Retinal disorders

Abstract

The present invention relates to retinal disorders and to genetic constructs and recombinant vectors containing such constructs, and their use in methods of gene therapy for treating, preventing or ameliorating a wide range of retinal disorders. The constructs and vectors are particularly useful for treating wet age-related macular degeneration (wet AMD), i.e., neovascular age-related macular degeneration. The present invention extends to the use of the constructs and vectors to reduce vascular leakage and retinal cell damage. The present invention also extends to the pharmaceutical composition itself, and its use in treating, preventing or ameliorating retinal disorders and for reducing vascular leakage and retinal cell damage.

Description

[Technical field]

[0001] The present invention relates to retinal disorders and to genetic constructs and recombinant vectors containing such constructs, and their use in methods of gene therapy for treating, preventing or ameliorating a wide range of retinal disorders. The constructs and vectors are particularly, but not exclusively, useful for treating wet age-related macular degeneration (wet AMD), i.e., neovascular age-related macular degeneration. The present invention also extends to the use of the constructs and vectors for reducing vascular leakage and retinal cell damage. The present invention also extends to the pharmaceutical composition itself, and its use in treating, preventing or ameliorating retinal disorders and for reducing vascular leakage and retinal cell damage. [Background technology]

[0002] Macular degeneration, also known as age-related macular degeneration (AMD), is a common eye condition among people over 50 years of age that affects the macula, a small portion of the retina.[1-3] The macula is used for central and detailed vision, necessary for activities such as reading and driving. AMD is the leading cause of permanent and irreversible blindness in adult populations across developed countries.[4] It affects approximately 600,000 people in the UK.[5] In 2013, it was estimated to be the fourth most common cause of blindness after cataracts, premature birth and glaucoma.[6] In 2015, AMD was estimated to affect 6.2 million people worldwide.[7] Approximately 0.4% of people between the ages of 50 and 60 have AMD, rising to 0.7% in people aged 60-70, 2.3% in people aged 70-80, and almost 12% in people over 80 years of age.[1] Thus, the incidence of AMD is also rising in line with the shift towards an aging population.[8] AMD can be diagnosed as either "dry" (non-neovascular) or "wet" (exudative or neovascular) AMD.[1,2] Dry AMD or geographic atrophy is the more common form of AMD, accounting for 90% of cases. Dry AMD generally results in more gradual vision loss, whereas wet AMD causes relatively sudden changes in vision, resulting in substantial blindness.[1,2]

[0003] A Canadian study concluded that moderate AMD caused a 40% decrease in quality of life, a decrease similar to that associated with permanent kidney dialysis or severe angina. Very severe AMD causes a 63% decline in quality of life, a decrease comparable to that of individuals with advanced prostate cancer who suffer from uncontrolled pain, or a severe stroke that leaves a person bedridden, incontinent, and requiring constant nursing care. [9] Symptoms may develop slowly, especially if only one eye is affected, but as the condition progresses, vision will worsen and result in gaps or dark spots (like a stain on glasses) appearing in the individual's vision. In addition, words may disappear when reading, straight lines, such as door frames and lampposts, may appear distorted or crooked, and colors may appear faded. Subjects may also find it difficult to adapt when moving from a dark to a light environment. Although AMD does not result in total blindness, loss of central vision makes everyday activities extremely challenging, such as difficulty recognizing faces, driving and reading [1-3], and some individuals have been reported to experience visual hallucinations

[10] .

[0004] Wet AMD is the result of new blood vessels growing under the macula (angiogenesis), which as they form leak blood and fluid into the retina, causing tissue damage.

[11] Prevention efforts include exercising, eating a good diet, and not smoking.

[12] Antioxidant vitamins and minerals have limited effectiveness,

[13] and there are currently no cures or treatments that can restore lost vision.[1-3]

[0005] VEGF-A is a homodimeric glycoprotein produced and secreted by glial, ganglion and epithelial cells, including the retinal pigment epithelium (RPE) of the eye, as well as by astrocytes [14, 15]. Multiple isoforms of VEGF-A exist, resulting from alternative splicing of mRNA from a single 8-exon VEGF-A, including four principle forms VEGF-121, VEGF-165, VEGF-189 and VEGF-206 that display varying levels of heparin binding [14, 15]. VEGF-A isoforms display essential physiological roles in vascular development and are important for neuronal survival.

[0006] Current treatments targeting the vascular endothelial growth factor (VEGF) pathway include monoclonal antibodies or other VEGF neutralizing fragments (ranibizumab, Lucentis® / bevacizumab, Avastin® / brolucizumab / Beovu®), DNA aptamers (pegaptanib / Macugen®), or recombinant VEGF capture proteins (aflibercept; Eylea®), which have been shown to limit vision loss [16-19]. These treatments are currently preferred over more invasive laser coagulation or photodynamic early treatment options.

[0007] However, some patients show inherent refractory state to anti-VEGF therapy, with persistent fluid or recurrent exudation

[20] . Moreover, advanced stage disease is associated with subretinal fibrosis along with focal areas of retinal pigment epithelium (RPE) loss, subretinal or sub-RPE hemorrhage. Current strategies targeting the VEGF pathway alone do not address these issues, and therefore there is considerable room for improvement [20, 21].

[0008] Moreover, a recent multicenter study testing the effects of anti-VEGF therapy in 1185 patients with wet AMD noted that by the first year, subretinal scarring occurred in approximately one-third of eyes treated with anti-VEGF drugs, and this increased to approximately half of patients by the end of the second year

[21] . Induction of angiogenesis can result in the recruitment of inflammatory cells and fibroblasts, and injury induces the transformation of epithelial cells into myofibroblasts

[22] . Collectively, these cells undergo epithelial-mesenchymal transition (EMT) where they can proliferate and cover the area of ​​damage

[22] . Chronic inflammation leads to persistent scarring.

[0009] Macular fibrosis causes irreversible vision loss in neovascular age-related macular degeneration (nAMD), even with anti-vascular endothelial growth factor (VEGF) therapy. A factor implicated in fibrosis pathophysiology is connective tissue growth factor / cellular signaling network-2 (CTGF / CCN2)

[23] . CTGF is a 38 kDa secreted cysteine-rich protein first identified in human umbilical vein endothelial cells and a member of the CCN family of growth factors [24-27]. CTGF is composed of four linked cysteine-rich domains (I-IV) spanning an insulin-like growth factor binding protein domain (IGFBP; domain I) at the N-terminus, a von Willebrand C-type repeat (vWC; domain II), followed by thrombospondin type 1 repeats (TSP; domain III) and finally a C-terminal domain IV with a cysteine ​​knot motif [24-27].

[0010] CTGF has been shown to induce contraction of fibroblast-assembled collagen matrix and increase components of the extracellular matrix, including collagen and fibronectin [26, 27]. CTGF production and release is induced by transforming growth factor beta (TGF-β) and results in fibrosis in conditions including biliary fibrosis and diabetic retinopathy [28–30]. Although the specific CTGF receptor responsible for the profibrotic effect has not been identified, CTGF has been shown to bind nonspecifically to several other growth factor receptors, such as insulin growth factor-2 receptor

[31] , fibroblast growth factor receptor-2

[32] , epidermal growth factor receptor

[33] , integrins [34–36], and TrkA receptor

[37] . CTGF is consistently found in fluids that accumulate in the subretinal space after retinal detachment

[38] . For full activity, full-length CTGF appears to need to be cleaved by extracellular endopeptidases. Cleavage releases the C-terminal part of the protein, which contains domains III–IV, and the N-terminal part, which consists of domains I–II. The N-terminal domain may act as an inhibitor of CTGF activity

[39] .

[0011] There is growing evidence of the involvement of complement in the development of macular fibrosis. Increased plasma levels of C3a, C3d, Bb and C5a have been observed in AMD patients [40-42]. Notably, in a study of 96 patients with nAMD, plasma levels of complements C3a, C4a and C5a were shown to be significantly higher in individuals with subretinal fibrosis than in controls

[42] . In addition, higher plasma levels of C3a were detected in nAMD that partially responded to anti-VEGF therapy.

[0012] Furthermore, the complement system (CS) is a part of the innate immune system to defend against foreign pathogens such as microorganisms [43-45]. The complement system (CS) consists of three biochemical pathways: the classical pathway (CP), the lectin pathway (LP) and the alternative pathway (AP), each of which has a distinct trigger. Although each pathway can be activated by separate components, the pathways converge to involve a crucial protein component called complement factor 3 (C3).

[0013] The activity of AP can be modulated by complement factor I (CFI), also called C3b / C4b inactivator, since it cleaves cell-bound or fluid-phase C3b and C4b [46, 47]. Another modulating factor is CD55 or decay-accelerating factor (DAF), a membrane-bound protein that also protects cells from complement-mediated lysis [48-50]. The main function of CD55 is to inactivate the C3 convertase by dissociating it into its component proteins

[48] . Yet another modulating protein called complement factor H-related protein-1 (CFHR1), a splice variant of complement factor H [51-53], is also involved in reducing complement activation by targeting the decay of the C3 convertase composed of C3b and factor B. Another factor that can reduce complement activation is called membrane cofactor protein or CD46, which is membrane-bound. Upon binding to C3b, factor H and CFHL-1 occupy the factor B binding site on C3b, promoting its decay and preventing the formation of new C3 convertases. In addition, factor H, CFHL-1 and CD46 act as cofactors for the protease factor I, which cleaves C3b to iC3b, and, in the case of CD46, as a cofactor for C4b. CFHR-1 does not mediate the decay-promoting activity of factor I cofactor. Like factor H, CFHR-1 binds C3b and recognizes self-surfaces by binding to glycosaminoglycans, inhibiting C5 convertases and terminal complement complex formation

[53] . Summary of the Invention [Problem to be solved by the invention]

[0014] Thus, there is a great need for improved therapies for the treatment of retinal disorders, such as AMD, in particular wet age-related macular degeneration (wet AMD), that can neutralize VEGF and prevent the development of inflammation and subretinal fibrosis.

[0015] Using extensive inventive efforts, the inventors have carefully designed and constructed a novel genetic construct that encodes an anti-VEGF protein and an anti-fibrotic protein under the control of a single promoter, i.e., it is bicistronic. The promoters of the construct can be used to ensure that both the anti-VEGF protein and the anti-fibrotic protein are maximally expressed to reduce vascular leakage, fibrosis, scarring and inflammation. [Means for solving the problem]

[0016] Thus, in a first aspect of the present invention there is provided a genetic construct comprising a promoter operably linked to a first coding sequence encoding an anti-VEGF protein and a second coding sequence encoding an anti-fibrotic protein.

[0017] As described in the Examples, the inventors have surprisingly demonstrated that it is possible to combine open reading frames (ORFs) encoding both anti-VEGF and anti-fibrotic proteins in a single genetic construct. This was particularly challenging given the large size of each component. It could not have been predicted that both of these large proteins could be co-expressed at physiologically useful concentrations from a single expression cassette under the control of a single promoter, and that this expression cassette could be housed by an AAV vector (such as the rAAV-2 vector). Advantageously, the construct of the present invention does not require the injection of recombinant proteins, as described in the prior art. Moreover, in the prior art, it is still necessary to perform regular injections of proteins into the eye, which is obviously inconvenient, whereas the construct of the present invention surprisingly requires only a single administration to achieve a long-term therapeutic effect, thereby providing a great benefit to the patient.

[0018] Preferably, the genetic construct of the present invention comprises an expression cassette, one embodiment of which is shown in Figure 2. As can be seen from Figure 2, in one embodiment, the construct comprises a promoter, a first nucleotide sequence encoding an anti-VEGF protein, and a second nucleotide sequence encoding an anti-fibrotic protein. Thus, preferably, the genetic construct and the expression cassette can be referred to as being bicistronic.

[0019] The first and second coding sequences encoding the anti-VEGF protein and the anti-fibrotic protein can be arranged in any order from 5' to 3'. For example, in one embodiment, the coding sequence of the anti-VEGF protein is arranged 5' of the coding sequence of the anti-fibrotic protein, preferably with a spacer sequence between them. Alternatively, in another embodiment, the coding sequence of the anti-fibrotic protein can be arranged 5' of the coding sequence of the anti-VEGF protein, preferably with a spacer sequence between them.

[0020] The promoter in the genetic construct of the first aspect may be any nucleotide sequence that is capable of inducing RNA polymerase to bind and transcribe the first and second coding sequences.

[0021] Promoters can be constitutive or tissue specific.

[0022] A suitable constitutive promoter may be the cytomegalovirus promoter. One embodiment of a nucleotide sequence (508 bp) encoding the cytomegalovirus (CMV) promoter is as follows, and is referred to herein as SEQ ID NO:1:

[0023] [ka]

[0024] In another embodiment, the promoter is preferably a truncated form of the CMV promoter. One embodiment of a nucleotide sequence (60 bp) encoding a truncated form of the CMV promoter is as follows, and is referred to herein as SEQ ID NO:2:

[0025] [ka]

[0026] In another embodiment, the promoter is a fusion of the cytomegalovirus (CMV) early enhancer element and the first intron of the chicken beta-actin gene (CAG). One embodiment of a nucleotide sequence (583 bp) encoding the cytomegalovirus early enhancer element and the first intron of the chicken beta-actin gene is as follows, and is referred to herein as SEQ ID NO:3:

[0027] [ka]

[0028] A suitable tissue-specific promoter may be the vitelloid macular dystrophy protein-2 (VMD2) promoter (sometimes referred to as bestrophin-1). Advantageously, this promoter restricts transgene expression to RPE cells. One embodiment of a nucleotide sequence (2039 bp) encoding the VMD2 promoter is referred to herein as SEQ ID NO:4, as follows:

[0029] [ka]

[0030] In yet a further preferred embodiment, the promoter is a truncated form of the VMD2 promoter. One embodiment of a nucleotide sequence (623 bp) encoding the truncated form of the VMD2 promoter is as follows, and is referred to herein as SEQ ID NO:5:

[0031] [ka]

[0032] In yet another preferred embodiment, the nucleotide sequence (462 bp) encoding a truncated form of the VMD2 promoter is as follows, and is referred to herein as SEQ ID NO:6:

[0033] [ka]

[0034] In another embodiment, the promoter is the human phosphoglycerate kinase-1 (PGK) promoter. One embodiment of a nucleotide sequence (500 bp) encoding the human PGK-1 promoter is as follows, and is referred to herein as SEQ ID NO:7:

[0035] [ka]

[0036] In a further embodiment, the promoter is EF1α, derived from the human EEF1A1 gene, which expresses the alpha subunit of eukaryotic elongation factor 1. One embodiment of a nucleotide sequence (1182 bp) encoding the EF1α promoter is as follows, and is referred to herein as SEQ ID NO:8:

[0037] [ka]

[0038] In a further embodiment, the promoter is EF1α without the large intron. One embodiment of a nucleotide sequence (230 bp) encoding the EF1α promoter is as follows, and is referred to herein as SEQ ID NO:9:

[0039] [ka]

[0040] Thus, preferably the promoter comprises a nucleic acid sequence substantially as set out in SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8 or 9, or a fragment or variant thereof.

