of 10-week-old C57BL / 6 male mice. Successful subretinal delivery was verified using spectral domain optical coherence tomography (SD-OCT). The contralateral eye of each animal served as a non-injected control. Four weeks later, mice were sacrificed, and ocular fluid and posterior eye cups were collected.

[0827] The study consisted of eight treatment groups:

[0828] Group 1: AAV8-RC313, 5x10 7 vg / eye (n=10 mice).

[0829] Group 2. AAV8-RC313, 5x10 8 vg / eye (n=10).

[0830] Group 3: AAV8-RC289, 5x10 7 vg / eye (n=10).

[0831] Group 4: AAV8-RC289, 5x10 8 vg / eye (n=10).

[0832] Group 5: AAV8-RC304, 5x10 7 vg / eye (n=9).

[0833] Group 6: AAV8-RC304, 5x10 8 vg / eye (n=10).

[0834] Group 7: AAV8-RC312, 5x10 7 vg / eye (n=9).

[0835] Group 8: AAV8-RC312, 5x10 8 vg / eye (n=10).

[0836] RNA extraction and quantitative RT-PCR

[0837] The posterior eye cup was placed in a new tube containing 50 μL RNAlater solution and further dissected with dissecting scissors. The dissected eye cup was then snap frozen in liquid nitrogen for 30 seconds and 50 μL of RLT buffer containing β-mercaptoethanol was added, followed by further homogenization with a homogenizer for 2 minutes. The eye cup homogenate was added with 200 μL of RLT buffer containing β-mercaptoethanol and mixed by repeated pipetting. All eye cup homogenates were stored at -80°C for at least 24 hours before RNA extraction.

[0838] RNA was isolated from rat posterior eye cup tissue using the RNeasy Mini kit (Qiagen) according to the manufacturer's instructions. Frozen eye cup homogenate was thawed on ice and mechanically minced using QIAshredder. After mincing, 70% EtOH was added to the homogenate and the mixture was transferred to an RNeasy column. The column was centrifuged and washed with RW1 buffer. The column membrane was then treated with DNaseI, incubated at room temperature for 15 minutes, and then further washed with RW1 and RPE buffer. The RNA was then eluted with RNase-free water in a final volume of 50 μL. The RNA concentration of each sample was then measured using a NeoDot nanospectrometer (Generon).

[0839] Reverse transcription was performed using the SuperScript III Reverse Transcriptase Kit (Invitrogen). For mouse samples, transgene-derived mRNA was quantified by using the following primers targeting the bGHpA sequence: bGHpAFR 5′-CATCGCATTGTCTGAGTAGGT-3′ and bGHpARv 5′-AGCATGCCTGCTATTGTCTT-3′. All qRT-PCRs were performed using the CFX96 Touch Real-Time PCR Detection System (BioRad) and SYBR Green chemistry. As standards, linearized transgenic plasmids diluted to known concentrations were used. SYBR Green was applied as follows: initial denaturation at 95°C for 3 min, followed by 40 cycles of 95°C for 10 s and 56°C for 30 s.

[0840] Fig.14 Shows the expression of aflibercept mRNA from a bicistronic vector

[0841] Quantitative analysis of aflibercept and complement expression

[0842] Aflibercept

[0843] Unbound aflibercept in ocular fluid was quantitatively measured using a sandwich ELISA following the manufacturer's instructions (ImmunoGuide). Briefly, diluted aflibercept standards and ocular fluid samples were incubated in microtiter plates coated with recombinant human vascular endothelial growth factor-A (rhVEGF-A). After incubation, the wells were washed and horseradish peroxidase (HRP)-conjugated anti-human IgG monoclonal antibodies were added to bind to the Fc portion of aflibercept pre-captured by rhVEGF-A on the well surface. After incubation, the wells were washed and bound enzyme activity was detected by adding a chromogenic substrate. Color development was proportional to the amount of aflibercept in the sample or standard, and the optical density was measured at 450 nm using a photometer (OD620 nm reference value is optional).

