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 to treat, prevent or ameliorate a wide range of retinal disorders. The constructs and vectors are particularly useful for treating geographic atrophy (GA) and dry age-related macular degeneration (dry AMD). The present invention extends to the use of the constructs and vectors to reduce complement activation and retinal cell damage and loss. The present invention also extends to pharmaceutical compositions themselves and their use in treating, preventing or ameliorating retinal disorders, and reducing complement activation and retinal cell damage and loss.

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 gene therapy methods for treating, preventing or ameliorating a wide range of retinal disorders. The constructs and vectors are particularly, but not exclusively, useful for treating geographic atrophy (GA) and dry age-related macular degeneration (dry AMD). The present invention also extends to the use of the constructs and vectors for reducing complement activation and retinal cell damage and loss. The present invention also extends to the pharmaceutical composition itself and its use in treating, preventing or ameliorating retinal disorders, and reducing complement activation and retinal cell damage and loss. [Background technology]

[0002] It is estimated that there are approximately 5 million people worldwide with geographic atrophy (GA) [1], with over 1 million in the United States alone [2], equating to an average of 1 in 29 people over the age of 75. GA is a chronic progressive degeneration of the macula as part of late-stage age-related macular degeneration (AMD). The disease is characterized by focal atrophy of retinal tissue and choriocapillaris, resulting in a central scotoma and permanent vision loss. Aging and a family history of AMD are the most widely recognized risk factors for GA [3]. Past or current smoking also significantly increases an individual's risk of developing GA [3, 4], and to date, no studies have found any gender differences in GA disease prevalence [3, 5]. Age-related eye disease studies have found an increased risk of GA in users of thyroid hormone or antacid drugs, and other studies have shown that patients with coronary heart disease, lens opacities, or previous cataract surgery have a greater risk of developing GA [6]. Nevertheless, the pathogenesis of GA remains unclear.

[0003] The natural history of AMD begins early, characterized by pigment translocation and the presence of drusen, yellow deposits that form between the retinal pigment epithelium (RPE) and Bruch's membrane [7, 8]. Reticular pseudodrusen are particularly associated with the development of GA [9, 10]. Later stages of AMD are characterized by either choroidal neovascularization or GA. GA is recognized as a well-defined area in the posterior pole with atrophy of the RPE, overlying photoreceptors, and choriocapillaris.

[0004] One of the main components of drusen is the accumulated autofluorescent membrane-bound lipofuscin

[10] . Lipofuscin is often found in the aging retina and is significantly elevated within the RPE cell layer in patients with GA [11-13]. Lipofuscin is primarily responsible for autofluorescence in the human fundus in normal aging retina and more severely in patients with GA [14, 15]. Around the age of 90, lipofuscin granules occupy approximately 20% of the area of ​​macular RPE cells

[12] . As the atrophic area expands, visual function decreases [10, 12, 13, 16]. Clinically, wet and non-exudative AMD are very different, but these late stages of AMD are by no means exclusive. Individuals with GA carry a higher risk of developing choroidal neovascularization, and patients with wet AMD are at high risk of developing atrophic areas.

[0005] Lipofuscin contains a complex mixture of pigments, but the major component has been shown to be A2E (N-retinylidene-N-retinylethanolamine; and iso-A2E, which readily interconvert) formed from two molecules of all-trans-retinal or 11-cis-retinal found in the photoreceptor outer segment membrane and ethanolamine [17-22]. Lipofuscin levels have been shown to directly correlate with the histopathological damage of GA [12, 13, 22], indicating that lipofuscin is one of the major factors in GA. Not only does A2E act as a surfactant, thereby interfering with normal RPE lysosomal activity, A2E can also interfere with cholesterol metabolism

[23] and cause oxidative damage.

[0006] Several unsuccessful approaches to reduce A2E levels and drusen accumulation have been attempted using drugs that slow the biochemical visual cycle. For example, fenretinide (ReVision Therapeutics), an oral retinal binding protein 4 (RBP4)-transthyretin-retinol complex inhibitor, failed to slow GA progression in a phase 2 clinical study. In addition, the RPE65 isomerase inhibitor, ACU-4426 (emixustat hydrochloride, Acucela), reduced A2E accumulation in an ABCA4 / RDH8 double knockout mouse model [24, 25] but failed to halt GA progression in a phase 2b / 3 clinical study

[26] .

[0007] The complement system (CS) is part of the innate immune system to defend against foreign pathogens such as microorganisms [27-29] and may play a role in reducing drusen. For example, complement factor H variants, Y402H and ARMS2, have been associated with an increased risk of developing GA [30, 31], and drusen have been shown to contain multiple complement components in addition to lipofuscin [27-33]. This indicates that localized inflammation mediated by the complement system may be an important element in AMD.

[0008] CS consists of three biochemical pathways: the classical pathway (CP), the lectin pathway (LP) and the alternative pathway (AP), each of which has distinct triggers.[27-29] Although each pathway can be activated by separate components, the pathways converge to involve a crucial protein component called complement factor 3 (C3).

[0009] The classical pathway (CP) is activated by antigen-antibody complexes, particularly through the interaction between C1q and antibodies. After activation of the C1 complex, complements C2 and C4 are cleaved into C2a, C2b, C4a and C4b. The C4b and C2a proteins then combine into the C4b2a complex, which is the C3 convertase. The C4b2a complex further binds to C3b to form the C5 convertase.