[0041] The inventors have carefully considered the sequence of the anti-VEGF protein and have created several preferred embodiments of the protein which may be encoded by the first coding sequence in the genetic construct of the first aspect.

[0042] Preferably, the anti-VEGF protein is capable of capturing all soluble forms of VEGF, including VEGF-A, VEGF-B, VEGF-C, VEGF-D and / or placenta growth factor (PIGF). More preferably, the anti-VEGF protein specifically captures VEGF-A. Preferably, in this embodiment, the anti-VEGF protein captures all isoforms of VEGF-A, including VEGF-121, VEGF-145, VEGF-165, VEGF-183, VEGF-189 and / or VEGF-206.

[0043] Preferably, the anti-VEGF protein is an anti-VEGF antibody or an antigen-binding fragment thereof.

[0044] The antigen-binding fragment may comprise or consist of any of the fragments selected from the group consisting of VH, VL, Fd, Fv, Fab, Fab', scFv, F(ab')2 and Fc fragments that bind VEGF. The antigen-binding fragment may comprise a complementarity determining region (CDR) that binds to a VEGF epitope.

[0045] In a preferred embodiment, the anti-VEGF protein is a single chain variable fragment (SCVF). In other words, in this preferred embodiment, the anti-VEGF protein is an anti-VEGF single chain variable fragment.

[0046] Thus, in a first embodiment, the first coding sequence comprises a nucleotide sequence encoding an anti-VEGF single chain variable fragment (SCVF-1) capable of neutralizing the most common isoform of VEGF-A. Preferably, the SCVF-1 comprises the amino acid sequence referred to herein as SEQ ID NO: 10, or a fragment or variant thereof, as follows:

[0047] [ka]

[0048] Preferably, in this embodiment, the first coding sequence comprises the nucleotide sequence referred to herein as SEQ ID NO:11 (756 bp), or a fragment or variant thereof, as follows:

[0049] [ka]

[0050] In a second preferred embodiment, the first coding sequence comprises a nucleotide sequence encoding an anti-VEGF single chain variable fragment (SCVF-2) capable of neutralizing the most common isoform of VEGF-A. Preferably, the SCVF-2 comprises the amino acid sequence referred to herein as SEQ ID NO: 12, or a fragment or variant thereof, as follows:

[0051] [ka]

[0052] Preferably, in this second embodiment, the first coding sequence comprises the nucleotide sequence referred to herein as SEQ ID NO: 13 (756 bp), or a fragment or variant thereof, as follows:

[0053] [ka]

[0054] In a third preferred embodiment, the first coding sequence comprises a nucleotide sequence encoding an anti-VEGF single chain variable fragment (SCFV-3) capable of neutralizing the most common isoform of VEGF-A. Preferably, the SCFV-3 comprises the amino acid sequence referred to herein as SEQ ID NO: 14, or a fragment or variant thereof, as follows:

[0055] [ka]

[0056] Preferably, in this third preferred embodiment, the first coding sequence comprises the nucleotide sequence referred to herein as SEQ ID NO: 15 (765 bp), or a fragment or variant thereof, as follows:

[0057] [ka]

[0058] In a fourth preferred embodiment, the first coding sequence comprises a nucleotide sequence encoding an anti-VEGF single chain variable fragment (SCVF-4) capable of neutralizing the most common isoform of VEGF-A. Preferably, the SCVF-4 comprises the amino acid sequence referred to herein as SEQ ID NO: 16, or a fragment or variant thereof, as follows:

[0059] [ka]

[0060] Preferably, in this fourth embodiment, the first coding sequence comprises the nucleotide sequence referred to herein as SEQ ID NO: 17 (747 bp), or a fragment or variant thereof, as follows:

[0061] [ka]

[0062] In a fifth preferred embodiment, the first coding sequence comprises a nucleotide sequence encoding an anti-VEGF protein (VEGF trapping protein-1), a protein capable of neutralizing all soluble forms of VEGF. Preferably, the VEGF trapping protein-1 comprises the amino acid sequence referred to herein as SEQ ID NO: 18, or a fragment or variant thereof, as shown below:

[0063] [ka]

[0064] Preferably, in this fifth embodiment, the first coding sequence comprises the nucleotide sequence referred to herein as SEQ ID NO: 19 (1293 bp), or a fragment or variant thereof, as follows:

[0065] [ka]

[0066] In a sixth preferred embodiment, the first coding sequence comprises a nucleotide sequence encoding an anti-VEGF protein (VEGF trapping protein-2), a protein capable of neutralizing all soluble forms of VEGF. Advantageously and preferably, VEGF trapping protein-2 has a lower affinity for binding to human IgG-Fc-gamma receptors I, II and III. Preferably, VEGF trapping protein-2 comprises the amino acid sequence referred to herein as SEQ ID NO: 20, or a fragment or variant thereof, as shown below:

[0067] [ka]

[0068] Preferably, in this sixth embodiment, the first coding sequence comprises the nucleotide sequence referred to herein as SEQ ID NO:21 (1293 bp), or a fragment or variant thereof, as follows:

[0069] [ka]

[0070] Thus, in a preferred embodiment, the first coding sequence comprises a nucleotide sequence substantially as set out in any one of SEQ ID NOs: 11, 13, 15, 17, 19 or 21, or a fragment or variant thereof. Preferably, the anti-VEGF protein comprises an amino acid sequence substantially as set out in SEQ ID NOs: 10, 12, 14, 16, 18 or 20, or a fragment or variant thereof.

[0071] It will be appreciated that the second coding sequence encodes an anti-fibrotic protein.

[0072] In one embodiment, the anti-fibrotic protein is an anti-complement protein. Preferably, the anti-complement protein is capable of neutralizing or attenuating complement activation. Even more preferably, the anti-complement protein is capable of targeting the alternative pathway (AP) of the complement system. Preferably, the anti-complement protein minimally affects the classical pathway (CP) and / or the lectin pathway (LP) of the complement system. Preferably, the anti-complement protein does not target the classical pathway (CP) and / or the lectin pathway (LP) of the complement system.

[0073] Preferably, the anti-complement protein is capable of neutralizing complement factors C3b and / or Bb. Thus, in this embodiment, the anti-complement protein is an anti-C3b or anti-Bb antibody, or an antigen-binding fragment thereof.

[0074] The antigen-binding fragment may comprise or consist of any of the fragments selected from the group consisting of VH, VL, Fd, Fv, Fab, Fab', scFv, F(ab')2 and Fc fragments that bind to C3b and / or Bb. The antigen-binding fragment may comprise a complementarity determining region (CDR) that binds to a C3b and / or Bb epitope.

[0075] Even more preferably, the anti-complement protein is a single chain variable fragment (SCVF). In other words, in this preferred embodiment, the anti-complement protein is an anti-C3b single chain variable fragment or an anti-Bb single chain variable fragment.

[0076] Alternatively, in another preferred embodiment, the anti-complement protein is CD55 (Decay Accelerating Factor; DAF). Preferably, the anti-complement protein is non-membrane-associated CD55. CD55 (DAF) destabilizes the complement protein complex, thereby reducing the activity of this biochemical pathway.

[0077] In another preferred embodiment, the anti-complement protein is complement factor H related protein-1 (CFHR1). Preferably, CFHR1 attenuates the complement system activation cascade.

[0078] In another preferred embodiment, the anti-complement protein is CD46. Preferably, in this embodiment, the anti-complement protein is the soluble (non-membrane-associated) human complement regulatory protein CD46 (sCD46).

[0079] In another preferred embodiment, the anti-complement protein is complement factor H-like protein 1 (CFHL1), a splice variant of factor H that contains a regulatory domain and inhibits complement activation at the level of core complement component C3 and beyond.

[0080] In a preferred embodiment, the amino acid sequence of the anti-C3b single chain variable fragment is as follows, referred to herein as SEQ ID NO: 22, or a fragment or variant thereof:

[0081] [ka]

[0082] In a preferred embodiment, the nucleic acid sequence (726 bp) encoding the anti-C3b single chain variable fragment is as follows, referred to herein as SEQ ID NO: 23, or a fragment or variant thereof:

[0083] [ka]

[0084] In another embodiment, the amino acid sequence of the anti-Bb single chain variable fragment is as follows, referred to herein as SEQ ID NO:24, or a fragment or variant thereof:

[0085] [ka]

[0086] In a preferred embodiment, the nucleic acid sequence (750 bp) encoding the anti-Bb single chain variable fragment is as follows, referred to herein as SEQ ID NO: 25, or a fragment or variant thereof:

[0087] [ka]

[0088] In another embodiment, the amino acid sequence of the soluble (non-membrane bound) form of CD55 (sCD55, sometimes also referred to as decay accelerating factor; DAF) is as follows, referred to herein as SEQ ID NO:26, or a fragment or variant thereof:

[0089] [ka]

[0090] In a preferred embodiment, the 960 nucleic acid sequence (960 bp) encoding the soluble (non-membrane bound) form of CD55 (sCD55) (sometimes known as decay accelerating factor; DAF) is referred to herein as SEQ ID NO:27, or a fragment or variant thereof, as follows:

[0091] [ka]

[0092] In a further embodiment, the amino acid sequence of human complement factor H related protein-1 (CFHR1) is referred to herein as SEQ ID NO:28, or a fragment or variant thereof, as follows:

[0093] [ka]

[0094] In a preferred embodiment, the nucleic acid sequence (936 bp) encoding human complement factor H related protein-1 (CFHR1) is referred to herein as SEQ ID NO:29, or a fragment or variant thereof, as follows:

[0095] [ka]

[0096] In another embodiment, the codon-optimized nucleic acid sequence (936 bp) encoding human complement factor H related protein-1 (CFHR1) is as follows, referred to herein as SEQ ID NO:30, or a fragment or variant thereof:

[0097] [ka]

[0098] In a further embodiment, the amino acid sequence of the soluble (non-membrane bound) form of CD46 (sCD46) is referred to herein as SEQ ID NO:31, or a fragment or variant thereof, as follows:

[0099] [ka]

[0100] In a preferred embodiment, the nucleic acid sequence (930 bp) encoding the soluble (non-membrane bound) form of CD46 (sCD46) is referred to herein as SEQ ID NO: 32, or a fragment or variant thereof, as follows:

[0101] [ka]

[0102] In a preferred embodiment, the amino acid sequence of CFHL1 is referred to herein as SEQ ID NO:97, or a fragment or variant thereof, as follows:

[0103] [ka]

[0104] In a preferred embodiment, the nucleic acid sequence encoding CFHL1 (1278 bp) is referred to herein as SEQ ID NO:98, or a fragment or variant thereof, as follows:

[0105] [ka]

[0106] Alternatively, in another embodiment, the anti-fibrotic protein is capable of neutralizing connective tissue growth factor (CTGF). Preferably, the anti-fibrotic protein is an anti-connective tissue growth factor (anti-CTGF) antibody or an antigen-binding fragment thereof. Preferably, the anti-CTGF antibody or an antigen-binding fragment thereof is capable of neutralizing connective tissue growth factor (CTGF).

[0107] The antigen-binding fragment may comprise or consist of any of the fragments selected from the group consisting of VH, VL, Fd, Fv, Fab, Fab', scFv, F(ab')2 and Fc fragments that bind to CTGF. The antigen-binding fragment may comprise a complementarity determining region (CDR) that binds to a CTGF epitope.

[0108] Most preferably, the anti-CTGF antibody is an anti-CTGF single chain variable fragment (anti-CTGF SCVF).

[0109] Since neither the specific receptor nor the binding site for CTGF has yet been identified, we utilized single-chain variable fragments (SCVFs) capable of neutralizing the entire CTGF sequence (anti-CTGF SCVF-1) or SCVFs capable of neutralizing the C-terminal CTGF fragment (anti-CTGF SCVF-2).

[0110] The inventors have carefully considered the sequences of SCVF that are capable of neutralising CTGF and have created preferred embodiments of proteins that may be encoded by the second coding sequence in the genetic construct of the first aspect.

[0111] In a preferred embodiment, the amino acid sequence of the anti-CTGF single chain variable fragment (anti-CTGF SCVF-1) is as follows, referred to herein as SEQ ID NO:33, or a fragment or variant thereof:

[0112] [ka]

[0113] In a preferred embodiment, the nucleic acid sequence (747 bp) encoding anti-CTGF SCVF-1 is referred to herein as SEQ ID NO: 34, or a fragment or variant thereof, as follows:

[0114] [ka]

[0115] In another preferred embodiment, the amino acid sequence of the anti-CTGF single chain variable fragment (anti-CTGF SCVF-2) is as follows, referred to herein as SEQ ID NO:35, or a fragment or variant thereof:

[0116] [ka]

[0117] In a preferred embodiment, the nucleic acid sequence (723 bp) encoding the anti-CTGF SCVF-2 is referred to herein as SEQ ID NO: 36, or a fragment or variant thereof, as follows:

[0118] [ka]

[0119] Thus, in a preferred embodiment, the second coding sequence comprises a nucleotide sequence substantially as set out in any one of SEQ ID NOs: 23, 25, 27, 29, 30, 32, 34, 36 or 98, or a fragment or variant thereof. Preferably, the anti-complement protein comprises an amino acid sequence substantially as set out in SEQ ID NOs: 22, 24, 26, 28, 31, 33, 35 or 97, or a fragment or variant thereof.

[0120] Many gene therapy constructs expressing two or more genes published in the scientific literature have either (i) dual promoters to drive the expression of two or more genes separately, or (ii) an internal ribosome entry site (IRES) to link the genes, such as that from encephalomyocarditis virus (EMCV), to allow translation of the genes from a single transcript driven by a single promoter in a recombinant viral vector. However, the efficiency of IRES-dependent translation varies dramatically in different cells and tissues, and IRES-dependent translation can be significantly lower than cap-dependent translation, meaning that lower expression of the gene downstream of the IRES often occurs compared to the gene at position 1 of the expression cassette. Furthermore, the limited coding capacity of rAAV vectors (generally <5 kb) prevents the incorporation of large genes / ORFs, such as the coding sequences of one of the anti-VEGF and anti-fibrotic proteins, using dual promoters and / or IRES linkers (in this regard, the EMCV IRES is 553 nucleotides long).

[0121] Thus, in a preferred embodiment, the genetic construct comprises a spacer sequence disposed between the first and second coding sequences. For example, see FIG. 3, where a spacer sequence (v2A) is disposed between the first and second coding sequences. This spacer sequence encodes a peptide spacer configured to produce the anti-VEGF protein and the anti-fibrotic protein as separate molecules. This is possible because the spacer is configured to skip linear ribosomal sequence transcription to produce separate molecules or peptides. It will be appreciated that the separate molecules are active.

[0122] Preferably, the spacer sequence comprises and encodes a viral peptide spacer sequence, most preferably a viral-2A peptide spacer sequence, hi one embodiment, the viral-2A peptide spacer sequence comprises an F2A, E2A, T2A or P2A sequence.