[0844] Expression of CFI protein in ocular fluid

[0845] Human CFI in ocular fluid was quantified using electrochemiluminescence detection technology from MSD. Biotinylated anti-CFI antibody was coated on MSD Gold 96-well small spot streptavidin plates and shaken (850 RPM) for 1 hour at room temperature. CFI standards (Complement Technology) and samples were diluted accordingly. Plates were washed with PBS-Tween 0.05% and 25uL of standards, controls and samples were added. Plates were sealed and incubated for 1 hour at room temperature with shaking (850 RPM). Plates were then washed as before and 25uL of anti-FI (anti-FI, A231) detection antibody was added to the wells and incubated for 1 hour at room temperature with shaking (850 RPM). After incubation, plates were washed as before and 150ul of 2X Read Buffer T was added to each well and read on the MSD reader.

[0846] Expression of FHL-1 protein in ocular fluid

[0847] Human FHL-1 in ocular fluid was quantified using electrochemiluminescence detection technology from MSD. Biotinylated anti-FHL-1 antibody was coated on MSD Gold 96-well small spot streptavidin plates and shaken (850 RPM) for 1 hour at room temperature. FHL-1 standards and samples were diluted accordingly. The plates were washed with PBS-Tween 0.05% and 25uL of standards, controls, and samples were added. The plates were sealed and incubated for 1 hour at room temperature with shaking (850 RPM). The plates were then washed as before and 25uL of anti-FH (anti-FH, MA170057, Invitrogen) detection antibody was added to the wells and incubated for 1 hour at room temperature with shaking (850 RPM). After incubation, the plates were washed as before and 150ul of 2X Read Buffer T was added to each well and then read on the MSD reader.

[0848] Fig.15 The expression of aflibercept, CFI and FHL1 proteins on a bicistronic vector is shown.

[0849] Example 7 - In vivo efficacy in a mouse laser-induced choroidal neovascularization model

[0850] Five groups of mice received unilateral (right eye) subretinal injections of different AAV vectors. Spectral domain optical coherence tomography (SD-OCT) was used immediately after AAV administration to confirm the success of subretinal treatment. Four weeks after the administration of the vector, CNV was induced by laser photocoagulation. Briefly, anesthetized animals received a drop of 0.5% tropicamide (Oftan Tropicamid, Santen Oy) to dilate the pupil. A drop of Viscotears (Dr. Gerhard Mann Chem.-Pharm., Germany) was placed on the eye and the cornea was flattened with a coverslip. Three laser injuries were performed around the optic nerve head of the unilateral right eye using a 532 nm diode laser (spot size: 100 microns; power: 130 mW; time: 120 ms. Oculight TX. Iridex Corp., USA). Successful generation of CNV lesions was confirmed by using SD-OCT and fluorescein angiography (FA). Another group of mice received intravitreal injections of aflibercept immediately after CNV induction. On days 4 and 7 after laser treatment, mice were imaged again with SD-OCT and FA and then sacrificed.

[0851] Laser-irradiated eyes were enucleated and dissected. Choroidal flat mounts were prepared and stained with fluorescein-labeled Griffonia Simplicifolia Lectin I (GSLI) isolectin B4 (FL-1201, Vector Laboratories, USA) to assess neovascularization. Briefly, flat mounts were washed with Tris-buffered saline (TBS) and blocked with 10% normal goat serum (NGS) and 0.5% Triton X-100 (TBST) in TBS pH 7.4 for 1 hour at room temperature. Samples were washed with TBS and incubated overnight with fluorescein-labeled Isolectin GS-IB4 (1:200, Vector Laboratories) in 1% NGS diluted in 0.1% TBST at +4°C. Then, samples were washed 3x 10 minutes with 1% NGS diluted in 0.1% TBST, counterstained with DAPI, and mounted on microscope slides with FluoroshieldTM mounting medium (Sigma-Aldrich). Choroidal samples were imaged using a DMi8 THUNDER 3D microscope (Leica Microsystems, Germany). The stained area was outlined and measured using the image processing software FIJI (Schindelin et al., 2012).