[0010] The lectin pathway (LP) is usually activated by binding to mannose residues on microbial surfaces via either mannose-binding lectins or ficolins, which resemble C1q, C1r, and C1s, to form mannan-binding lectin-associated serine proteases (MASPs). Three MASPs have been characterized to date; MASP-1, MASP-2, and MASP-3. MASP-2 has a catalytic specificity similar to C1s and can cleave C2 and C4.

[0011] The alternative pathway (AP) is closely related to the pathogenesis of AMD. In AP, complement factors B, P (properdin) and D are involved in the activation of C3 through C3b and activator factor B (Bb), which form the C3 convertase complex. C3 then splits into C3a and C3b. C3b binds to the C3a receptor in immune cells, causing inflammation and thus allowing the infiltration of immune cells to further neutralize pathogens. The C3b component further amplifies C3 convertase activity and also forms complexes with either C4b and C2a (C4b-C2a-C3b) or Bb (C3b-Bb-C3b), both of which act as C5 convertases to generate C5a and C5b from the C5 protein. C5a, like C3a, can then bind to the C5b receptor on immune cells, while C5b complexes with C6, C7, C8 and C9 (S5b-9) to form the membrane attack complex (MAC) or terminal complement complex (TCC), which is capable of destroying invading microorganisms through cell swelling and eventual lysis.

[0012] 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 [34, 35]. Another modulating factor is CD55 or decay-accelerating factor (DAF), a membrane-bound protein that protects cells from complement-mediated lysis [36-39]. The main function of CD55 is to inactivate the C3 convertase by dissociating it into its component proteins [36-39]. Yet another modulating protein called complement factor H-related protein-1 (CFHR1), a splice variant of complement factor H [40-43], is also involved in reducing complement activation. A prominent regulator is complement factor H and its splice product FHL-1, which regulates fluid-phase and surface complement activity by decay-accelerating and cofactor activity. Factor H and FHL-1 target and decay the C3 convertase, which is composed of C3b and factor B. Another factor capable of reducing complement activation is membrane-associated, called membrane cofactor protein or CD46 [44, 46]. When bound to C3b, factor H and CFHL-1 occupy the factor B binding site on C3b, promoting 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 decay promotion or factor I cofactor activity. Like factor H, CFHR-1 binds C3b, recognizes self-surfaces by binding to glycosaminoglycans, and inhibits C5 convertase and terminal complement complex formation

[43] .

[0013] Despite evidence of a crucial role for the complement system in mediating GA, clinical trials of drugs modulating the complement system have shown inconsistent results. For example, the monoclonal antibodies eculizumab (Aexion) and lampalizumab

[44] (Genentech / Roche) have failed to slow GA progression in the clinic. Moreover, Gyroscope Therapeutics has produced a complement factor I gene therapy that has entered clinical trials for GA (GT005)

[47] . However, boosting CFI in patients with low or absent CFI would theoretically reduce complement activation, but all three pathways, including CP and LP, responsible for antimicrobial activity, would be affected by this treatment. Thus, systemic reduction of all complement system pathways may leave the eye vulnerable to bacterial infections, or infected eyes would take longer to clear. Moreover, early clinical trials with anti-complement monotherapy, e.g., avacincaptad pegol and complement I factor gene therapy, have demonstrated that some GA patients develop wet AMD, which necessitates patients requiring monthly anti-VEGF injections of ranibizumab or aflibercept to prevent rapid vision loss.

[0014] PEDF is a 50 kDa protein that is released apicolaterally in large quantities from RPE cells [48, 49] and displays retinal neuroprotective properties through interaction with PEDF receptors [50, 51]. PEDF has several functions in the retina, namely, it is antiangiogenic, in part through inhibition of endothelial cell migration

[54] , and displays antitumorigenic and neurotrophic properties [50-56]. There is a high density of PEDF receptors in the RPE cell layer

[57] , suggesting an autocrine and paracrine neuroprotective role in the retina. In addition, PEDF was demonstrated to protect human RPE cells from oxidative stress through upregulation of uncoupling protein-2 (UCP-2), a phenomenon proposed in the retina of GA patients as a result of lipofuscin accumulation [55, 58]. Thus, we reasoned that reduced retinal concentrations of PEDF in GA patients would leave the retinal pigment epithelium and photoreceptors extremely vulnerable to damage from oxidative stress and complement attack. Summary of the Invention [Problem to be solved by the invention]

[0015] Thus, there is a great need for improved therapies for the treatment of retinal disorders such as geographic atrophy (GA) and dry age-related macular degeneration (dry AMD) that can activate PEDF receptors and neutralize or attenuate the complement system.

[0016] With great inventive efforts, the present inventors have carefully designed and constructed a novel genetic construct that encodes PEDF receptor agonist and anti-complement protein under the control of a single promoter, i.e., it is bicistronic.The promoter of the construct can be used to ensure that both PEDF receptor agonist and anti-complement protein are maximally expressed to reduce retinal cell damage and loss through two independent pathophysiological pathways, i.e., increasing activity at PEDF receptor and reducing complement activation. [Means for solving the problem]

[0017] 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 agonist of the PEDF receptor and a second coding sequence encoding an anti-complement protein.

[0018] PEDF receptor agonists restore the concentration of PEDF, thereby reducing inflammation, reducing the level of toxic lipofuscin components, and preserving RPE and photoreceptor cells. Moreover, anti-complement proteins can neutralize or attenuate the alternative complement pathway, thereby preventing further RPE cell loss. Advantageously, the genetic constructs of the present invention target the AP pathway, which means that the classical and lectin pathways of the complement system are preserved so that an antimicrobial defense system is maintained that can facilitate the destruction of invading pathogens. Importantly, the combination of anti-complement and PEDF receptor activation will limit retinal VEGF release that, if left untreated, has the potential to transform dry AMD into the much more aggressive wet AMD pathophysiology. Furthermore, the promoters of the constructs can be used to ensure that both the PEDF receptor agonist and the anti-complement protein are maximally expressed to restore the concentration of PEDF, reduce inflammation, neutralize the complement pathway, and prevent further RPE cell loss.