[0123] Preferably, the viral-2A peptide sequence connects the first coding sequence to the second coding sequence, which allows the construct to overcome size limitations associated with expression in various vectors and allows expression of all peptides encoded by the construct of the first aspect to occur under the control of a single promoter as a single mRNA transcript.

[0124] Thus, in one embodiment, following transcription of a single mRNA transcript encoding the sequences of the anti-VEGF protein, the viral-2A peptide and the anti-fibrotic protein, translational skipping can occur in the viral-2A peptide sequence between the terminal glycine-proline of the viral-2A peptide, which would thereby generate two proteins, the anti-VEGF protein and the anti-fibrotic protein (see FIG. 3).

[0125] The inventors have generated four embodiments of the spacer sequence. One important section of the peptide spacer sequence that is common to all the embodiments described herein is the C-terminus.

[0126] In one embodiment, the peptide spacer sequence is P2A. Preferably, the P2A peptide spacer sequence encodes the amino acid sequence referred to herein as SEQ ID NO: 37, or a fragment or variant thereof, as follows:

[0127] [ka]

[0128] Preferably, the digestion or cleavage site of the peptide spacer sequence is located between the terminal glycine and the terminal proline in SEQ ID NO:37.

[0129] In this first embodiment, the P2A peptide spacer sequence comprises the nucleotide sequence designated herein as SEQ ID NO:38 (57 bp), or a fragment or variant thereof, as follows:

[0130] [ka]

[0131] In a second embodiment, the peptide spacer sequence is E2A. Preferably, the E2A peptide spacer sequence encodes the amino acid sequence referred to herein as SEQ ID NO:39, or a fragment or variant thereof, as follows:

[0132] [ka]

[0133] Preferably, the digestion or cleavage site of the peptide spacer sequence is located between the terminal glycine and the terminal proline in SEQ ID NO:39.

[0134] In this second embodiment, the E2A peptide spacer sequence comprises the nucleotide sequence designated herein as SEQ ID NO:40 (60 bp), or a fragment or variant thereof, as follows:

[0135] [ka]

[0136] In a third embodiment, the peptide spacer sequence is T2A. Preferably, the T2A peptide spacer sequence encodes the amino acid sequence referred to herein as SEQ ID NO:41, or a fragment or variant thereof, as follows:

[0137] [ka]

[0138] Preferably, the digestion or cleavage site of the peptide spacer sequence is located between the terminal glycine and the terminal proline in SEQ ID NO:41.

[0139] In this third embodiment, the T2A peptide spacer sequence comprises the nucleotide sequence designated herein as SEQ ID NO:42 (54 bp), or a fragment or variant thereof, as follows:

[0140] [ka]

[0141] In a fourth preferred embodiment, the peptide spacer sequence is F2A. Preferably, the F2A peptide spacer sequence encodes the amino acid sequence referred to herein as SEQ ID NO:43, or a fragment or variant thereof, as follows:

[0142] [ka]

[0143] Preferably, the digestion or cleavage site of the peptide spacer sequence is located between the terminal glycine and the terminal proline in SEQ ID NO:43.

[0144] In this fourth embodiment, the F2A peptide spacer sequence comprises the nucleotide sequence designated herein as SEQ ID NO:44 (66 bp), or a fragment or variant thereof, as follows:

[0145] [ka]

[0146] Thus, in a preferred embodiment, the peptide spacer sequence comprises a nucleotide sequence substantially as set out in any one of SEQ ID NOs: 38, 40, 42 or 44, or a fragment or variant thereof. Preferably, the peptide spacer sequence encodes an amino acid sequence substantially as set out in SEQ ID NOs: 37, 39, 41 or 43, or a fragment or variant thereof.

[0147] After translation skipping, the viral-2A peptide sequence remains fused to the C-terminus of the upstream protein (such as the anti-VEGF protein), while the proline remains fused to the N-terminus of the downstream protein (such as the anti-fibrotic protein). This may impose an immunogenic risk and potentially interfere with intracellular signaling capabilities. Therefore, we introduced an enzyme cleavage coding sequence directly upstream of the viral-2A peptide sequence, such that the remaining viral-2A peptide sequence is removed from both the encoded proteins (i.e., the anti-VEGF protein and the anti-fibrotic protein). The introduction of an enzyme cleavage site has the effect of removing the viral-2A peptide either intracellularly prior to the release of the secreted protein, in the case of the enzyme furin, or immediately after the protein is secreted from the target (retinal) cell, in the case of the enzyme recognition sites for matrix metalloprotein-2 (MMP-2) or renin. MMP-2 and furin are enzymes known to be secreted by Müller glial cells and therefore available for cleavage and removal of viral-2A peptide sequences from anti-VEGF and anti-fibrotic proteins within the neural retina after secretion.

[0148] Thus, in one embodiment, the construct further comprises a viral-2A deletion sequence. Preferably, the viral-2A deletion sequence is located 5' of the viral-2A sequence. Preferably, the viral-2A deletion sequence is separated from the viral-2A sequence by a linker sequence comprising the tripeptide glycine-serine-glycine sequence (GSG).

[0149] The inventors introduced a furin recognition sequence to enzymatically remove the viral-2A peptide sequence from the C-terminus of the protein. Thus, in one embodiment, the viral-2A removal sequence is a furin recognition sequence.

[0150] Currently, the furin recognition sequence is generally recognized to contain three or four basic amino acids (arginine or lysine), with an optional non-basic amino acid at position 2, and is cleaved by the enzyme furin after the last basic amino acid. However, using various plasmid constructs, the inventors have determined that this basic furin recognition sequence does not necessarily result in enzyme activity and separation of the viral-2A sequence. As such, the inventors have generated preferred furin recognition sequences for use in the genetic constructs of the present invention.

[0151] Thus, in a preferred embodiment, the genetic construct comprises a viral-2A excision sequence encoding the amino acid sequence referred to herein as SEQ ID NO:45, or a fragment or variant thereof, as follows, where B=basic amino acid, X=hydrophilic amino acid and S=serine:

[0152] [ka]

[0153] Preferably, the hydrophilic amino acid (X) is either serine (S) or threonine (T).Thus, in one embodiment, the viral-2A excision sequence encodes an amino acid sequence substantially as set forth in SEQ ID NO: 45, or a fragment or variant thereof.

[0154] In one embodiment, the viral-2A removal sequence encodes the amino acid sequence referred to herein as SEQ ID NO:46, or a fragment or variant thereof, as follows:

[0155] [ka]

[0156] In this first embodiment, the viral-2A excision sequence comprises the nucleotide sequence referred to herein as SEQ ID NO:47, or a fragment or variant thereof, as follows:

[0157] [ka]

[0158] In a second embodiment, the viral-2A excision sequence encodes the amino acid sequence referred to herein as SEQ ID NO:48, or a fragment or variant thereof, as follows:

[0159] [ka]

[0160] In this second embodiment, the viral-2A excision sequence comprises the nucleotide sequence referred to herein as SEQ ID NO:49, or a fragment or variant thereof, as follows:

[0161] [ka]

[0162] In a third embodiment, the viral-2A excision sequence encodes the amino acid sequence referred to herein as SEQ ID NO:50, or a fragment or variant thereof, as follows:

[0163] [ka]

[0164] In this third embodiment, the viral-2A excision sequence comprises the nucleotide sequence referred to herein as SEQ ID NO:51, or a fragment or variant thereof, as follows:

[0165] [ka]

[0166] Thus, in one embodiment, the viral-2A removal sequence comprises a nucleotide sequence substantially as set forth in any of SEQ ID NOs: 47, 49 or 51, or a fragment or variant thereof. Preferably, the viral-2A removal sequence encodes an amino acid sequence substantially as set forth in SEQ ID NOs: 46, 48 or 50, or a fragment or variant thereof.

[0167] Alternatively, in another embodiment, the virus-2A removal sequence is a gelatinase MMP-2 recognition sequence.Preferably, in this embodiment, the virus-2A removal sequence encodes the amino acid sequence GPQGIAGQ [SEQ ID NO: 52], GPLGIAGA [SEQ ID NO: 53] or GPQGLLGQ [SEQ ID NO: 54], or a fragment or variant thereof.Cleavage preferably occurs after the second glycine residue.

[0168] The present inventors have generated a preferred amino acid sequence, designated herein as SEQ ID NO:55, which contains the gelatinase MMP-2 recognition sequence and the tripeptide GSG linker sequence.

[0169] Thus, in one embodiment, the viral-2A excision sequence encodes the amino acid sequence referred to herein as SEQ ID NO:55, or a fragment or variant thereof, as follows:

[0170] [ka]

[0171] In this embodiment, the viral-2A removal sequence comprises a nucleotide sequence designated herein as SEQ ID NO:56, or a fragment or variant thereof, as follows:

[0172] [ka]

[0173] Thus, in one embodiment, the viral-2A removal sequence comprises a nucleotide sequence substantially as set forth in SEQ ID NO: 56, or a fragment or variant thereof. Preferably, the viral-2A removal sequence encodes an amino acid sequence substantially as set forth in SEQ ID NO: 55, or a fragment or variant thereof.

[0174] Alternatively, in another embodiment, the virus-2A excision sequence is a renin recognition sequence.Preferably, in this embodiment, the virus-2A excision sequence encodes the amino acid sequence HPFHLVYS [SEQ ID NO: 57] or HPFHLLVYS [SEQ ID NO: 58], or a fragment or variant thereof.Cleavage preferably occurs after the leucine residue(s).

[0175] The inventors have generated a preferred amino acid sequence, designated herein as SEQ ID NO:59, which contains a renin recognition sequence and a tripeptide GSG linker sequence.

[0176] Thus, in one embodiment, the viral-2A excision sequence encodes the amino acid sequence referred to herein as SEQ ID NO:59, or a fragment or variant thereof, as follows:

[0177] [ka]

[0178] In this embodiment, the viral-2A removal sequence comprises a nucleotide sequence designated herein as SEQ ID NO:60, or a fragment or variant thereof, as follows:

[0179] [ka]

[0180] Thus, in one embodiment, the viral-2A removal sequence comprises a nucleotide sequence substantially as set forth in SEQ ID NO: 60, or a fragment or variant thereof. Preferably, the viral-2A removal sequence encodes an amino acid sequence substantially as set forth in SEQ ID NO: 59, or a fragment or variant thereof.

[0181] As illustrated in Figure 2, the expression cassette further comprises a sequence encoding a Hepatitis virus post-transcriptional regulatory element (WPRE), a sequence encoding a polyA tail, and left and right inverted terminal repeats (ITRs). Preferably, the genetic construct comprises a nucleotide sequence encoding a Woodchuck Hepatitis virus (WHP) post-transcriptional regulatory element (WPRE), which enhances expression of the transgenes, i.e., anti-VEGF and anti-fibrotic proteins. Preferably, the WPRE coding sequence is located 3' of the transgene coding sequence.

[0182] One embodiment of the Woodchuck Hepatitis Virus Post-transcriptional Regulatory Element (WPRE) is 592 nucleotides in length containing a gamma-alpha-beta element, and is referred to herein as SEQ ID NO:61, as follows:

[0183] [ka]

[0184] Preferably, the WPRE comprises a nucleic acid sequence substantially as set forth in SEQ ID NO: 61, or a fragment or variant thereof.

[0185] However, in a preferred embodiment, a truncated WPRE is used which is 247 nucleotides in length due to deletion of the β-element and is referred to herein as SEQ ID NO:62, as follows:

[0186] [ka]

[0187] Preferably, therefore, the truncated WPRE comprises a nucleic acid sequence substantially as set out in SEQ ID NO: 62, or a fragment or variant thereof.

[0188] Advantageously, the truncated WPRE sequence used in the construct omits a total of about 300 nucleotides without negatively affecting transgene expression. Preferably, the WPRE thus comprises a nucleic acid sequence substantially as set forth in SEQ ID NO: 62, or a fragment or variant thereof.

[0189] Preferably, the genetic construct comprises a nucleotide sequence encoding a polyA tail. Preferably, the polyA tail coding sequence is located 3' of the transgene coding sequence, preferably 3' of the WPRE coding sequence. The polyA tail is important for the nuclear export, translation and stability of the mRNA. The tail shortens over time and, when it is short enough, the mRNA is enzymatically degraded.

[0190] Preferably, the polyA tail comprises the simian virus-40 polyA 224 nucleotide sequence. One embodiment of a polyA tail is designated herein as SEQ ID NO:63, as follows:

[0191] [ka]

[0192] In another embodiment, the polyA tail comprises a 169 nucleotide sequence polyA component, referred to herein as SEQ ID NO:64, as follows:

[0193] [ka]

[0194] In a further embodiment, the poly A tail comprises the bovine growth hormone poly A 225 nucleotide sequence, referred to herein as SEQ ID NO:99, as follows:

[0195] [ka]

[0196] Preferably, therefore, the poly A tail comprises a nucleic acid sequence substantially as set out in SEQ ID NO: 63, 64 or 99, or a fragment or variant thereof.

[0197] Preferably, the genetic construct comprises a left and / or right inverted terminal repeat (ITR). Preferably, each ITR is located at the 5' and / or 3' end of the construct. The ITR can be any sequence, as long as it is specific to the virus (e.g., AAV or lentivirus) serotype and forms a hairpin loop in its secondary structure.

[0198] The DNA sequence of one embodiment of the ITR (left ITR sequence obtained from a commercially available recombinant AAV genomic plasmid) is represented herein as SEQ ID NO:65, as follows:

[0199] [ka]

[0200] The DNA sequence of another embodiment of the ITR (right ITR sequence obtained from a commercially available recombinant AAV genomic plasmid) is represented herein as SEQ ID NO:66, as follows:

[0201] [ka]

[0202] Preferably, the left and / or right inverted terminal repeat comprises a nucleic acid sequence substantially as set out in SEQ ID NO: 65 or 66, or a fragment or variant thereof.

[0203] Recently, it has been discovered that non-coding introns located between the promoter and the gene (next to the 3' end of the promoter and the 5' end of the gene) can facilitate gene expression in certain genomic sequences through mRNA accumulation [51, 52]. Thus, the inclusion of introns in the genetic constructs of viral vectors can facilitate better transgene expression and subsequent production of mature proteins when used in combination with constitutive or regulated promoters.

[0204] Thus, in one embodiment, the genetic construct comprises a non-coding intron. Preferably, the non-coding intron is located between the promoter and the first coding sequence. In other words, the non-coding intron is located 3' of the promoter and 5' of the first coding sequence.

[0205] In one embodiment, the non-coding intron is the Minute Virus of Mice (MVM) small (121 bp) intron

[53] , referred to herein as SEQ ID NO: 67, as follows:

[0206] [ka]

[0207] In another embodiment, the non-coding intron is a sequence (133 bp) from the 5'-donor site of the first intron of the human β-globin gene and a branch from an intron of an immunoglobulin gene heavy chain variable region and a 3'-acceptor site, referred to herein as SEQ ID NO:68, as follows:

[0208] [ka]

[0209] In another embodiment, the non-coding intron is a fusion of the 5' and 3' nucleotide components of the splice acceptor of rabbit β-globin gene 1 (210 bp), designated herein as SEQ ID NO:69, as follows:

[0210] [ka]

[0211] Thus, preferably, the non-coding intron comprises a nucleic acid sequence substantially as set out in SEQ ID NO: 67, 68 or 69, or a fragment or variant thereof.