[0852] Group 1: RC268, empty, 5E8, and CNV induction at day 28;

[0853] Group 2: RC289, 5E7, and CNV induction on day 28;

[0854] Group 3: RC289, 5E8, and CNV induction on day 28;

[0855] Group 4: RC304, 5E7, and CNV induction on day 28;

[0856] Group 5: RC304, 5E8, and CNV induction on day 28;

[0857] Group VI: Aflibercept (80 μg / eye) treatment on day 0, CNV induction.

[0858] Quantitative analysis of CNV leakage area and CNV lesion size

[0859] Fluorescein angiography (FA): Heidelberg Spectralis HRA system (Heidelberg Engineering, Germany) was used to examine vascular leakage at the choroidal level. Briefly, a drop of 0.5% tropicamide (Oftan Tropicamid. Santen Oy) was dripped on the cornea of ​​anesthetized mice to dilate the pupil, and the mice were placed on a mouse stand. After aligning the optic nerve head at the level of the retina using an infrared reflectance camera, a 2.5% sodium fluorescein (Sigma-Aldrich, Finland) solution (30 μl / 10 g) was administered subcutaneously. Within 5 minutes after fluorescein administration, continuous fluorescent images were taken from the retinal and choroidal focal levels every 60 seconds (sensitivity: 45; ART average: 5 frames).

[0860] Spectral Domain Optical Coherence Tomography (SD-OCT). SD-OCT was performed to verify subretinal drug administration before and after CNV induction, and on days 4 and 7 after CNV induction. Mice were examined using the SD-OCT system Envisu R2200 (Bioptigen Inc. / Leica Microsystems, USA) immediately after FA imaging. The scan area covered 1.4 x 1.4 mm centered on the optic nerve. 2 Each scan contains 100 B scans, and each scan contains 1000 A scans.

[0861] CNV leakage area was assessed by FA fundus imaging and subsequent AI image analysis. FA scans were analyzed by a proprietary algorithm that employed a combination of convolutional neural networks (CNNs) designed for semantic segmentation and traditional computer vision algorithms. The neural network was trained to identify and quantify CNV lesions using a transfer learning approach. The model's results were reviewed and adjusted if necessary by a scientist who was blind to the treatment.

[0862] Based on the results of FA scans and SD-OCT scans, the laser-irradiated areas were qualitatively graded as leakage or no leakage.

[0863] Data analysis

[0864] Quantitative data were plotted and analyzed and expressed as mean ± standard deviation (SD). Statistical analysis was performed using GraphPad Prism software (v8.0.1GraphPad Software.USA). Differences were considered statistically significant at the level of P < 0.05.

[0865] The results are shown in Fig.16 middle.

[0866] All publications mentioned in the above description 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 the skilled person without departing from the scope and spirit of the present invention. Although the present invention has been disclosed in conjunction with specific preferred embodiments, it should be understood that the claimed invention should not be unduly limited to these specific embodiments. In fact, various modifications to the disclosed modes of implementing the present invention will be apparent to the skilled person and are intended to be within the scope of the following claims.

Claims

1. An isolated polynucleotide comprising a nucleotide sequence encoding (i) an anti-VEGF entity and (ii) a negative complement regulator.

2. The isolated polynucleotide of claim 1, wherein the anti-VEGF entity is selected from the group consisting of an Ig fusion protein, an antibody, a polypeptide, a peptide, a non-antibody scaffold, an antisense oligonucleotide, siRNA, shRNA, a CRISPR guide strand, and an aptamer.

3. The isolated polynucleotide of claim 2, wherein the anti-VEGF entity is selected from the group consisting of aflibercept, ranibizumab, bevacizumab, brocuzumab and pegaptanib.