[0019] The present inventors have demonstrated in the examples that it is surprisingly possible to combine open reading frames (ORFs) encoding both PEDF receptor agonists and anti-complement proteins in a single genetic construct. This was particularly difficult considering the large size of PEDF receptor agonists and anti-complement proteins. It was not possible to predict 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 accommodated by an AAV vector (such as 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 patients.

[0020] 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 a PEDF receptor agonist, and a second nucleotide sequence encoding an anti-complement protein. Thus, preferably, the genetic construct and expression cassette can be referred to as bicistronic.

[0021] As illustrated in Figure 2, the first and second coding sequences encoding the PEDF receptor agonist and the anti-complement protein can be arranged in any order from 5' to 3'. For example, in one embodiment, the coding sequence of the PEDF receptor agonist is arranged 5' of the coding sequence of the anti-complement protein, preferably with a spacer sequence between them. Alternatively, in another embodiment, the coding sequence of the anti-complement protein can be arranged 5' of the coding sequence of the PEDF receptor agonist, preferably with a spacer sequence between them.

[0022] 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.

[0023] Promoters can be constitutive or tissue specific.

[0024] 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: CGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTG GAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGG ACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCAT TGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCT [SEQ ID NO:1] 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: AGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTGGTTTAGTGAACCGTCAGAT [SEQ ID NO:2] 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: CGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATGGGACTTTCCATTGACGTCAATGGGTGGACTATTTACGGTAAACT GCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCAT CGCTATTACCATGGTCGAGGTGAGCCCCACGTTCTGCTTCACTCTCCCCATCTCCCCCCCTCCCCACCCCCAATTTTGTATTTATTTATTTTTTAATTATTTTGTGCAGCGATGGGGGCGGGGGGGGGGGGGGCGCGCGCCAG GCGGGGCGGGGCGGGGCGAGGGGCGGGGCGGGGCGAGGCGGAGAGGTGCGGCGGCAGCCAATCAGAGCGGCGCGCTCCGAAAGTTTCCTTTTATGGCGAGGCGGCGGCGGCGGCGGCCCTATAAAAAGCGAAGCGCGCGGCGGGCG [SEQ ID NO:3] A suitable tissue-specific promoter may be the vitelloid macular degeneration 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: [SEQ ID NO:4] 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: AATTCTGTCATTTTACTAGGGTGATGAAATTCCCAAGCAACACCATCCTTTTCAGATAAGGGCACTGAGGCTGAGAGAGGAGCTGAAACCTACCCGGCGTCACCACACACAGGTGGCAAGGCTGGGACCAGAAACCAGGACTGTTGACTGCAGCC CGGTATTCATTCTTTCCATAGCCCACAGGGCTGTCAAAGACCCCAGGGCCTAGTCAGAGGCTCCTCCTTCCTGGAGAGTTCCTGGCACAGAAGTTGAAGCTCAGCACAGCCCCCTAACCCCCAACTCTCTCTGCAAGGCCTCAGGGGTCAGAACAC TGGTGGAGCAGATCCTTTAGCCTCTGGATTTTAGGGCCATGGTAGAGGGGGTGTTGCCCTAAATTCCAGCCCTGGTCTCAGCCCAACACCCTCCAAGAAGAAATTAGAGGGGCCATGGCCAGGCTGTGCTAGCCGTTGCTTCTGAGCAGATTACAA GAAGGGACTAAGACAAGGACTCCTTTGTGGAGGTCCTGGCTTAGGGAGTCAAGTGACGGCGGCTCAGCACTCACGTGGGCAGTGCCAGCCTCTAAGAGTGGGCAGGGGCACTGGCCACAGAGTCCCAGGGAGTCCCACCAGCCTAGTCGCCAGACC [SEQ ID NO:5] 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: TCATTCTTTCCATAGCCCACAGGGCTGTCAAAGACCCCAGGGCCTAGTCAGAGGCTCCTCCTTCCTGGAGAGTTCCTGGCACAGAAGTTGAAGCTCAGCACAGCCCCCTAACCCCCAACTCTCTCTGCAAGGCCTCAGGGGTCAGAACACTGGTGGAGCAGATCCTTTAGCCTCTGGATTTTAGGGCCATGGTAGAGGGGGTGTTGCCCTAAATTCCAGCCCTGGTCTCAG CCCAACACCCTCCAAGAAGAAATTAGAGGGGCCATGGCCAGGCTGTGCTAGCCGTTGCTTCTGAGCAGATTACAAGAAGGGACTAAGACAAGGACTCCTTTGTGGAGGTCCTGGCTTAGGGAGTCAAGTGACGGCGGCTCAGCACTCACGTGGGCAGTGCCAGCCTCTAAGAGTGGGCAGGGGCACTGGCCACAGAGTCCCAGGGAGTCCCACCAGCCTAGTCGCCAGACC [SEQ ID NO:6] 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: GGGTAGGGGAGGCGCTTTTCCCAAGGCAGTCTGGAGCATGCGCTTTAGCAGCCCGCTGGGCACTTGGCGCTACACAAGTGGCCTCTGGCCTCGCACACATTCCACATCCACCGGTAGGCGCCAA CCGGCTCCGTTCTTTGGTGGCCCCTTCGCGCCACCTTCTACTCCTCCCTAGTCAGGAAGTTCCCCCCCGCCCCGCAGCTCGCGTCGTGCAGGACGTGACAAATGGAAGTAGCACGTCTCACTAG TCTCGTGCAGATGGACAGCACCGCTGAGCAATGGAAGCGGGTAGGCCTTTGGGGCAGCGGCCAATAGCAGCTTTGCTCCTTCGCTTTCTGGGCTCAGAGGCTGGGAAGGGGTGGTCCGGGGGCG GGCTCAGGGGCGGGCTCAGGGGCGGGGGCGGGCGCCCGAAGGTCCTCCGGAGGCCCGGCATTCTGCACGCTTCAAAAAGCGCACGTCTGCCGCGCTGTTCTCCTCTTCCTCATCTCCGGGCCTTTCG [SEQ ID NO: 7] 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: [SEQ ID NO:8] 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: GCTCCGGTGCCCGTCAGTGGGCAGAGCGCACATCGCCCACAGTCCCCGAGAAGTTGGGGGAGGGGTCGGCAATTGAACCGGTGCCTAGAGAAGGTGGCGCGGGGTAAACTGGGAAAGTGATGTCGTGTACTGGCTCCGCCTTTTTCCCGAGGGTGGGGGAGAACCGTATATAAGTGCAGTAGTCGCCGTGAACGTTCTTTTTCGCAACGGGTTTGCCGCCAGAACACAG [SEQ ID NO: 9] 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.