[0212] To allow correct folding of the polypeptides encoded by the genetic constructs, intracellular trafficking and secretion of the anti-VEGF and anti-fibrotic proteins from target cells, the coding sequences of these proteins are preceded by novel N-terminal minimal signal peptide coding sequences derived from the sequences of known secreted human proteins. Secretory signal peptides consist of a methionine initiator amino acid, a string of two or more basic amino acids (arginine or lysine), followed by a string of hydrophobic amino acids (leucine, isoleucine, valine or phenylalanine) and finally a cutting sequence that allows cleavage of the signal peptide from the final mature secreted protein.

[0213] Thus, in one embodiment, the genetic construct comprises a signal peptide coding sequence. Advantageously, this novel signal peptide coding sequence generated by the present inventors optimizes the intracellular cleavage and transport of secretory proteins within the cell. Preferably, the genetic construct comprises a first signal peptide coding sequence located in front of the first coding sequence and a second signal peptide coding sequence located in front of the second coding sequence. Preferably, the first and second signal peptide coding sequences are located 5' of the first and second coding sequences, respectively.

[0214] In one embodiment, the signal peptide coding sequence encodes the amino acid sequence referred to herein as SEQ ID NO:70, or a fragment or variant thereof, as shown below:

[0215] [ka]

[0216] Preferably, in this embodiment, the signal peptide coding sequence is derived from human trypsin and preferably comprises the nucleotide sequence referred to herein as SEQ ID NO:71 (57 bp), or a fragment or variant thereof, as shown below:

[0217] [ka]

[0218] In an alternative embodiment, the signal peptide coding sequence is modified to enhance secretion from a target cell. In this embodiment, the signal peptide coding sequence encodes the amino acid sequence referred to herein as SEQ ID NO: 72, or a fragment or variant thereof, as shown below:

[0219] [ka]

[0220] Preferably, in this embodiment, the signal peptide coding sequence comprises the nucleotide sequence referred to herein as SEQ ID NO:73, or a fragment or variant thereof, as shown below:

[0221] [ka]

[0222] In another embodiment, the signal peptide coding sequence encodes the amino acid sequence referred to herein as SEQ ID NO:74, or a fragment or variant thereof, as shown below:

[0223] [ka]

[0224] Preferably, in this embodiment, the signal peptide coding sequence comprises the nucleotide sequence referred to herein as SEQ ID NO:75, or a fragment or variant thereof, as shown below:

[0225] [ka]

[0226] In another embodiment, the signal peptide coding sequence encodes the amino acid sequence referred to herein as SEQ ID NO:76, or a fragment or variant thereof, as shown below:

[0227] [ka]

[0228] Preferably, in this embodiment, the signal peptide coding sequence comprises the nucleotide sequence referred to herein as SEQ ID NO:77, or a fragment or variant thereof, as shown below:

[0229] [ka]

[0230] In another embodiment, the signal peptide coding sequence encodes the amino acid sequence referred to herein as SEQ ID NO:78, or a fragment or variant thereof, as shown below:

[0231] [ka]

[0232] Preferably, in this embodiment, the signal peptide coding sequence comprises the nucleotide sequence referred to herein as SEQ ID NO:79, or a fragment or variant thereof, as shown below:

[0233] [ka]

[0234] Thus, preferably, the signal peptide coding sequence comprises a nucleotide sequence substantially as set out in any one of SEQ ID NOs: 71, 73, 75, 77 or 79, or a fragment or variant thereof. Preferably, the signal peptide coding sequence encodes an amino acid sequence substantially as set out in SEQ ID NOs: 70, 72, 74, 76 or 78, or a fragment or variant thereof.

[0235] In a preferred embodiment, the genetic construct may include, in this designated order, a 5' promoter; a first coding sequence encoding an anti-VEGF protein; and a 3' second coding sequence encoding an anti-fibrotic protein. The use of 5' and 3' indicates that the feature is either upstream or downstream, and is not intended to indicate that the feature is necessarily a terminal feature. Furthermore, one skilled in the art will understand that the first and second coding sequences encoding the anti-VEGF protein and the anti-fibrotic protein may be arranged in any 5' to 3' order.

[0236] In certain embodiments, the genetic construct can include, in this specified order: a 5' promoter; a first coding sequence encoding an anti-VEGF protein; a spacer sequence; and a 3' second coding sequence encoding an anti-fibrotic protein.

[0237] In certain embodiments, the genetic construct can include, in this specified order: a 5' promoter; a first coding sequence encoding an anti-VEGF protein; a viral-2A excision sequence; a spacer sequence; and a 3' second coding sequence encoding an anti-fibrotic protein.

[0238] In certain embodiments, the genetic construct may include, in this specified order: a 5'ITR; a promoter; a first coding sequence encoding an anti-VEGF protein; a viral-2A excision sequence; a spacer sequence; a second coding sequence encoding an anti-fibrotic protein; a sequence encoding a WPRE; a sequence encoding a polyA tail; and a 3'ITR.

[0239] In certain embodiments, the genetic construct may include, in this specified order: a 5'ITR; a promoter; a non-coding intron; a first coding sequence encoding an anti-VEGF protein; a viral-2A excision sequence; a spacer sequence; a second coding sequence encoding an anti-fibrotic protein; a sequence encoding a WPRE; a sequence encoding a polyA tail; and a 3'ITR.

[0240] In certain embodiments, the genetic construct may include, in this specified order: a 5'ITR; a promoter; a non-coding intron; a first signal peptide coding sequence; a first coding sequence encoding an anti-VEGF protein; a viral-2A excision sequence; a spacer sequence; a second signal peptide coding sequence; a second coding sequence encoding an anti-fibrotic protein; a sequence encoding a WPRE; a sequence encoding a polyA tail; and a 3'ITR.

[0241] From the foregoing, the skilled artisan will recognize the nucleotide sequence of the embodiment of the construct of the first aspect, as well as the amino acid sequence of the encoded transgene. However, for the avoidance of doubt, in one embodiment, the amino acid sequence of [VEGF capture protein-2-furin-P2A-anti-CTGF SCVF-1] is referred to herein as SEQ ID NO: 80, as follows:

[0242] [ka]

[0243] Preferably, in this embodiment, the construct comprises the 2241 nucleotide sequence (contained in plasmid IKC153P), referred to herein as SEQ ID NO:81, or a fragment or variant thereof, as follows:

[0244] [ka]

[0245] In another embodiment, the amino acid sequence of [VEGF capture protein-2-furin-P2A-anti-CTGF SCVF-2] is as follows, referred to herein as SEQ ID NO:82, or a fragment or variant thereof:

[0246] [ka]

[0247] Preferably, in this embodiment, the construct comprises the 2217 nucleotide sequence (as contained in plasmid IKC154P), referred to herein as SEQ ID NO:83, or a fragment or variant thereof, as follows:

[0248] [ka]

[0249] In another embodiment, the amino acid sequence of [VEGF capture protein 2-furin-P2A-anti-C3b SCVF] is as follows, referred to herein as SEQ ID NO:84, or a fragment or variant thereof:

[0250] [ka]

[0251] Preferably, in this embodiment, the construct comprises the 2220 nucleotide sequence (as contained in plasmid IKC129P), referred to herein as SEQ ID NO:85, or a fragment or variant thereof, as follows:

[0252] [ka]

[0253] In another embodiment, the amino acid sequence of [VEGF capture protein 2-furin-P2A-anti-Bb SCVF] is as follows, referred to herein as SEQ ID NO:86, or a fragment or variant thereof:

[0254] [ka]

[0255] Preferably, in this embodiment, the construct comprises the 2244 nucleotide sequence (as contained in plasmid IKC130P), referred to herein as SEQ ID NO:87, or a fragment or variant thereof, as follows:

[0256] [ka]

[0257] In another embodiment, the amino acid sequence of [VEGF capture protein 2-furin-P2A-sCD55] is as follows, referred to herein as SEQ ID NO:88, or a fragment or variant thereof:

[0258] [ka]

[0259] Preferably, in this embodiment, the construct comprises the 2454 nucleotide sequence designated herein as SEQ ID NO:89 (as contained in plasmid IKC131P), or a fragment or variant thereof, as follows:

[0260] [ka]

[0261] In another embodiment, the amino acid sequence of [sCD55-Furin-P2A-VEGF capture protein 2] is as follows, referred to herein as SEQ ID NO:90, or a fragment or variant thereof:

[0262] [ka]

[0263] Preferably, in this embodiment, the construct comprises the 2454 nucleotide sequence designated herein as SEQ ID NO:91 (as contained in plasmid IKC132P), or a fragment or variant thereof, as follows:

[0264] [ka]

[0265] In another embodiment, the amino acid sequence of [VEGF capture protein 2-furin-P2A-CFHR1] is as follows, referred to herein as SEQ ID NO:92, or a fragment or variant thereof:

[0266] [ka]

[0267] Preferably, in this embodiment, the construct comprises the 2430 nucleotide sequence designated herein as SEQ ID NO:93 (as contained in plasmid IKC133P), or a fragment or variant thereof, as follows:

[0268] [ka]

[0269] In another embodiment, the amino acid sequence of [CFHR1-Furin-P2A-VEGF capture protein 2] is as follows, referred to herein as SEQ ID NO:94, or a fragment or variant thereof:

[0270] [ka]

[0271] Preferably, in this embodiment, the construct comprises the 2430 nucleotide sequence designated herein as SEQ ID NO:95 (as contained in plasmid IKC134P), or a fragment or variant thereof, as follows:

[0272] [ka]

[0273] In another embodiment, the amino acid sequence of [VEGF capture protein 2-furin-P2A-CFHL1] is as follows, referred to herein as SEQ ID NO: 100, or a fragment or variant thereof:

[0274] [ka]

[0275] Preferably, in this embodiment, the construct comprises the 2769 nucleotide sequence designated herein as SEQ ID NO:101 (as contained in plasmid IKC144P), or a fragment or variant thereof, as follows:

[0276] [ka]

[0277] In another embodiment, the amino acid sequence of [CFHL1-Furin-P2A-VEGF capture protein 2] is as follows, referred to herein as SEQ ID NO: 102, or a fragment or variant thereof:

[0278] [ka]

[0279] Preferably, in this embodiment, the construct comprises the 2766 nucleotide sequence designated herein as SEQ ID NO: 103 (as contained in plasmid IKC175P), or a fragment or variant thereof, as follows:

[0280] [ka]

[0281] In another embodiment, the amino acid sequence of [VEGF capture protein 2-furin-P2A-sCD46] is as follows, referred to herein as SEQ ID NO: 104, or a fragment or variant thereof:

[0282] [ka]

[0283] Preferably, in this embodiment, the construct comprises the 2424 nucleotide sequence designated herein as SEQ ID NO: 105 (as contained in plasmid IKC176P), or a fragment or variant thereof, as follows:

[0284] [ka]

[0285] In another embodiment, the amino acid sequence of [sCD46-Furin-P2A-VEGF capture protein 2] is as follows, referred to herein as SEQ ID NO: 106, or a fragment or variant thereof:

[0286] [ka]

[0287] Preferably, in this embodiment, the construct comprises the 2421 nucleotide sequence designated herein as SEQ ID NO: 107 (as contained in plasmid IKC177P), or a fragment or variant thereof, as follows:

[0288] [ka]

[0289] Thus, in a preferred embodiment the construct encodes an amino acid sequence substantially as set out in SEQ ID NO: 80, 82, 84, 86, 88, 90, 92, 94, 100, 102, 104 or 106, or a fragment or variant thereof.

[0290] Preferably, the construct comprises a nucleotide sequence substantially as set out in SEQ ID NO: 81, 83, 85, 87, 89, 91, 93, 95, 101, 103, 105 or 107, or a fragment or variant thereof.

[0291] The present inventors have created a series of recombinant expression vectors which contain the constructs of the present invention.

[0292] Thus, in a second aspect there is provided a recombinant vector comprising a genetic construct according to the first aspect.

[0293] In one embodiment, the recombinant vector (e.g., known as “IKC153P”) comprises a nucleotide sequence designated herein as SEQ ID NO:96, or a fragment or variant thereof, as follows:

[0294] [ka]

[0295] [ka]

[0296] Thus, in one embodiment, the recombinant vector comprises a nucleotide sequence substantially as set out in SEQ ID NO: 96, or a fragment or variant thereof.

[0297] The recombinant vector can be a recombinant AAV (rAAV) vector. The rAAV can be a naturally occurring vector or a vector with a hybrid AAV serotype. The rAAV can be AAV-1, AAV-2, AAV-2.7m8, AAV-3A, AAV-3B, AAV-4, AAV-5, AAV-6, AAV-7, AAV-8, AAV-9, AAV-10 and AAV-11. Preferably, the rAAV is rAAV serotype-2.

[0298] Advantageously, recombinant AAV2 elicits a minimal immune response in the host organism and mediates long-term transgene expression in the retina that can persist for at least one year following vector administration.

[0299] The term "recombinant AAV (rAAV) vector" can refer to a recombinant AAV-derived nucleic acid, which can contain at least one terminal repeat sequence.

[0300] The capsid coat of AAV and recombinant vectors is known to be composed of three capsid proteins called VP1, VP2 and VP3, all of which contain a significant amount of overlapping amino acids between them, but contain unique N-terminal sequences. The AAV virus contains 60 subunits of each of the VP1, VP2 and VP3 capsid proteins in a 1:1:10 ratio, which together form an icosahedral structure. Many AAV serotypes have been identified that differ in amino acid composition and thus confer different binding properties to receptors in host cells. There are several naturally occurring AAV serotypes identified, such as AAV1, AAV2, AAV2.7m8, AAV3, AAV4, AAV5, AAV6, AA7, AAV8, AAV9, AAV10, AAV11 and AAV12, as well as many artificial variants, where further modifications to the amino acid sequence have been identified by screening DNA variants from libraries of capsid coding sequences. Thus, different serotypes can display tropism, and the exchange of various amino acids in one pseudotype can change the tropism or infectivity to different target cells. Thus, specific AAV pseudotypes can be designed to target one specific cell type or to largely restrict infectivity to a specific organ. In some embodiments, the rAAV vector is a vector derived from an AAV serotype, including AAV1, AAV2, AAV2.7m8, AAV3, AAV4, AAV5, AAV6, AA7, AAV8, AAV9, AAV10, AAV11, or AAV12. The rAAV particle can include capsid proteins derived from any AAV serotype, including AAV1, AAV2, AAV2.7m8, AAV3, AAV4, AAV5, AAV6, AA7, AAV8, AAV9, AAV10, AAVrhlO, AAV11, or AAV12 capsid. The rAAV particles can contain viral proteins and viral nucleic acids of the same serotype or mixed serotypes.