4. The isolated polynucleotide according to claim 3, wherein the anti-VEGF entity is aflibercept, for example wherein aflibercept is encoded by a polynucleotide sequence comprising any one of SEQ ID NOs: 3 to 11.

5. An isolated polynucleotide according to any preceding claim, wherein the negative complement regulator is selected from complement factor I (CFI), complement factor H-like protein 1 (FHL1), complement factor H (CFH), complement receptor type 1 (CR1), membrane cofactor protein (MCP), complement decay accelerating factor (DAF), MAC-inhibitory protein (MAC-IP), C1-inhibitor, hypersensitive protein inhibitor, C4b binding protein (C4BP), clusterin, vitronectin or variants or fragments thereof.

6. The isolated polynucleotide according to claim 5, wherein the negative complement regulator is selected from the group consisting of CFI and FHL1 or variants or fragments thereof.

7. The isolated polynucleotide according to any one of claims 3 to 6, wherein the nucleotide sequence encoding aflibercept has at least 75% sequence identity with SEQ ID NO:

11.

8. The isolated polynucleotide according to claim 6 or 7, wherein the nucleotide sequence encoding CFI has at least 75% sequence identity with SEQ ID NO: 35 or 36, or the nucleotide sequence encoding FHL-1 has at least 75% sequence identity with SEQ ID NO:

41.

9. The isolated polynucleotide according to any one of claims 3 to 8, wherein the polynucleotide comprises the nucleotide sequence of SEQ ID NO: 45 to 54, or a nucleotide sequence having at least 75% sequence identity thereto.

10. The isolated polynucleotide of any one of claims 1 to 9, wherein the polynucleotide is less than or equal to 4.7 kb.

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

12. The vector of claim 11, wherein the vector is an adeno-associated virus (AAV) vector, optionally in the form of a viral vector particle, preferably an AAV vector particle comprising an AAV2 genome and AAV2 or AAV8 capsid proteins.

13. A pharmaceutical composition comprising the isolated polynucleotide or vector of any one of claims 1 to 12 in combination with a pharmaceutically acceptable carrier, diluent or excipient.

14. The isolated polynucleotide, vector or pharmaceutical composition of any one of claims 1 to 13 for use in therapy.

15. The isolated polynucleotide, vector or pharmaceutical composition of any one of claims 1 to 14 for use in treating or preventing a complement-mediated disorder of the eye, such as age-related macular degeneration (AMD), diabetic retinopathy, glaucoma, Stargardt's disease, central serous choroidopathy, retinitis pigmentosa, polypoidal choroidal vasculopathy, diabetic macular edema, branch retinal vein occlusion or uveitis, preferably AMD.

16. The isolated polynucleotide, vector or pharmaceutical composition according to claim 15 for use in treatment or prevention, wherein the AMD is wet AMD and / or dry AMD.

17. The isolated polynucleotide, vector or pharmaceutical composition of claim 15 for use in treatment or prevention, wherein i) the AMD is wet AMD and the use further prevents and / or treats the onset of dry AMD in a subject, or ii) the AMD is dry AMD and the use further prevents and / or treats the onset of wet AMD in a subject, or iii) the use prevents and / or treats both dry AMD and wet AMD.

18. The isolated polynucleotide, vector or pharmaceutical composition according to claim 17 for use in treatment or prevention, wherein AMD is wet AMD, and said use further prevents and / or treats the onset of dry AMD in the subject.

19. An isolated polynucleotide, vector or pharmaceutical composition according to any one of claims 15 to 18 for use in treatment or prevention, wherein: (i) the onset of geographic shrinkage is prevented or reduced, and / or the total amount of geographic shrinkage is reduced, or the progression of geographic shrinkage is slowed; (ii) the increase in geographic atrophy in the treated eye of the subject is reduced by at least 10% over the 12 months following administration of the drug relative to the untreated eye during the same period; or (iii) administering the isolated polynucleotide, vector or pharmaceutical composition increases the level of C3b inactivation and iC3b degradation activity in a subject or in the eye of a subject, such as in retinal pigment epithelial cells (RPE), optionally above normal levels in the subject or in its eye or RPE.