[0025] In order to ensure the survival of damaged and fragile RPE cells in GA patients, the present inventors have incorporated PEDF receptor agonists into the genetic construct of the present invention. The present inventors have carefully considered the sequence of PEDF receptor agonists and created several preferred embodiments of proteins that can be encoded by the first coding sequence in the genetic construct of the first aspect.

[0026] In one embodiment, the first coding sequence comprises a nucleotide sequence encoding a PEDF protein. Preferably, the PEDF protein is a human PEDF protein. Preferably, the human PEDF protein comprises the amino acid sequence (418 residues) referred to herein as SEQ ID NO: 10, or a fragment or variant thereof, as follows: MQALVLLLCIGALLGHSSCQNPASPPEEGSPDPDSTGALVEEEDPFFKVPVNKLAAAVSNFGYDLYRVRSSTSPTTNVLLSPLSVATALSALSLGAEQRTESIIHRALYYDLISSPDIHGTYKELLDTVTAPQKNLKSASRIVFEKKLRIKSFVAPLEKSYGTRPRVLTGNPRLDLQEINNWVQAQMKGKLARSTKEIPDEISILLLG VAHFKGQWVTKFDSRKTSLEDFYLDEERTVRVPMMSDPKAVLRYGLDSDLSCKIAQLPLTGSMSIIFFLPLKVTQNLTLIEESLTSEFIHDIDRELKTVQAVLT VPKLKLSYEGEVTKSLQEMKLQSLFDSPDFSKITGKPIKLTQVEHRAGFEWNEDGAGTTPSPGLQPAHLTFPLDYHLNQPFIFVLRDTTDTGALLFIGKILDPRGP [SEQ ID NO: 10] Preferably, in this embodiment, the first coding sequence comprises the nucleotide sequence referred to herein as SEQ ID NO:11 (1254 bp) as follows: [SEQ ID NO: 11] Codon analysis of the endogenous PEDF 1254 nucleotide sequence (SEQ ID NO: 11) using online rare-codon analysis tools (www.jcaj.de and https: / / www.genscript.com / tools / rare-codon-analysis) revealed that the sequence displayed a poor codon adaptation index (CAI) (JCAT CAI = 0.40 and Genscript CAI = 0.78). Highly expressed transgenes should ideally display a CAI between 0.80 and 1.00 for efficient gene expression. Thus, rare codons contained within the endogenous PEDF transgene were identified and replaced with codons more frequently used in the expression host (i.e., human eye), thus generating a codon-optimized 1254 sequence (JCAT CAI = 0.96; Genscript CAI = 1.00).

[0027] Thus, in another embodiment, the first coding sequence comprises the nucleotide sequence referred to herein as SEQ ID NO: 12 (1254 bp), or a fragment or variant thereof, as follows: [SEQ ID NO: 12] In yet another preferred embodiment, the first coding sequence comprises a nucleotide sequence (1254 bp) containing a modified signal peptide sequence, referred to herein as SEQ ID NO: 13, or a fragment or variant thereof, as follows: [SEQ ID NO: 13] Thus, in a preferred embodiment, the first coding sequence comprises a nucleotide sequence substantially as set forth in any one of SEQ ID NO: 11, 12 or 13, or a fragment or variant thereof. Preferably, the PEDF receptor agonist comprises an amino acid sequence substantially as set forth in SEQ ID NO: 10, or a fragment or variant thereof.

[0028] It will be appreciated that the second coding sequence encodes 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.

[0029] Preferably, the anti-complement protein is capable of neutralizing complement factors C3b, Bb and / or C5. Thus, in this embodiment, the anti-complement protein is an anti-C3b, anti-Bb and / or anti-C5 antibody, or an antigen-binding fragment thereof.

[0030] 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.

[0031] 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, an anti-Bb single chain variable fragment or an anti-C5 single chain variable fragment.