[0301] The capsid protein of the recombinant viral particles of the invention can comprise or consist of the amino acid sequence of a naturally occurring protein (e.g., a naturally occurring AAV capsid protein, such as the capsid protein of AAV serotypes AAV1, AAV2, AAV2.7m8, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAVrhlO, AAV11, or AAV12), or can be a derivative or chimera of a naturally occurring capsid protein that contains one or more amino acid substitutions, deletions, or additions compared to the amino acid sequence of the naturally occurring capsid protein, for example, to confer tropism for a desired tissue or cell type (such as retinal ganglion cells, photoreceptor or retinal pigment epithelial cells) or to reduce immunogenicity of the recombinant viral particle.

[0302] In some embodiments, the capsid protein of a recombinant viral particle of the invention has an amino acid sequence along its entire length that has at least 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98% or 99% amino acid identity to the amino acid sequence of a naturally occurring capsid protein, e.g., a naturally occurring AAV capsid protein of serotypes AAV1, AAV2, AAV2.7m8, AAV3, AAV4, AAV5, AAV6, AA7, AAV8, AAV9, AAV10, AAVrhlO, AAV11, or AAV12.

[0303] The constructs and expression vectors described herein can be used to treat retinal disorders, in particular wet age-related macular degeneration or diabetic retinopathy, including diabetic macular edema (DMO), and more generally to reduce vascular leakage and retinal cell damage.

[0304] Thus, in a third aspect there is provided a genetic construct according to the first aspect or a recombinant vector according to the second aspect for use as a medicament or in therapy.

[0305] In a fourth aspect, there is provided a genetic construct according to the first aspect or a recombinant vector according to the second aspect for use in the treatment, prevention or amelioration of retinal disorders or for reducing vascular leakage and retinal cell damage.

[0306] In a fifth aspect, there is provided a method for treating, preventing or ameliorating a retinal disorder or reducing vascular leakage and retinal cell damage in a subject, the method comprising administering or having administered to a subject in need of such treatment a therapeutically effective amount of a genetic construct according to the first aspect or a recombinant vector according to the second aspect.

[0307] Preferably, the genetic constructs or recombinant vectors according to the invention are used in gene therapy techniques. The anti-VEGF and anti-fibrotic proteins encoded by the constructs or vectors neutralize both VEGF and either CTGF or complement proteins, thereby preventing angiogenesis, reducing vascular leakage, and reducing inflammation, fibrosis and scarring.

[0308] In one embodiment, the retinal disorder to be treated can be wet age-related macular degeneration.Alternatively, in another embodiment, the retinal disorder to be treated can be diabetic retinopathy or any other retinal disorder associated with diabetes, such as diabetic macular edema (DMO).In addition, the retinal disorder to be treated can be any pathophysiological condition that involves vascular leakage and the resulting damage to retinal structure.

[0309] In another embodiment, the constructs and vectors can be used to reduce vascular leakage activation and retinal cell damage.The constructs and vectors can be used to treat vascular leakage and retinal cell damage associated with the following conditions: diabetic retinopathy, cancer, systemic capillary leak syndrome (SCLS) / Clarkson syndrome, angioedema, severe trauma, shock, sepsis, multiple organ dysfunction syndrome (MODS), chronic kidney disease, end-stage renal disease, Kawasaki disease, severe Ebola virus disease, dengue virus infection and / or mycobacteria infection.

[0310] It will be appreciated that the genetic construct according to the first aspect or the recombinant vector according to the second aspect may be used in a medicament (i.e. use of the genetic construct according to the first aspect or the vector according to the second aspect of the invention) that may be used as a sole therapy for treating, ameliorating or preventing retinal disorders or for reducing vascular leakage and retinal cell damage. Alternatively, the genetic construct or recombinant vector according to the invention may be used as an adjunct to or in combination with known therapies for treating, ameliorating or preventing retinal disorders or for reducing vascular leakage and retinal cell damage.

[0311] An effective amount of recombinant viral vector is administered depending on the purpose of treatment. For example, if a low percentage of transduction can achieve the desired therapeutic effect, the purpose of treatment is generally to meet or exceed this level of transduction. In some instances, this level of transduction can be achieved by transduction of only about 1-5% of the target cells, in some embodiments, at least about 20% of the cells of the desired tissue type, in some embodiments, at least about 50% of the cells of the desired tissue type, in some embodiments, at least about 80%, in some embodiments, at least about 95%, and in some embodiments, at least about 99%. In some embodiments of the present invention, the dose of viral particles administered to the subject is about 1×10 8 ~1×10 14 Between genome copies.

[0312] The recombinant viral particles can be administered by one or multiple injections in the same procedure or spaced apart by days, weeks, months or years. In some embodiments, multiple vectors can be used to treat a subject. In some embodiments, at least 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70% or 75% to 100% of the cells of a target tissue (e.g., retinal cells of the eye) are transduced. Methods for identifying cells transduced by recombinant viral particles, including recombinant viral particle capsids, are known in the art. For example, immunohistochemistry or the use of markers (market), such as enhanced green fluorescent protein, can be used to detect transduction of recombinant viral particles.

[0313] In some embodiments, the recombinant vector is administered (e.g., by injection or infusion) to one or more locations in the desired tissue (e.g., eye). In some embodiments, the recombinant vector is administered (e.g., by injection or infusion) to any one of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more than 10 locations in the tissue. In some embodiments, the recombinant vector is administered simultaneously or sequentially to more than one location. In some embodiments, multiple injections of the recombinant vector are separated by 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 9 hours, 12 hours, or 24 hours or less.

[0314] The genetic constructs or recombinant vectors according to the invention can be combined in compositions having many different forms, depending in particular on the manner in which the composition is to be used. Thus, for example, the composition can be in the form of a powder, tablet, capsule, liquid, ointment, cream, gel, hydrogel, aerosol, spray, micellar solution, transdermal patch, liposomal suspension, or any other suitable form that can be administered to a person or animal in need of treatment. It will be appreciated that the pharmaceutical vehicle according to the invention should be a vehicle that is well tolerated by the subject to which it is given.

[0315] The genetic construct or recombinant vector of the present invention can also be incorporated into a slow or delayed release device. Such a device can be inserted, for example, above or below the skin, and can release the drug over a period of weeks or even months. The device can be located at least adjacent to the treatment site. Such a device can be particularly advantageous when long-term treatment with the genetic construct or recombinant vector is required, which would normally require frequent administration (e.g., at least daily injections).

[0316] In a preferred embodiment, the medicament of the present invention can be administered to a subject by injection into the bloodstream, into a nerve, or directly to the site requiring treatment.For example, the medicament can be injected at least adjacent to the retina.The injection can be intravitreal, suprachoroidal, subretinal, intraretinal, intravenous (bolus or injection), or subcutaneous (bolus or injection), or intradermal (bolus or injection).

[0317] It will be appreciated that the amount of genetic construct or recombinant vector required will be determined by its biological activity and bioavailability, which in turn will depend on the mode of administration, the physiochemical properties of the genetic construct or recombinant vector, and whether it is used as a monotherapy or in a combination therapy. The frequency of administration will also be affected by the half-life of the transgene protein in the subject being treated. The optimal dosage to be administered can be determined by those skilled in the art and will vary with the specific genetic construct or recombinant vector being used, the strength of the pharmaceutical composition, the mode of administration, and the progression of retinal edema or resulting retinal damage or loss in retinal neurons. Additional factors depending on the specific subject being treated, including the subject's age, weight, sex, diet, and time of administration, will result in the need to adjust the dosage.

[0318] The genetic construct or recombinant vector may be administered before, during or after the onset of the retinal disorder. The daily dose may be given as a single administration (e.g., a single daily injection or inhalation of a nasal spray). Alternatively, the genetic construct or recombinant vector may require administration two or more times during the day. By way of example, the genetic construct or recombinant vector may be between 0.001 μg / kg (body weight) and 10 mg / kg (body weight) of DNA plasmid, or 1×10 8 GC / mL ~ 1 × 10 13 The viral vector may be administered as two (or more, depending on the severity of the retinal disorder or retinal capillary dysfunction being treated) daily doses of between 1000 and 10000 GC / mL (i.e. assuming a body weight of 70 kg). The patient undergoing treatment may take the first dose upon waking and then the second dose at night (in the case of a two-dose regime), or at intervals of every 3 or 4 hours thereafter. Alternatively, a slow release device may be used to provide the patient with an optimal dose of the genetic construct or recombinant vector of the present invention without the need for repeated doses.

[0319] Known procedures, such as those conventionally used by the pharmaceutical industry (e.g., in vivo experimental methods, clinical trials, etc.), can be used to create specific formulations and precise treatment regimens (such as daily doses of drugs and frequency of administration) of the genetic constructs or recombinant vectors of the present invention. The inventors consider themselves the first to suggest a bicistronic genetic construct that encodes a promoter operably linked to a coding sequence that reduces VEGF concentrations below levels that cause pathophysiology while at the same time reducing or eliminating subretinal fibrosis via CTGF neutralization or attenuating complement activation.

[0320] In a sixth aspect, there is provided a pharmaceutical composition comprising a genetic construct according to the first aspect or a recombinant vector according to the second aspect and a pharma- ceutically acceptable vehicle.

[0321] In a seventh aspect there is provided a method of preparing a pharmaceutical composition according to the sixth aspect, the method comprising contacting a genetic construct according to the first aspect or a recombinant vector according to the second aspect with a pharma- ceutically acceptable medium.

[0322] A "subject" may be a vertebrate, a mammal or a domestic animal. Thus, the compositions and medicaments of the present invention may be used to treat any mammal, such as livestock (e.g., horses), pets, or in other veterinary applications. Most preferably, however, the subject is a human being.

[0323] A "therapeutically effective amount" of a genetic construct, recombinant vector, or pharmaceutical composition is any amount that, when administered to a subject, is the amount described above required for the treatment of dry age-related macular edema or GA.

[0324] For example, a therapeutically effective amount of the genetic construct, recombinant vector or pharmaceutical composition used may be about 1×10 8 vector particles ~ approx. 1×10 15 vector particles, preferably about 1 x 10 11 vector particles ~ approx. 1×10 12 It may be a vector particle.

[0325] In some embodiments, the viral titer of the pharmaceutical composition of the invention is 5×10 per milliliter or less. 10 ~5×10 13 Between genome copies.

[0326] In some embodiments, the viral titer of the pharmaceutical composition of the invention is 5×10 per milliliter or less. 10 ~5×10 13 The term "transducing unit" as used in reference to viral titer refers to the number of infectious recombinant vector particles that result in the production of a functional transgene product as measured in a functional assay such as those described in [57, 58].

[0327] A "pharmaceutically acceptable vehicle" as referred to herein is any known compound or combination of known compounds known to those of skill in the art to be useful in formulating a pharmaceutical composition.

[0328] In one embodiment, the pharma- ceutically acceptable vehicle may be solid and the composition may be in the form of a powder or tablet. A solid pharma- ceutically acceptable vehicle may contain one or more substances that may act as flavorings, lubricants, solubilizers, suspending agents, dyes, fillers, glidants, compression aids, inert binders, sweeteners, preservatives, pigments, coatings or tablet disintegrants. The vehicle may also be an encapsulating material. In a powder, the vehicle is a finely divided solid in admixture with a finely divided active agent according to the invention. In a tablet, the active agent (e.g., a genetic construct or recombinant vector according to the invention) may be mixed with a vehicle having the necessary compression properties in a suitable ratio and compacted into the desired shape and size. Powders and tablets preferably contain up to 99% of the active agent. Suitable solid vehicles include, for example, calcium phosphate, magnesium stearate, talc, sugars, lactose, dextrin, starch, gelatin, cellulose, polyvinylpyrrolidine, low melting waxes and ion exchange resins, hi another embodiment, the pharmaceutical vehicle may be a gel and the composition may be in the form of a cream or the like.

[0329] However, the pharmaceutical medium may be liquid and the pharmaceutical composition is in the form of a suspension of particles in the solution. Liquid media are used in the preparation of solutions, suspensions, emulsions, syrups, elixirs and pressurized compositions. The genetic construct or recombinant vector of the present invention may be dissolved or suspended in a pharma- ceutically acceptable liquid medium, such as water, an ionic buffer solution, an organic solvent, a mixture of both, or a pharma- ceutically acceptable oil or fat. The liquid medium may contain other suitable pharmaceutical excipients, such as solubilizers, emulsifiers, buffers, preservatives, sweeteners, flavorings, suspending agents, thickeners, colorants, viscosity regulators, stabilizers or osmo regulators. Suitable examples of liquid media for oral and parenteral administration include water (partially containing additives as described above, e.g., cellulose derivatives, preferably sodium carboxymethylcellulose solution), alcohols (including monohydric and polyhydric alcohols, e.g., glycols) and their derivatives, and oils (e.g., fractionated coconut oil and arachis oil). For parenteral administration, the vehicle can also be an oily ester, such as ethyl oleate and isopropyl myristate. Sterile liquid vehicles are useful in sterile liquid form compositions for parenteral administration. The liquid vehicle for pressurized compositions can be a halogenated hydrocarbon or other pharma- ceutically acceptable propellant.

[0330] Liquid pharmaceutical compositions that are sterile solutions or suspensions can be used, for example, by intravitreal, suprachoroidal, subretinal, intraretinal, intracameral, intramuscular, intrathecal, epidural, intraperitoneal, intravenous and especially subcutaneous injection. Genetic constructs or recombinant vectors can be prepared as sterile solid compositions that can be dissolved or suspended at the time of administration using sterile water, saline or other suitable sterile injectable medium.

[0331] Pharmaceutically acceptable carriers, excipients and diluents are relatively inert or pharma- ceutically effective substances that facilitate administration and can be provided as liquid solutions or suspensions, as emulsions, or as solid forms suitable for dissolution or suspension in liquid prior to use. For example, excipients can provide a suitable form for or consistency, or act as diluents. Suitable excipients include, but are not limited to, stabilizing agents, wetting agents and emulsifying agents, salts for varying osmolality, encapsulating agents, pH buffering agents, and buffers. Such excipients include any pharmaceutical agent suitable for direct delivery to a subject (e.g., intravitreal or subretinal) that can be administered without undue toxicity. Pharmaceutically acceptable excipients include, but are not limited to, sorbitol, any of the various TWEEN compounds, and liquids such as water, saline, glycerol, and ethanol. Pharmaceutically acceptable salts may be included therein, for example mineral acid salts such as hydrochloride, hydrobromide, phosphate, sulfate, etc.; and salts of organic acids such as acetates, propionates, malonates or benzoates.