20. An isolated polynucleotide, vector or pharmaceutical composition according to any one of claims 15 to 19 for use in treatment or prevention, wherein i) neovascularization is prevented or reduced, ii) vascular leakage is prevented or reduced and / or iii) retinal oedema is prevented or reduced.

21. An isolated polynucleotide, vector or pharmaceutical composition according to any one of claims 15 to 20 for use in treatment or prevention, wherein the polynucleotide, vector, cell or pharmaceutical composition is administered intraocularly, such as by subretinal, direct retinal, suprachoroidal or intravitreal injection.

22. A product comprising (i) an anti-VEGF entity; and (ii) a complement negative regulator, or nucleotide sequences encoding the same, as a combined preparation for i) simultaneous, separate or sequential use in the treatment of wet AMD, and wherein the use further prevents and / or treats the onset of dry AMD in a subject; or ii) simultaneous, separate or sequential use in the treatment of dry AMD, and wherein the use further prevents and / or treats the onset of wet AMD in a subject, or iii) simultaneous, separate or sequential use in the treatment and / or prevention of both dry AMD and wet AMD.

23. A product according to claim 22 for use in treating for simultaneous, separate or sequential use in treating wet AMD, wherein the use further prevents and / or treats the onset of dry AMD in the subject.

24. The product according to claim 22 or 23 for use in therapy, wherein the anti-VEGF entity is selected from Ig fusion proteins, antibodies, polypeptides, peptides, non-antibody scaffolds, antisense oligonucleotides, siRNA, shRNA, CRISPR guide strands and aptamers, preferably wherein the anti-VEGF entity is selected from aflibercept, ranibizumab, bevacizumab, brocuzumab and pegaptanib.

25. A product for use in treatment according to any one of claims 22 to 24, wherein the negative complement regulator is selected from complement factor I (CFI), complement factor H-like protein 1 (FHL1), complement factor H (CFH), complement receptor type 1 (CR1), membrane cofactor protein (MCP), complement decay accelerating factor (DAF), MAC-inhibitory protein (MAC-IP), C1-inhibitor, allergic protein inhibitor, C4b binding protein (C4BP), clusterin, vitronectin or variants or fragments thereof, preferably, wherein the negative complement regulator is selected from CFI or FHL1 or variants or fragments thereof.

26. A product comprising (i) an anti-VEGF entity; and (ii) Factor I, or a nucleotide sequence encoding the same, as a combined preparation for simultaneous, separate or sequential use as pharmaceuticals.

27. The product according to claim 26 for use as a medicament for the treatment or prevention of complement-mediated ocular disorders such as age-related macular degeneration (AMD), diabetic retinopathy, glaucoma, Stargardt's disease, central serous choroidopathy, retinitis pigmentosa, polypoidal choroidal vasculopathy, diabetic macular edema, branch retinal vein occlusion or uveitis, preferably AMD.

28. The product according to any one of claims 22 to 27 for use, wherein: (i) the onset of geographic shrinkage is prevented or reduced, and / or the total amount of geographic shrinkage is reduced, or the progression of geographic shrinkage is slowed; (ii) the increase in geographic atrophy in the treated eye of the subject is reduced by at least 10% over the 12 months following administration of the drug relative to the untreated eye during the same period; or (iii) administering the isolated polynucleotide, vector or pharmaceutical composition increases the level of C3b inactivation and iC3b degradation activity in a subject or in the eye of a subject, such as in retinal pigment epithelial cells (RPE), optionally above normal levels in the subject or in its eye or RPE.

29. A product according to any one of claims 22 to 28 for use, wherein i) neovascularization is prevented or reduced, ii) vascular leakage is prevented or reduced and / or iii) retinal oedema is prevented or reduced.

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