[0032] Alternatively, in another preferred embodiment, the anti-complement protein is a soluble form of the normally membrane-bound CD55 (sCD55) (sometimes also known as decay-accelerating factor; DAF). Preferably, the anti-complement protein is non-membrane-associated CD55 (sCD55). Soluble CD55 (DAF) destabilizes the complement protein complex, thereby reducing the activity of this biochemical pathway.

[0033] In another preferred embodiment, the anti-complement protein is complement factor H related protein-1 (CFHR1). Preferably, CFHR1 attenuates the complement system activation cascade.

[0034] In another preferred embodiment, the anti-complement protein is a soluble form of the normally membrane-bound CD46 (sCD46). Preferably, in this embodiment, the anti-complement protein is the soluble (non-membrane-associated) human complement regulatory protein CD46 (sCD46).

[0035] 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.

[0036] 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: 14, or a fragment or variant thereof: DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYATLPTFEQGTKVEIKRGGGGGSGGGGSGG GGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFSFTSSSVSPGKGLEWVGLIYPYNGFNYYADSVKGRFTISADTSLQMNSLRAEDTAVYYCARNALYGSGGYYAMDYWGQGTLVTVSS [SEQ ID NO: 14] 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: 15, or a fragment or variant thereof: GACATCCAGATGACCCAGAGCCCCAGCAGCCTGAGCGCCAGCGTGGGCGACCGCGTGACCATCACCTGCCGCGCCAGCCAGGACGTAAGCACCGCCGTGGCCTGGTACCAGCAGAAGCCCGCAAGGCCCCCAAGCTGCTGATCTACAGCGCCAGCTTCCTGTACAGCGGCGTGCCCAGCC GCTTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGACCATCAGCAGCCTGCAGCCCGAGGACTTCGCCACCTACTACTGCCAGCAGGCTACGCCACCCTGCCCACCTTCGAGCAGGGCACCAAGGTGGAGATCAAGCGCGGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGC GGCGGCAGCGGCGGCGGCGGCAGCGAGGTGCAGCTGGTGGAGAGCGGCGGCGGCCTGGTGCAGCCCGGCGGCAGCCTGCGCCTGAGCTGCGCCGCCAGCGGCTTCAGCTTCACCAGCAGCAGCGTGAGCCCCGGCAAGGGCCTGGAGTGGGTGGGCCTGATCTACCCCTACAACGGCTTCA ACTACTACGCCGACAGCGTGAAGGGCCGCTTCACCATCAGCGCCGACACCAGCCTGCAGATGAACAGCCTGCGCGCCGAGGACACCGCCGTGTACTACTGCGCCCGCAACGCCCTGTACGGCAGCGGCGGCTACTACGCCATGGACTACTGGGGCCAGGGCACCCTGGTGACCGTGAGCAGC [SEQ ID NO: 15] 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: 16, or a fragment or variant thereof: DVQITQSPSYLAASPGETITINCRASKSISKYLAWYQDKPGKTNKLLIYSGSTLQSGIPSRFSGSGSGTDFTLTISSLEPEDFAMYYCQQHDEYPWTFGGGTKLEIKRGGGGGSGGGGSGGGGSG GGGSQVQLQQSGAELAKPGASVRMSCKASGYTFTNYWIHWVKQRPGQGLEWIGYINPNTGYNDYNQKFKDKATLTADKSSSTVYMQLSSLTSEDSAVYYCARGGQLGLRRAMDYWGQGTSVTVSS [SEQ ID NO: 16] 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: 17, or a fragment or variant thereof: GACGTGCAGATCACCCAGAGCCCCAGCTACCTGGCCGCCAGCCCCGGCGAGACCATCACCATCAACTGCCGCGCCAGCAAGAGCATCAGCAAGTACCTGGCCTGGTACCAGGACAAGCCCGGCAAGACCAAACAAGCTGCTGATCTCAGCGGCAGCACCCTGCAGAGCGGCATCCCCAGCCGCTTCA GCGGCAGCGGCAGCGGCACCGACTTCACCCTGACCATCAGCAGCCTGGAGCCCGAGGACTTCGCCATGTACTACTGCCAGCAGCACGACGAGTACCCCTGGACCTTCGGCGGCGGCACCAAGCTGGAGATCAAGCGCGGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCAGCGGCAGCGGC GGCGGCGGCAGCCAGGTGCAGCTGCAGCAGAGCGGCGCCGAGCTGGCCAAGCCCGGCGCCAGCGTGCGCATGAGCTGCAAGGCCAGCGGCTACACCTTCACCAACTACTGGATCCACTGGGTGAAGCAGCGCCCCGCCAGGGCCTGGAGTGGATCGGCTACATCAACCCCAACACCGGCTACAACG