[0332] In some embodiments, the pharmaceutical acceptable excipients can include pharmaceutical acceptable carriers. Such pharmaceutical acceptable carriers can be sterile liquids such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, and the like. Saline solutions and dextrose water, polyethylene glycol (PEG) and glycerol solutions can also be used as liquid carriers, particularly for injectable solutions. Additional ingredients, such as preservatives, buffers, isotonicity agents, antioxidants and stabilizers, non-ionic wetting or clarifying agents, or viscosity-increasing agents, can also be used. A thorough discussion of pharmaceutical acceptable excipients and carriers can be found in Remington's Pharmaceutical Sciences (Ed Remington JP and Gennaro AR; Mack Pub. Co. Easton, Pa 1990).

[0333] It will be appreciated that the invention extends to any nucleic acid or peptide comprising substantially the amino acid or nucleic acid sequence of any of the sequences referenced herein, or variants, derivatives or analogs thereof, including variants or fragments thereof. The terms "substantially an amino acid / nucleotide / peptide sequence", "variant" and "fragment" may refer to a sequence having at least 40% sequence identity with the amino acid / nucleotide / peptide sequence of any one of the sequences referenced herein, for example 40% identity with the sequences identified as SEQ ID NOs: 1-107, etc.

[0334] Also envisaged are amino acid / polynucleotide / polypeptide sequences having sequence identity of greater than 65%, more preferably greater than 70%, even more preferably greater than 75%, and even more preferably greater than 80% sequence identity to any of the referenced sequences. Preferably, the amino acid / polynucleotide / polypeptide sequence has at least 85% identity to any of the referenced sequences, more preferably at least 90% identity, even more preferably at least 92% identity, even more preferably at least 95% identity, even more preferably at least 97% identity, even more preferably at least 98% identity, and most preferably at least 99% identity to any of the sequences referenced herein.

[0335] Those skilled in the art will know how to calculate the percentage identity between two amino acid / polynucleotide / polypeptide sequences.To calculate the percentage identity between two amino acid / polynucleotide / polypeptide sequences, it is necessary to first make an alignment of the two sequences, and then calculate sequence identity value.The percentage identity of two sequences can take different values ​​depending on: (i) the method used to align sequences, such as ClustalW, BLAST, FASTA, Smith-Waterman (implemented in different programs), or structural alignment from 3D comparison; and (ii) the parameters used by alignment method, such as local vs. global alignment, the pair-score matrix used (such as BLOSUM62, PAM250, Gonnet, etc.), and gap-penalty, such as function type and constant.

[0336] Once aligned, there are many different ways to calculate the percentage identity between two sequences. For example, the number of identities can be divided by (i) the length of the shortest sequence; (ii) the length of the alignment; (iii) the average length of the sequences; (iv) the number of non-gap positions; or (iv) the number of equivalenced positions excluding overhangs. Furthermore, it will be appreciated that the percentage identity is also strongly length-dependent. Thus, the shorter the sequence pair, the higher the sequence identity that can be expected to occur by chance.

[0337] It will therefore be appreciated that accurate alignment of protein or DNA sequences is a complex process. The popular multiple alignment program ClustalW [59, 60] is the preferred way to generate protein or DNA multiple alignments according to the present invention. Suitable parameters for ClustalW may be as follows: for DNA alignment: gap opening penalty = 15.0, gap extension penalty = 6.66 and matrix = identity. For protein alignment: gap opening penalty = 10.0, gap extension penalty = 0.2 and matrix = Gonnet. For DNA and protein alignment: ENDGAP = -1 and GAPDIST = 4. Those skilled in the art will be aware that the above and other parameters may need to be varied for optimal sequence alignment.

[0338] Preferably, the calculation of the percentage identity between two amino acid / polynucleotide / polypeptide sequences is then carried out as follows: (N / T) *Percentage identity between two sequences can be calculated from alignments such as 100, where N is the number of positions where the sequences share identical residues and T is the total number of positions being compared, including gaps and including or excluding overhangs. Preferably, overhangs are included in the calculation. Thus, the most preferred method for calculating the percentage identity between two sequences involves (i) creating a sequence alignment using, for example, the ClustalW program with a suitable set of parameters, as set forth above; and (ii) calculating the percentage identity using the formula: sequence identity=(N / T). * Inserting the values ​​of N and T into 100.

[0339] Alternative methods for identifying similar sequences will be known to those skilled in the art. For example, a substantially similar nucleotide sequence will be encoded by a sequence that hybridizes to a DNA sequence or its complement under stringent conditions. Stringent conditions mean that the nucleotide hybridizes to filter-bound DNA or RNA in 3x sodium chloride / sodium citrate (SSC) at approximately 45°C, followed by at least one wash in 0.2x SSC / 0.1% SDS at approximately 20-65°C. Alternatively, a substantially similar polypeptide can differ from the sequences shown herein by at least one, but less than 5, 10, 20, 50 or 100 amino acids.

[0340] Due to the degeneracy of the genetic code, it is clear that any nucleic acid sequence described herein can be varied or changed without substantially affecting the sequence of the protein encoded thereby to provide a functional variant thereof. Suitable nucleotide variants are those that have a sequence that is altered by the substitution of different codons that code for the same amino acid within the sequence, thus resulting in a silent change. Other suitable variants are those that have a homologous nucleotide sequence but contain all or part of the sequence, altered by the substitution of different codons that code for amino acids with side chains of similar biophysical properties to the amino acid that is substituted, to produce a conservative change. For example, small non-polar, hydrophobic amino acids include glycine, alanine, leucine, isoleucine, valine, proline and methionine. Large non-polar, hydrophobic amino acids include phenylalanine, tryptophan and tyrosine. Polar neutral amino acids include serine, threonine, cysteine, asparagine and glutamine. Positively charged (basic) amino acids include lysine, arginine and histidine. Negatively charged (acidic) amino acids include aspartic acid and glutamic acid. It is therefore recognized that any amino acid may be replaced with an amino acid having similar biophysical properties, and one of ordinary skill in the art would know the nucleotide sequences encoding such amino acids. All of the features described herein (including any accompanying claims, abstracts and drawings) and / or all of the steps of any method or process so disclosed may be combined in any combination with any of the above-described embodiments, except combinations in which at least some of such features and / or steps are mutually exclusive.

[0341] All of the features described in this specification (including any accompanying claims, abstract and drawings) and / or all of the steps of any method or process so disclosed may be combined in any combination with any of the above-described aspects, except combinations in which at least some of such features and / or steps are mutually exclusive.

[0342] For a better understanding of the invention and to show how embodiments thereof may be carried into effect, reference will now be made, by way of example, to the accompanying drawings in which: [Brief description of the drawings]

[0343] [Figure 1] Figure 1 is an illustration of one embodiment of gene therapy viral vectors according to the invention (top of figure) expressing various transgene proteins, namely anti-VEGF and anti-fibrotic proteins, and their biological effects in reducing the pathophysiology associated with retinal disorders such as wet AMD. In the figure, the anti-fibrotic proteins are shown to be either anti-CTGF (connective tissue growth factor) proteins or anti-complement proteins. [Diagram 2] Figure 2 is a schematic diagram of one embodiment of a genetic construct according to the present invention. The transgene cassette essentially codes for a single mRNA transcript from which two independently secreted proteins, a VEGF neutralizing component and an anti-fibrotic component, are produced. These components can be in either position (orientation), linked via the enzyme target / viral 2A cleavage / skipping site. [Diagram 3] Figure 3 illustrates the intracellular biochemical processing of the bicistronic construct from a single mRNA transcript into two mature therapeutic proteins. Step 1 is the transcription of messenger RNA by a single promoter followed by translation of a single large coding sequence. Step 2 is translational skipping by the ribosome guided by the viral 2A sequence resulting in two separate proproteins. Step 3 occurs at the level of the Golgi where the viral-2A sequence is cleaved from the proprotein by the activity of furin / enzyme at the upstream cleavage site. Step 4 is the removal of the secretory protein signal peptide, which removes the remaining proline amino acid from the N-terminus of the downstream component prior to secretion from the target retinal cells. [Figure 4]FIG. 4 shows images of enhanced green fluorescent protein (eGFP) reporter gene expression in HEK293 cells harvested 24 hours after transduction with rAAV2 vectors containing different promoter sequences: chicken beta-actin promoter / cytomegalovirus enhancer promoter (sCAG), the sCAG promoter followed by the addition of an intron created from the fusion of a short stretch of nucleotides derived from the 5' and 3' ends of a rabbit beta-globulin intron (sCAG-intron), the cytomegalovirus promoter (CMV), the mouse phosphoglycerate kinase promoter (mPGK), and the human synaptin-1 promoter (hSYN1). [Diagram 5] Figure 5 shows both cross-sections and flat-mount images of mouse retinas to illustrate the level of eGFP expression 3 weeks after intravitreal injection with rAAV2 vectors containing different promoters: small chicken beta-actin promoter / cytomegalovirus enhancer promoter (sCAG); cytomegalovirus enhancer element, plus a short stretch of nucleotides from the 5' and 3' of the rabbit beta-globulin intron (sCAG-intron); cytomegalovirus promoter (CMV); mouse phosphoglycerate kinase-1 promoter (mPGK); and human synaptin-1 (hSYN1) promoter. The symbol "*" indicates the ganglion cell layer. [Figure 6]FIG. 6 shows Western blots illustrating expression of VEGF capture-2 protein in supernatants collected from HEK293T cells 24 hours after transfection with a series of expression plasmids (A and B) or transduction with a series of rAAV2 / 2 vectors (C and D). FIG. 6A and FIG. 6C show the results of supernatants probed with an IgG-Fc antibody that detects secreted VEGF capture-2 protein. FIG. 6B and FIG. 6D show the results of supernatants probed with an antibody that detects viral-2A protein. VEGF capture-2 protein with the viral-2A amino acid sequence still attached is shown above the dotted line, which is present in some of the lanes. [Figure 7] Figure 7A shows a Western blot illustrating the intracellular processing of expression cassettes from IKC153P and IKC144P plasmid transfected HEK293T cells, which secrete VEGF capture-2 protein and anti-fibrotic components anti-CTGF SCVF-1 or CFHL1, respectively, by translation skipping at the viral-2A sequence and subsequent removal by furin enzymatic cleavage. Figure 7B shows immunocytochemistry in HEK293T cells transfected with either control (IKC166P) or IKC154P (VEGFCap-2-Furin-P2A-Anti-CTGF SCVF-2), showing the detection of VEGF capture-2 protein and anti-CTGF SCVF. Examples of anti-fibrotic components are anti-CTGF SCVF-1 and anti-CTGF SCVF-2 (IKC153P and IKC154P), and CFHL1 protein (IKC144P). [Figure 8]Figure 8 illustrates VEGF-165 concentrations measured by ELISA in cell culture medium produced by HEK293T cells 24 hours after transfection with a series of plasmids. Secreted proteins capable of VEGF-165 neutralization were compared to medium from cells that did not receive any plasmid (no plasmid) or cells transfected with IKC036P (null-control plasmid). IKC112P contains only novel VEGF capture protein-2, IKC115P contains VEGF capture protein-2-furin-viral P2A-anti-CTGF-SCVF-1, and IKC116P contains VEGF capture protein-2-furin-viral P2A-anti-CTGF-SCVF-2. [Figure 9] Figure 9 demonstrates the ability of VEGF-165A (Figure 9A) or VEGF-121B (Figure 9B) to induce proliferation of human umbilical vascular endothelial cells (HUVEC) as measured by increased MTS absorbance. Addition of culture medium from HEK293T cells previously transfected with various plasmid constructs shows that transgene proteins released from HRK293T cells transfected with IKC053P, IKC112P, IKC115P and IKC116P plasmids are able to prevent exogenous VEGF-165A or VEGF-121B induced HUVEC cell proliferation compared to control untransfected HEK293T cells or HEK293T cells transfected with a null control plasmid (IKC036P) or a plasmid expressing only anti-CTGF SCVF-1 (IKC118P). The mechanism for HUVEC anti-proliferative efficacy is through the production and release of VEGF-neutralizing proteins from IKC053P, IKC112P, IKC115P and IKC116P transfected HEK293T cells. [Figure 10]Figure 10 further illustrates the ability of the plasmid construct to produce a protein that can prevent HUVEC proliferation when grown on a bed of human fibroblasts (HUVEC-fibroblast co-culture). Figure 10A shows that HUVEC express eGFP marker protein and the signal-to-noise is improved by staining with anti-eGFP antibody. Longer tubules are formed in the IKC036P control group compared to the IKC115P and IKC116P exposed groups, both of which express anti-VEGF transgene protein. Figures 10B and 10C show the formation of longer HUVEC tubules with branches in the presence (under) 10 ng / mL VEGF-165A (control and IKC036P null control), which is significantly inhibited by the addition of culture medium from HEK293T cells transfected with plasmids producing VEGF-neutralizing proteins (IKC112P, IKC115P and IKC116P). [Figure 11] FIG. 11 is a quantification of Western blots to compare the levels of fibrotic markers in ARPE-19 cells after transfection with IKC153P (precursor is IKC115P) or IKC154P (precursor is IKC116P) versus IKC036P null plasmids. FIG. 11A shows the levels of the profibrotic enzyme MMP2 secreted from cells under serum starvation conditions. FIG. 11B shows the levels of MMP2 secreted from cells after stimulation with transforming growth factor-beta 2 (TGF-β2). FIG. 11C shows the secreted levels of fibronectin in the culture medium, and FIG. 11D shows the levels of α-smooth muscle actin (α-SMA) in cells (cell lysates) normalized to β-actin. [Figure 12] Figure 12: Confluent and serum-starved ARPE-19 monolayers stained for fibronectin protein (grey) after transduction with IKC036V (null control), IKC115V or IKC116V. Note the reduction in fibronectin scaffolds placed in IKC115V and IKC116V treated cells versus the IKC036V null control. [Figure 13]FIG. 13 shows an embodiment of a plasmid map of a vector of the present invention (IKC153P). [Figure 14] Figure 14 is a C3b cleavage assay to demonstrate the activity of the CFHL1 component in the IKC144P plasmid. HEK293T cells were transfected with either the null control plasmid (IKC036P) or the plasmid of the CFHL1 bicistronic plasmid (IKC144P), and non-transfected HEK293T cells were used as an additional control (PBS). After transfection, HEK293T supernatants were collected and incubated with recombinant C3b and recombinant CFI proteins, then Western blots were performed and probed with C3 antibody. Figure 14 shows that C3b cleavage occurred only in the presence of the IKC144P plasmid, but not in the presence of any of the controls (IKC036P or PBS only). [Figure 15] FIG. 15 shows an embodiment of a plasmid map of a vector of the present invention (IKC115P). [Figure 16] Figure 16 shows the results of mouse laser treatment-induced choroidal neovascularization (CNV) study, in which mice were intravitreally treated with PBS control or IKC115V or IKC116V vector. Figure 16A shows sequential images at the choroidal focal plane at 1, 2 and 3 minutes after fluorescein administration to demonstrate the level of leakage from each laser treatment site (circled). Figure 16B is a graph to show the fluorescein leakage area at 14 days after CNV, showing that the IKC115V and IKC116V treatment groups have significantly lower CNV leakage area compared to the PBS treatment group. [Figure 17]Figure 17 illustrates that intravitreal injection of either rAAV2 IKC151V or IKC152V bicistronic vectors in mice significantly increases vitreous concentrations of VEGF trapping-2 protein compared to IKC166V null vector-treated mice, as measured by IgG ELISA in samples taken 3 weeks after vector administration (****P<0.0001; one-way ANOVA). In addition, the vitreous concentrations of VEGF trapping-2 produced by bicistronic vectors IKC151V and IKC152V were similar to those produced by monocistronic vector IKC163V, which produces only aflibercept. Importantly, the levels of expressed VEGF trapping-2 protein exceed the baseline modeled aflibercept pharmacokinetic range after a single aflibercept (2 mg) dose in patients, measured 6 weeks after bolus injection

[61] . This study demonstrates that inclusion of a secondary coding component, which attenuates the onset and potentially reverses subretinal fibrosis, does not reduce the ability of the vector to neutralize common isoforms of soluble VEGF. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0344] [Example] With reference to Figures 1 and 2, the present inventors have designed and constructed novel genetic constructs encoding (i) anti-VEGF proteins and (ii) anti-fibrotic proteins (either anti-complement proteins or anti-CTGF proteins, such as antibodies or antigen-binding fragments thereof) under the control of a single promoter. As illustrated in Figure 2, the present inventors have also advantageously introduced spacer sequences into the genetic constructs (e.g., viral-2A peptide spacer sequences) that allow expression of all of the peptides encoded by the constructs to occur as a single mRNA transcript under the control of a single promoter. Moreover, in order to enzymatically remove the viral-2A peptide sequence from the C-terminus of the protein, the present inventors have introduced viral-2A removal sequences, such as a furin recognition sequence, into the constructs.

[0345] As illustrated in Figure 3, the bicistronic expression cassette produces two mature therapeutic proteins, an anti-VEGF protein and an anti-fibrotic protein. The anti-VEGF protein acts to prevent angiogenesis and reduce vascular leakage. The anti-fibrotic protein reduces fibrosis, scarring and inflammation.

[0346] Next, the inventors introduced the genetic construct into a recombinant expression vector such as rAAV2 (see, e.g., Figures 13 and 15).

[0347] Materials and Methods DNA plasmid design and production DNA sequences were codon-optimized using the tool (http: / / www.jcat.de) or Genscript online tool. Synthetic DNA blocks and cloning were performed using standard molecular biology techniques. All DNA plasmids were scaled up in SURE competent cells (Agilent Technologies) overnight after maxi-prep purification with minimal endotoxin present.

[0348] IKC036P is a null control and IKC053P is a reference plasmid designed to produce and secrete only VEGF capture protein-1. IKC112P contains only the novel VEGF capture protein-2, IKC115P contains VEGF capture protein-2-furin-viral P2A-anti-CTGF-SCVF-1, IKC116P contains VEGF capture protein-2-furin-viral P2A-anti-CTGF-SCVF-2, IKC097P contains VEGF capture protein-2-furin-viral P2A-anti-CTGF-SCVF (non-optimized), IKC102P contains VEGF capture protein-2-furin-viral P2A-anti-CTGF-SCVF-1 (non-optimized), and IKC118P contains anti-CTGF-SCVF-1.

[0349] Recombinant AAV vector production The DNA plasmids were used to produce recombinant AAV2 vectors. HEK293T cells (2.5 × 10 8 HEK293T cells (1000 x 1000 cells) were transduced with a total of 500 μg of the three plasmids (Rep-2-Cap2, pHelper, and ORF- and ITR-containing plasmids). Freezing and thawing of HEK293T cells to release viral vector particles was followed by iodixanol gradient ultracentrifugation and desalting. After obtaining vector titers by qPCR using primers recognizing the ITR region, the vectors were suspended in Dulbecco's Phosphate Buffered Saline (DPBS) buffer from Thermo Fisher / Gibco manufactured to cGMP standards (catalog no. 14190250, consisting of 8 g / L NaCl, 1.15 g / L Na2HPO4, 0.2 g / L KCl, and 0.2 g / L K2HPO4, without calcium or magnesium; pH 7.0-7.3, 270-300 mOsm / kg).

[0350] Figure 4 illustrates enhanced green fluorescent protein (eGFP) reporter gene expression in HEK293T cells harvested 24 hours after plasmid transfection with constructs containing different promoter sequences: sCAG, CMV, hSYN1, mPGK and sCAG-intron. As illustrated in Figure 4, both sCAG and CMV display high levels of eGFP transgene expression in HEK293T cells.

[0351] Figure 5 shows both cross-section and flat-mount images of mouse retina to illustrate the level of eGFP expression 3 weeks after intravitreal injection of 5x10^9GC / eye with rAAV2 vectors containing different promoter sequences: sCAG, CMV, hSYN1, mPGK and sCAG-intron. As can be seen in Figure 5, the addition of an intron in the sCAG-intron promoter increases retinal expression compared to the same promoter without the intron, i.e., sCAG. EXAMPLES

[0352] Detection of furin activity and viral-2A peptide cleavage Briefly, DNA plasmids were transfected into cells by mixing the plasmids with Opti-MEM (FisherSci) and Lipofectamine 3000 (FisherSci). HEK293T cells were optimally transfected at 80% confluency in 6-well plates such that each well received 2 μg of plasmid DNA and 3.75 μL of Lipofectamine. Cells were incubated for 24 hours at 37° C., 5% CO2.

[0353] Western blotting of collected supernatants was used to visualize separation of secreted VEGF capture-2 protein from anti-CTGF / complement proteins. VEGF capture proteins containing either wild type or modified human IgG-Fc moieties were detected using an HRP-conjugated anti-human IgG Fc antibody (goat anti-IgG Fc (HRP conjugated, ab98624; Abcam, diluted 1:7000). Antibody (NBP2-59627; NovusBio, 1:1000;) was used to test viral-2A peptide removal from the C-terminus of the first transgene protein.

[0354] HEK293T cells were transfected with the plasmids described above. To confirm the cleavage of viral-2A peptide from the C-terminus, IgG-Fc antibody was used to detect secreted VEGF capture-2 protein in all evaluated supernatants. As illustrated in Figure 6A and Figure 6C, the majority of VEGF capture-2 protein is cleaved at the furin site and detected without the presence of viral-2A. Importantly, the cleaved VEGF capture-2 protein migrates at the same size as the VEGF capture-2 protein derived from IKC112P transfected cells that does not contain viral-2A sequence.

[0355] Moreover, as shown in Figure 6B, the cells were also probed with an antibody that detects the viral-2A protein. Cross-reactivity (non-specific staining) can be seen at the same molecular weight as the VEGF capture-2 protein (IKC036P and IKC036V null vector lanes), which is derived from IKC112P transfected cells that do not contain the viral 2A sequence. The viral 2A antibody also detected (two) heavier molecular weight bands in IKC115P and IKC116P supernatants (Figure 6B), indicating that most, but not all, of the viral-2A sequence was cleaved at the furin site. The bands were weaker in the supernatants of the earlier expression constructs IKC097P and IKC102P, indicating that the viral-2A sequence was cleaved at the furin site.

[0356] Further Western blotting and immunocytochemistry studies (Figure 7) confirmed the expression and segregation of both transgenes from cells transfected with the bicistronic plasmid construct. In Figure 7A, HEK293T cells transfected with IKC153P and IKC144P plasmids express VEGF capture-2 protein (goat anti-IgG Fc (HRP conjugated, ab98624; Abcam, 1:2000 dilution), and either Ikarovec-commissioned anti-CTGF SCVF-1 (rabbit polyclonal (peptide 1); GenScript, 1:500 dilution) or CFHL-1 (rabbit anti-CFH, ab133536; Abcam, 1:1000 dilution), respectively. In Figure 7B, transfected cells were co-labeled with the above mentioned antibodies for IgG Fc (1:1000) or anti-CTGF SCVF-1 (1:1000). Anti-CTGF SCVF-1 (rabbit polyclonal (peptide 1) was used to express the anti-CTGF in plasmid IKC154P. Note that SCVF-2 was not immunolabeled and no non-specific staining was detected in the null control (IKC166P). EXAMPLES

[0357] VEGF concentrations in HEK293T cells after transfection with various plasmid constructs HEK293T cells were transfected with the plasmids described above. The HEK293T cell incubation medium was collected and centrifuged to remove any cell debris, and the VEGF-165 concentration produced by the cells was then measured using a commercially available human VEGF ELISA kit (ab222510; Abcam). The results were compared to the medium from cells not transfected with plasmid (no plasmid) and cells transfected with IKC036P (null plasmid). The VEGF-165 concentration was measured and the results were compared to the medium (supernatant) from cells not transfected with plasmid (no plasmid) and cells transfected with IKC036P (null plasmid). As illustrated in Figure 8, a significant reduction in VEGF concentration was observed in cells transfected with a plasmid containing the anti-VEGF protein component. EXAMPLES

[0358] Attenuation of VEGF-induced human umbilical vascular endothelial cell (HUVEC) growth by plasmid transgene products Media from HEK293T cell incubation media previously transfected with no plasmid (control) or test plasmid DNA in Opti-MEM for 6 h and then replaced with serum-free DMEM, high glucose, GlutaMAX (FisherSci) for 24 h was added to HUVECs grown in endothelial cell medium (ECM, C22010; PromoCell) containing 1× Pen-Strep in 96-well plates (3,000 cells / well). Recombinant supplemented VEGF (5-100 ng / mL; Caltag-Medsystems) was then added to each well and HUVECs were incubated for 72 h. Cell growth was measured by spectrophotometry using MTS (G3580, CellTitre 96® aqueous One solution; Promega). Absorbance was read at 490 nm.

[0359] As illustrated in Figure 9, culture medium from HEK293T cells previously transfected with the plasmids did not show HUVEC proliferation in the presence of 50 ng / mL VEGF-165A or VEGF-121B (Figure 9C and Figure 9D), demonstrating complete VEGF neutralization by the anti-VEGF plasmid constructs. EXAMPLES

[0360] HUVEC-fibroblast co-culture and generation of vascular networks We used the Caltag-Medsystems angiogenic endothelial / fibroblast co-culture kit, consisting of eGFP-expressing HUVECs and human fibroblasts at a 1:30 ratio. Endothelial cells initially form small islands within the culture matrix, then proliferate and eventually form thread-like tubule structures in the gel matrix to form a network of anastomosing tubules within 10 days of culture. Angiogenic co-cultures are known to be responsive to known micro- and macromolecular inhibitors and stimulators of angiogenesis. Starting on day 2, every 2–3 days, medium from HEK293T cells previously transfected with plasmid (using the protocol described above) was added to the co-cultures and supplemented with 10 μg / mL recombinant human VEGF (ab9571; Abcam). Control cultures received sumarin, which blocks the effects of fibroblast growth factor and other growth factors produced by fibroblasts. At the end of the experiment, tubular HUVEC structures were fixed and imaged using a fluorescent microscope for eGFP-expressing cells, which were then stained with an anti-GFP antibody (A11122; Invitrogen, 1:2,000 dilution).MetaLabs FastTrack tubule formation AI program was used to calculate average tube length and branching.

[0361] As illustrated in Figure 10A, HUVEC growth was significantly inhibited after exposure to medium from HEK293T cells transfected with expression plasmids IKC0115P and IKC116P, compared to the null control IKC036P. Moreover, as illustrated in Figures 10B and 10C, HUVEC tubule length and branching were significantly reduced after exposure to medium from HEK293T cells transfected with IKC115P and IKC116P, compared to the control. EXAMPLES

[0362] Reduction of fibrotic marker protein expression in ARPE-19 cells after transfection with various plasmids ARPE-19 cells were transfected with the plasmids described above in Opti-MEM for 24 hours. On day 2, the medium was replaced with DMEM / F12+5%FBS, then replaced with serum-free DMEM / F12 on day 3. On day 5, TGFbeta2 (10ng / mL, ab84070; Abcam) or fresh serum-free DMEM / F12 was added to confluent transfected cells, and on day 7, supernatants and lysates were collected. Western blotting of supernatants from serum-starved cells revealed a decrease in fibronectin (ab268020; Abcam, 1:500 dilution) (Figure 11A) and MMP2 (ab92536; Abcam, 1:1000 dilution) (Figure 11C), with significance shown for IKC154P (the precursor was known as IKC116P). A similar reduction in MMP2 was observed in supernatants from TGF beta 2 stimulated cells, with significance shown for both IKC153P (precursor was known as IKC115P) and IKC154P (precursor was known as IKC116P) (Figure 11B). Lysate samples revealed a slight reduction in alpha SMA (ab5694; Abcam, 1:500 dilution) in the TGF beta 2 stimulated group with plasmid IKC153P (precursor was known as IKC115P) and IKC154P (precursor was known as IKC116P) compared to IKC036P (null control) (Figure 11D).

[0363] As illustrated in FIG. 12, reduced fibronectin (ab268020; Abcam, diluted 1:200) immunolabeling is seen in ARPE-19 transduced with vectors IKC115V and IKC116V compared to IKC037V (null control) at day 5 post-transduction. EXAMPLES

[0364] C3b cleavage assay to assess CFHL1 HEK293T cells were transfected with the plasmids described above. The HEK293T cell incubation medium was collected and centrifuged to remove any cell debris, and then 50 μL of fresh supernatant was incubated with 42 nM recombinant C3b and 1.2 nM recombinant CFI at 37 ° C for 1 hour. After incubation, samples were then evaluated by Western blotting using C3 antibody (goat-anti-human-C3, AHP1752, 1:2000 and secondary donkey anti-goat, 705-035-147, 1:10,000).

[0365] As illustrated in Figure 14, C3b cleavage into iC3b 68 and 43 kDa fragments occurred in the presence of supernatant from HEK293T cells transfected with the IKC144P plasmid expressing the CFHL1 components, but not in the presence of control (IKC036P) or non-transfected supernatants. These results demonstrate that the CFHL1 components expressed by the bicistronic IKC144P plasmid have enzymatic activity. EXAMPLES

[0366] Efficacy of bicistronic rAAV vectors in a murine laser CNV model Mice were transfected with the rAAV vector IKC115V (8.7 × 10 9 genome copies (GC) / eye) or IKC116V (8.7 × 10 9Choroidal neovascularization was induced in treated eyes by laser photocoagulation using a 532 nm diode laser (spot size: 10 μm; power: 130 mW; time: 120 msec, Oculight TX. Iridex Corp) 3 weeks after receiving a unilateral intravitreal injection (2 μL) of GC / eye or control PBS. Mice eyes were imaged using fluorescein angiography (FA) 2 weeks after laser treatment using a Heidelberg Spectralis HRA system (Heidelberg Engineering) and a solution of 2.5% sodium fluorescein (Sigma-Aldrich) administered as a subcutaneous injection (30 μL / 10 g). Continuous fluorescent images (sensitivity: 45; ART average: 5 frames) were taken every 60 seconds from the choroidal focus level over a period of 5 minutes after fluorescein administration.

[0367] As illustrated in FIG. 16, intravitreal administration of the bicistronic rAAVs, IKC115V and IKC116V, significantly reduced the CNV leakage area detected from FA images 2 weeks after CNV compared to PBS control-treated mice. EXAMPLES

[0368] Detection of clinically relevant levels of VEGF-trapped-2 expressed from a bicistronic rAAV vector in vivo The concentration of VEGF trapping-2 protein in the mouse vitreous following intravitreal delivery of IKC151V and IKC152V rAAV vectors is shown in Figure 17, compared to the concentration of aflibercept protein (expressed from IKC163V).

[0369] Mouse, IKC151V (2.3 × 10 10 GC / Eye), IKC152V(2.7×10 10 GC / eye), IKC163V(7.8×10 9 GC / eye) or IKC166V (2.1 × 10 10GC / eye) was intravitreally injected (2 μL). After 21 days, the eyes were free dissected and vitreous samples (between 4-5 μL) were extracted. The concentration of VEGF trapping-2 or aflibercept protein in the vitreous was measured using a commercially available IgG ELISA kit (Abcam). Of note, the concentration of VEGF trapping-2 in the vitreous at 3 weeks after injection in bicistronic (IKC151V and IKC152V) treated eyes exceeds the predicted therapeutically relevant clinical levels of aflibercept based on data at 4 and 6 weeks of a 2 mg bolus of aflibercept in patients, represented in Figure 17 by the dotted line. This demonstrates that the inclusion of a secondary coding component to attenuate the onset and potentially reverse subretinal fibrosis does not reduce the vector's ability to neutralize common isoforms of soluble VEGF.

[0370] Discussion and Conclusion As illustrated in the Examples, the inventors have surprisingly demonstrated that it is possible to combine open reading frames (ORFs) encoding anti-VEGF and anti-fibrotic proteins in a single genetic construct. Given the large size of the genes, this is particularly challenging, and it could not have been predicted that these components would be co-expressed at physiologically useful concentrations from a single expression cassette, and that this expression cassette would be capable of being accommodated by the rAAV-2 vector.

[0371] As demonstrated in the examples, the anti-VEGF proteins of the claimed constructs and vectors can reduce VEGF concentrations, thereby preventing angiogenesis and reducing vascular leakage. Moreover, the presence of anti-fibrotic proteins reduces fibrosis, scarring and inflammation. Advantageously, the constructs of the present invention do not require the injection of recombinant proteins as described in the prior art, and the constructs require only a single administration to achieve a long-term therapeutic effect.

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Claims

1. A genetic construct comprising a promoter operably linked to a first coding sequence encoding an anti-VEGF protein and a second coding sequence encoding an anti-fibrotic protein.

2. 2. The genetic construct of claim 1, wherein the promoter is a cytomegalovirus (CMV) promoter, a fusion of a CMV early enhancer element and the first intron of the chicken beta-actin gene (CAG), a vitelloid macular dystrophy protein-2 (VMD2) promoter, a human phosphoglycerate kinase-1 (PGK-1) promoter, or an EF1 alpha promoter, and optionally the promoter comprises a nucleotide sequence substantially as set forth in SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8 or 9, or a fragment or variant thereof.

3. 2. The genetic construct of claim 1, wherein the first coding sequence comprises a nucleotide sequence encoding an anti-VEGF protein capable of capturing any soluble form of VEGF, including VEGF-A, VEGF-B, VEGF-C, VEGF-D and / or placenta growth factor (PIGF).

4. 2. The genetic construct of claim 1, wherein the first coding sequence comprises a nucleotide sequence encoding an anti-VEGF protein that specifically captures VEGF-A, and optionally the anti-VEGF protein is capable of capturing any isoform of VEGF-A, including VEGF-121, VEGF-145, VEGF-165, VEGF-183, VEGF-189 and / or VEGF-206.

5. 2. The genetic construct of claim 1, wherein the anti-VEGF protein is an anti-VEGF antibody or an antigen-binding fragment thereof, optionally wherein the anti-VEGF protein is a single chain variable fragment (SCVF).

6. 2. The genetic construct of claim 1, wherein the first coding sequence comprises a nucleotide sequence substantially as set forth in any one of SEQ ID NOs: 11, 13, 15, 17, 19 or 21, or a fragment or variant thereof, and / or the first coding sequence encodes an amino acid sequence substantially as set forth in SEQ ID NOs: 10, 12, 14, 16, 18 or 20, or a fragment or variant thereof.

7. The genetic construct of claim 1 , wherein the anti-fibrotic protein is an anti-complement protein.

8. The genetic construct of claim 7 , wherein the anti-complement protein is capable of neutralizing or attenuating complement activation.

9. 8. The genetic construct of claim 7, wherein the anti-complement protein is capable of targeting the alternative pathway (AP) of the complement system, and preferably, the anti-complement protein does not target the classical pathway (CP) and / or the lectin pathway (LP) of the complement system.

10. 8. The genetic construct of claim 7, wherein the anti-complement protein is an anti-C3b or anti-Bb antibody or an antigen-binding fragment thereof, and optionally the anti-complement protein is a single-chain variable fragment (SCVF).

11. 2. The genetic construct of claim 1, wherein the second coding sequence comprises a nucleotide sequence substantially as set forth in SEQ ID NO:23 or 25, or a fragment or variant thereof, and / or the second coding sequence encodes an amino acid sequence substantially as set forth in SEQ ID NO:22 or 24, or a fragment or variant thereof.

12. The genetic construct of claim 7, wherein the anti-complement protein is CD55, preferably soluble CD55 (sCD55).

13. 13. The genetic construct of claim 12, wherein the second coding sequence comprises a nucleotide sequence substantially as set forth in SEQ ID NO:27, or a fragment or variant thereof, and / or the second coding sequence encodes an amino acid sequence substantially as set forth in SEQ ID NO:26, or a fragment or variant thereof.

14. The genetic construct of claim 7, wherein the anti-complement protein is complement factor H related protein-1 (CFHR1).

15. 15. The genetic construct of claim 14, wherein the second coding sequence comprises a nucleotide sequence substantially as set forth in SEQ ID NO: 29 or 30, or a fragment or variant thereof, and / or the second coding sequence encodes an amino acid sequence substantially as set forth in SEQ ID NO: 28, or a fragment or variant thereof.

16. The genetic construct of claim 7, wherein the anti-complement protein is CD46, preferably soluble CD46 (sCD46).

17. 17. The genetic construct of claim 16, wherein the second coding sequence comprises a nucleotide sequence substantially as set forth in SEQ ID NO:32, or a fragment or variant thereof, and / or the second coding sequence encodes an amino acid sequence substantially as set forth in SEQ ID NO:31, or a fragment or variant thereof.

18. 8. The genetic construct of claim 7, wherein the anti-complement protein is complement factor H-like protein 1 (CFHL1).

19. 19. The genetic construct of claim 18, wherein the second coding sequence comprises a nucleotide sequence substantially as set forth in SEQ ID NO:98, or a fragment or variant thereof, and / or the second coding sequence encodes an amino acid sequence substantially as set forth in SEQ ID NO:97, or a fragment or variant thereof.

20. The genetic construct of claim 1 , wherein the anti-fibrotic protein is capable of neutralizing connective tissue growth factor (CTGF).

21. 21. The genetic construct of claim 20, wherein the anti-fibrotic protein is an anti-connective tissue growth factor (anti-CTGF) antibody or an antigen-binding fragment thereof, preferably the anti-CTGF antibody is an anti-CTGF single chain variable fragment (anti-CTGF SCVF).

22. 22. The genetic construct of claim 21 , wherein the second coding sequence comprises a nucleotide sequence substantially as set forth in SEQ ID NO: 34 or 36, or a fragment or variant thereof, and / or the second coding sequence encodes an amino acid sequence substantially as set forth in SEQ ID NO: 33 or 35, or a fragment or variant thereof.

23. 2. The genetic construct of claim 1, wherein the genetic construct comprises a spacer sequence disposed between the first and second coding sequences, the spacer sequence encoding a peptide spacer configured to produce the anti-VEGF protein and the anti-fibrotic protein as separate molecules.

24. 24. The genetic construct of claim 23, wherein said spacer sequence comprises and encodes a viral peptide spacer sequence, most preferably a viral-2A peptide spacer sequence.

25. 25. The genetic construct of claim 24, wherein the viral-2A peptide spacer sequence comprises an F2A, E2A, T2A or P2A sequence.

26. 24. The genetic construct of claim 23, wherein the spacer sequence comprises a nucleotide sequence substantially as set forth in SEQ ID NO: 38, 40, 42 or 44, or a fragment or variant thereof, and / or the spacer sequence encodes an amino acid sequence substantially as set forth in SEQ ID NO: 37, 39, 41 or 43, or a fragment or variant thereof.

27. 24. The genetic construct of claim 23, wherein the genetic construct comprises a viral-2A removal sequence, optionally positioned 5' to the viral-2A sequence.

28. 28. The genetic construct of claim 27, wherein the viral-2A excision sequence is separated from the viral-2A peptide spacer sequence by a linker sequence comprising the tripeptide glycine-serine-glycine sequence (G-S-G).

29. 28. The genetic construct of claim 27, wherein the viral-2A removal sequence is a furin recognition sequence, and optionally the viral-2A removal sequence encodes an amino acid sequence substantially as set forth in SEQ ID NO:45, or a fragment or variant thereof.

30. 30. The genetic construct of claim 29, wherein the viral-2A removal sequence comprises a nucleotide sequence substantially as set forth in SEQ ID NO: 47, 49 or 51, or a fragment or variant thereof, and / or wherein the viral-2A removal sequence encodes an amino acid sequence substantially as set forth in SEQ ID NO: 46, 48 or 50, or a fragment or variant thereof.

31. 28. The genetic construct of claim 27, wherein the viral-2A removal sequence is a gelatinase MMP-2 recognition sequence, and optionally wherein the viral-2A removal sequence comprises a nucleotide sequence substantially as set forth in SEQ ID NO:56, or a fragment or variant thereof, and / or wherein the viral-2A removal sequence encodes an amino acid sequence substantially as set forth in SEQ ID NO:55, or a fragment or variant thereof.

32. 28. The genetic construct of claim 27, wherein the viral-2A removal sequence is a renin recognition sequence, and optionally the viral-2A removal sequence comprises a nucleotide sequence substantially as set forth in SEQ ID NO:60, or a fragment or variant thereof, and / or the viral-2A removal sequence encodes an amino acid sequence substantially as set forth in SEQ ID NO:59, or a fragment or variant thereof.

33. 2. The genetic construct of claim 1, wherein the genetic construct comprises a nucleotide sequence encoding a Woodchuck Hepatitis Virus post-transcriptional regulatory element (WPRE), and optionally the WPRE comprises a nucleic acid sequence substantially as set forth in SEQ ID NO: 61 or 62, or a fragment or variant thereof.

34. 2. The genetic construct of claim 1, wherein the genetic construct comprises a nucleotide sequence encoding a polyA tail, and optionally the polyA tail comprises a nucleic acid sequence substantially as set forth in SEQ ID NO: 63, 64 or 99, or a fragment or variant thereof.

35. 2. The genetic construct of claim 1, wherein the genetic construct comprises a nucleotide sequence encoding a left and / or right inverted terminal repeat (ITR), and optionally the left and / or right inverted terminal repeat comprises a nucleic acid sequence substantially as set forth in SEQ ID NO: 65 or 66, or a fragment or variant thereof.

36. 2. The genetic construct of claim 1, wherein the genetic construct comprises a non-coding intron, optionally located between the promoter and the first coding sequence.

37. 37. The genetic construct of claim 36, wherein the non-coding intron comprises a nucleic acid sequence substantially as set forth in SEQ ID NO: 67, 68 or 69, or a fragment or variant thereof.

38. 2. The genetic construct of claim 1, wherein the genetic construct comprises a signal peptide coding sequence, optionally wherein the signal peptide coding sequence comprises a nucleotide sequence substantially as set forth in any one of SEQ ID NOs: 71, 73, 75, 77 or 70, or a fragment or variant thereof, and / or wherein the signal peptide coding sequence encodes an amino acid sequence substantially as set forth in SEQ ID NOs: 70, 72, 74, 76 or 78, or a fragment or variant thereof.

39. 2. The genetic construct of claim 1, wherein the genetic construct encodes an amino acid sequence substantially as set forth in SEQ ID NO: 80, 82, 84, 86, 88, 90, 92, 94, 100, 102, 104 or 106, or a fragment or variant thereof, and / or the genetic construct comprises a nucleotide sequence substantially as set forth in SEQ ID NO: 81, 83, 85, 87, 89, 91, 93, 95, 101, 103, 105 or 107, or a fragment or variant thereof.

40. A recombinant vector comprising the genetic construct of claim 1.

41. 41. The recombinant vector of claim 40, wherein the vector is a recombinant AAV (rAAV) vector.

42. The recombinant vector of claim 40, wherein the rAAV is AAV-1, AAV-2, AAV-3A, AAV-3B, AAV-4, AAV-5, AAV-6, AAV-7, AAV-8, AAV-9, AAV-10, AAV-11, or AAV-2.7m8.

43. The recombinant vector of claim 41, wherein the rAAV is rAAV serotype-2.

44. 41. The recombinant vector of claim 40, wherein the genetic construct comprises a nucleotide sequence substantially as set forth in SEQ ID NO:96, or a fragment or variant thereof.

45. 41. A genetic construct as claimed in claim 1 or a recombinant vector as claimed in claim 40 for use as a medicine or in therapy.

46. 41. A genetic construct as described in claim 1 or a recombinant vector as described in claim 40 for use in the treatment, prevention or amelioration of retinal disorders or for reducing vascular leakage and retinal cell damage.

47. 47. The genetic construct or vector for use according to claim 46, wherein the retinal disorder to be treated is wet age-related macular degeneration; diabetic retinopathy; any retinal disorder associated with diabetes; diabetic macular edema (DMO); or a pathophysiological condition involving vascular leakage and resulting damage to retinal structures.

48. 48. The genetic construct or vector for use according to claim 47, wherein the retinal disorder is wet age-related macular degeneration.

49. 47. The genetic construct or vector for use according to claim 46, wherein said construct or vector is used to reduce vascular leakage and retinal cell damage associated with any one of the following conditions: diabetic retinopathy, cancer, systemic capillary leak syndrome (SCLS) / Clarkson's syndrome, angioedema, severe trauma, shock, sepsis, multiple organ dysfunction syndrome (MODS), chronic kidney disease, end stage renal disease, Kawasaki disease, severe Ebola virus disease, Dengue virus infection and / or mycobacterial infection.

50. A pharmaceutical composition comprising the genetic construct of claim 1 or the recombinant vector of claim 40 and a pharma- ceutically acceptable vehicle.

51. A method for preparing a pharmaceutical composition according to claim 50, comprising contacting a genetic construct according to claim 1 or a recombinant vector according to claim 40 with a pharma- ceutically acceptable medium.