ACTACAACCAGAAGTTCAAGGACAAGGCCACCCTGACCGCCGACAAGAGCAGCAGCACCGTGTACATGCAGCTGAGCAGCCTGACCAGCGAGGACAGCGCCGTGTACTACTGCGCCCGCGGCGGCCAGCTGGGCCTGCGCCGCGCCATGGACTACTGGGGCCAGGGCACCAGCGTGACCGTGAGCAGC [SEQ ID NO: 17] In another embodiment, the amino acid sequence of the soluble form of normally cell membrane-bound CD55 (sCD55, sometimes known as decay accelerating factor; DAF) is as follows, referred to herein as SEQ ID NO: 18, or a fragment or variant thereof: DCGLPPDVPNAQPALEGRTSFPEDTVITYKCEESFVKIPGEKDSVICLKGSQWSDIEEFCNRSCEVPTRLNSASLKQPYITQNYFPVGTVVEYECRPGYRREPSLSPKLTCLQNLKWSTAVEFCKKKSCPNPGEIRNGQIDVPGGILFGATISFSCNTGY KLFGSTSSFCLISGSSVQWSDPLPECREIYCPAPPQIDNGIIQGERDHYGYRQSVTYACNKGFTMIGEHSIYCTVNNDEGEWSGPPPECRGKSLTSKVPPTVQKPTTVNVPTTEVSPTSQKTTTKTTTPNAQATRSTPVSRTTKHFHETTPNKGSGTTSG [SEQ ID NO: 18] In a preferred embodiment, the nucleic acid sequence (960 bp) encoding the soluble form of the normally cell membrane-bound CD55 (sCD55, sometimes also known as decay accelerating factor; DAF) is referred to herein as SEQ ID NO: 19, or a fragment or variant thereof, as follows: GACTGCGGCCTGCCCCCCGACGTGCCCAACGCCCAGCCCGCCCTGGAGGGCCGCACCAGCTTCCCCGAGGACACCGTGATCACCTACAAGTGCGAGGAGAGCTTCGTGAAGATCCCCGGCGAGAAGGACAGCGTGATCTGCCTGAAGGGCAGCCAGTGGAGCGACATCGAGGAGTTCTGCAACCGCAGCTGCGAGGTGCCCACCCGCCTGAACAGCGCCAGCCTGAAGCAGCCCTACATCACCCAGAACTACTTCCCCGTGGGCACCGTGGTGGAGTACGAGTGCCGCCCCGGCTACCGCCGCGAGCCCAGCCTGAGCCCCAAGCTGACCTGCCTGCAGAACCTGAAGTGGAGCACCGCCGTGGAGTTCTGCAAGAAGAAGAGCTGCCCCAACCCCGGCGAGATCCGCAACGGCCAGATCGACGTGCCCGGCGGCATCCTGTTCGGCGCCACCATCAGCTTCAGCTGCAACACCGGCTACAAGCTGTTCGGCAGCACCAGCAGCTTCTGCCTGATCAGCGGCAGCAGCGTGCAGTGGAGCGACCCCCTGCCCGAGTGCCGCGAGATCTACTGCCCCGCCCCCCCCCAGATCGACAACGGCATCATCCAGGGCGAGCGCGACCACTACGGCTACCGCCAGAGCGTGACCTACGCCTGCAACAAGGGCTTCACCATGATCGGCGAGCACAGCATCTACTGCACCGTGAACAACGACGAGGGCGAGTGGAGCGGCCCCCCCCCCGAGTGCCGAGGCAAGAGCCTGACCAGCAAGGTGCCCCCCACCGTGCAGAAGCCCACCACCGTGAACGTGCCCACCACCGAGGTGAGCCCCACCAGCCAGAAGACCACCACCAAGACCACCACCCCCAACGCCCAGGCCACCCGCAGCACCCCCGTGAGCCGCACCACCAAGCACTTCCACGAGACCACCCCCAACAAGGGCAGCGGCACCACCAGCGGC [SEQ ID NO: 19] 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:20, or a fragment or variant thereof, as follows: EATFCDFPKINHGILYDEEKYKPFSQVPTGEVFYYSCEYNFVSPSKSFWTRITCTEEGWSPTPKCLRLCFFPFVENGHSESSGQTHLEGDTVQIICNTGYRLQNNENNISCVERGWSTPPKCRSTDTSCVNPPTVQNAHILSRQMSKYPSGERVRY ECRSPYEMFGDEEVMCLNGNWTEPPQCKDSTGKCGPPPPIDNGDITSFPLSVYAPASSVEYQCQNLYQLEGNKRITCRNGQWSEPPKCLHPCVISREIMENYNIALRWTAKQKLYLRTGESAEFVCKRGYRLSSRSHTLRTTCWDGKLEYPTCAKR [SEQ ID NO:20] 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:21, or a fragment or variant thereof, as follows: GAAGCAACATTTTGTGATTTTCCAAAAATAAACCATGGAATTCTATATGATGAAGAAAAATATAAGCCATTTTCCCAGGTTCCTACAGGGGAAGTTTTCTATTACTCCTGTGAATATAATTTTGTGTCTCCTTCAAAATCATTTTGGACTCGCATAACATGCACAGAAGAAGGATGGTCACCAACACCAAAGTGTCTCAGACTGTGTTTCTTTCCTTTTGTGGAAAATGGTCATTCTGAATCTTCAGGACAAACACATCTGGAAGGTGATACTGTGCAAATTATTTGCAACACAGGATACAGACTTCAAAACAATGAGAACAACATTTCATGTGTAGAACGGGGCTGGTCCACCCCTCCCAAATGCAGGTCCACTGACACTTCCTGTGTGAATCCGCCCACAGTACAAAATGCTCATATACTGTCGAGACAGATGAGTAAATATCCATCTGGTGAGAGAGTACGTTATGAATGTAGGAGCCCTTATGAAATGTTTGGGGATGAAGAAGTGATGTGTTTAAATGGAAACTGGACAGAACCACCTCAATGCAAAGATTCTACGGGAAAATGTGGGCCCCCTCCACCTATTGACAATGGGGACATTACTTCATTCCCGTTGTCAGTATATGCTCCAGCTTCATCAGTTGAGTACCAATGCCAGAACTTGTATCAACTTGAGGGTAACAAGCGAATAACATGTAGAAATGGACAATGGTCAGAACCACCAAAATGCTTACATCCGTGTGTAATATCCCGAGAAATTATGGAAAATTATAACATAGCATTAAGGTGGACAGCCAAACAGAAGCTTTATTTGAGAACAGGTGAATCAGCTGAATTTGTGTGTAAACGGGGATATCGTCTTTCATCACGTTCTCACACATTGCGAACAACATGTTGGGATGGGAAACTGGAGTATCCAACTTGTGCAAAAAGA [SEQ ID NO: 21] In another embodiment, the codon-optimized nucleic acid sequence (936 bp) encoding human complement factor H related protein-1 (CFHR1) is referred to herein as SEQ ID NO:22, or a fragment or variant thereof, as follows: GAGGCCACCTTCTGCGACTTCCCCAAGATCAACCACGGCATCCTGTACGACGAGGAGAAGTACAAGCCCTTCAGCCAGGTGCCCACCGGCGAGGTGTTCTACTACAGCTGCGAGTACAACTTCGTGAGCCCCAGCAAGAGCTTCTGGACCCGCATCACCTGCACCGAGGAGGGCTGGAGCCCCACCCCAAGTGCCTGCGCCTTGCTTCTTCCCCTTCGTGGAGAACGGCCAC AGCGAGAGCAGCGGCCAGACCCACCTGGAGGGCGACACCGTGCAGATCATCTGCAACACCGGCTACCGCCTGCAGAACAACGAGAACAACATCAGCTGCGTGGAGCGCGGCTGGAGCACCCCCCCAAGTGCCCGCAGCACCGACACCAGCTGCGTGAACCCCCCACCGTGCAGAACGCCCACATCCTGAGCCGCCAGATGAGCAAGTACCCCAGCGGCGAGCGCGTGCGCTAC GAGTGCCGCAGCCCCTACGAGATGTTCGGCGACGAGGAGGTGATGTGCCTGAACGGCAACTGGACCGAGCCCCCCAGTGCAAGGACAGCACCGGCAAGTGCGGCCCCCCCCCCCATCGACAACGGCGACATCACCAGCTTCCCCCTGAGCGTGTACGCCCCCGCAGCAGCGTGGAGTACCAGTGCGAACCTGTACCAGCTGGAGGGGCAACAAGCGCATCACCTGCCGC AACGGCCAGTGGAGCGAGCCCCCCCAAGTGCCTGCACCCCTGCGTGATCAGCCGCGAGATCATGGAGAACTACAACATCGCCCTGCGCTGGACCGCCAAGCAGAAGCTGTACCTGCGCACCGGCGAGAGGCCCGAGTTCGTGTGCAAGCGCGGCTACCGCCTGAGCAGCGCAGCCACACCCTGCGCACCACCTGCTGGGACGGCAAGCTGGAGTACCCCACCTGCGCCAAGCGC [query number 22] In a further embodiment, the amino acid sequence of the soluble form of CD46 (sCD46), which is normally cell membrane bound, is as follows, referred to herein as SEQ ID NO:23, or a fragment or variant thereof: CEEPPTFEAMELIGKPKPYYEIGERVDYKCKKGYFYIPPLATHTICDRNHTWLPVSDDACYRETCPYIRDPLNGQAVPANGTYEFGYQMHFICNEGYYLIGEEILYCELKGSVAIWSGKPPICEKVLCTPPPKIKNGKHTFSEVEVFEYLDAVTY SCDPAPGPDPFSLIGESTIYCGDNSVWSRAAPECKVVKCRFPVVENGKQISGFGKKFYYKATVMFECDKGFYLDGSDTIVCDSNSTWDPPVPKCLKVLPPSSTKPPALSHSVSTSSTTKSPASSASGPRPTYKPVVSNYPGYPKPEEGILDSLDV [SEQ ID NO:23] In a preferred embodiment, the nucleic acid sequence (930 bp) encoding the soluble form of CD46 (sCD46), which is normally cell membrane bound, is as follows, referred to herein as SEQ ID NO: 24, or a fragment or variant thereof: TGCGAGGAGCCCCCCACCTTCGAGGCCATGGAGCTGATCGGCAAGCCCAAGCCCTACTACGAGATCGGCGAGCGCGTGGACTACAAGTGCAAGAAGGGCTACTTCTACATCCCCCCCCTGGCCACCCACACCATCTGCGACCGCAACCACACCTGGCTGCCCGTGAGCGACGACGCCTGCTACCGCGAGACCTGCCCCTACATCCGCGACCCCCTGAACGGCCAGGCCGTGCCCGCCAACGGCACCTACGAGTTCGGCTACCAGATGCACTTCATCTGCAACGAGGGCTACTACCTGATCGGCGAGGAGATCCTGTACTGCGAGCTGAAGGGCAGCGTGGCCATCTGGAGCGGCAAGCCCCCCATCTGCGAGAAGGTGCTGTGCACCCCCCCCCCCAAGATCAAGAACGGCAAGCACACCTTCAGCGAGGTGGAGGTGTTCGAGTACCTGGACGCCGTGACCTACAGCTGCGACCCCGCCCCCGGCCCCGACCCCTTCAGCCTGATCGGCGAGAGCACCATCTACTGCGGCGACAACAGCGTGTGGAGCCGCGCCGCCCCCGAGTGCAAGGTGGTGAAGTGCCGCTTCCCCGTGGTGGAGAACGGCAAGCAGATCAGCGGCTTCGGCAAGAAGTTCTACTACAAGGCCACCGTGATGTTCGAGTGCGACAAGGGCTTCTACCTGGACGGCAGCGACACCATCGTGTGCGACAGCAACAGCACCTGGGACCCCCCCGTGCCCAAGTGCCTGAAGGTGCTGCCCCCCAGCAGCACCAAGCCCCCCGCCCTGAGCCACAGCGTGAGCACCAGCAGCACCACCAAGAGCCCCGCCAGCAGCGCCAGCGGCCCCCGCCCCACCTACAAGCCCCCCGTGAGCAACTACCCCGGCTACCCCAAGCCCGAGGAGGGCATCCTGGACAGCCTGGACGTG [SEQ ID NO: 24] In a preferred embodiment, the amino acid sequence of CFHL1 is referred to herein as SEQ ID NO:80, or a fragment or variant thereof, as follows: EDCNELPPRRNTEILTGSWSDQTYPEGTQAIYKCRPGYRSLGNVIMVCRKGEWVALNPLRKCQKRPCGHPGDTPFGTFTLTGGNVFEYGVKAVYTCNEGYQLLGEI NYRECDTDGWTNDIPICEVVKCLPVTAPENGKIVSSAMEPDREYHFGQAVRFVCNSGYKIEGDEEMHCSDDGFWSKEKPKCVEISCKSPDVINGSPISQKIIYKENE RFQYKCNMGYEYSERGDAVCTESGWRPLPSCEEKSCDNPYIPNGDYSPLRIKHRTGDEITYQCRNGFYPATRGNTAKCTSTGWIPAPRCTLKPCDYPDIKHGGLYH ENMRRPYFPVAVGKYYSYYCDEHFETPSGSYWDHIHCTQDGWSPAVPCLRKCYFPYLENGYNQNYGRKFVQGKSIDVACHPGYALPKAQTTVTCMENGWSPTPRCIR [SEQ ID NO:80] In a preferred embodiment, the nucleic acid sequence encoding CFHL1 (1278 bp) is referred to herein as SEQ ID NO:81, or a fragment or variant thereof, as follows: [SEQ ID NO:81] In a preferred embodiment, the amino acid sequence of the anti-C5 single chain variable fragment is as follows, referred to herein as SEQ ID NO:82, or a fragment or variant thereof: DIQMTQSPSSLSASVGDRVTITCQASQSINNQLSWYQQKPGKAPKLLIYYASTLASGYPSRFSGSGSGTDFTLTISSLQPEDFATYYCQGSYYSGGWDYGFGQGTKVEIKRGGGGGSGGGSGGGGS GGGGSEVQLVESGGGLVQPGGSLRLSCAASGFSFSGRYWIQWVRQAPGKGLEWVASVWPGITGTNYANWAKGRFTISRDDSKNTLYLQMNSLRAEDTAVYYCAREPVAWGGGLDLWGQGTLVTVSS [SEQ ID NO:82] In a preferred embodiment, the nucleic acid sequence (759 bp) encoding the anti-C5 single chain variable fragment is as follows, referred to herein as SEQ ID NO: 83, or a fragment or variant thereof: GACATCCAGATGACCCAGAGCCCCAGCAGCCTGAGCGCCAGCGTGGGCGACCGCGTGACCATCACCTGCCAGGCCAGCCAGAGCATCAACAACCAGCTGAGCTGGTACCAGCAGAAGCCCGGCAAGGCCCCCAAGCTGCTGATCTACTACGCCAGCACCCTGGCCAGCGGCTACCCCAGCCGCTTCAGC GGCAGCGGCAGCGGCACCGACTTCACCCTGACCATCAGCAGCCTGCAGCCCGAGGACTTCGCCACCTACTACTGCCAGGGCAGCTACTACAGCGGCGGCTGGGACTACGGCTTCGGCCAGGGCACCAAGGTGGAGATCAAGCGCGGCGGCGGCGGCGGCAGCGGCGGCGGCAGCGGCGGCGGCGGCAGCG GCGGCGGCGGCAGCGAGGTGCAGCTGGTGGAGAGCGGCGGCGGCCTGGTGCAGCCCGGCGGCAGCCTGCGCCTGAGCTGCGCCGCCAGCGGCTTCAGCTTCAGCGGCCGCTACTGGATCCAGTGGGTGCGCCAGGCCCCCGGCAAGGGCCTGGAGTGGGTGGCCAGCGTGTGGCCCGGCATCACCGGCAC CAACTACGCCAACTGGGCCAAGGGCCGCTTCACCATCAGCCGCGACGACAGCAAGAACACCCTGTACCTGCAGATGAACAGCCTGCGCGCCGAGGACACCGCCGTGTACTACTGCGCCCGCGAGCCCGTGGCCTGGGGCGGCGGCCTGGACCTGTGGGGCCAGGGCACCCTGGTGACCGTGAGCAGCTGA [SEQ ID NO:83] Thus, in a preferred embodiment, the second coding sequence comprises a nucleotide sequence substantially as set out in any one of SEQ ID NOs: 15, 17, 19, 21, 22, 24, 81 or 83, or a fragment or variant thereof. Preferably, the anti-complement protein comprises an amino acid sequence substantially as set out in SEQ ID NOs: 14, 16, 18, 20, 23, 80 or 82, or a fragment or variant thereof.

[0037] Many genetic 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 (i.e., the 5' end) 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 for PEDF receptor agonists and anticomplement proteins, using dual promoters and / or IRES linkers (in this regard, the EMCV IRES is 553 nucleotides long).

[0038] 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 PEDF receptor agonist and the anti-complement 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 have activity.

[0039] 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.

[0040] 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.

[0041] Thus, in one embodiment, after transcription of a single mRNA transcript encoding the sequences of the PEDF receptor agonist, the viral-2A peptide and the anti-complement protein, a translational skip can occur in the viral-2A peptide sequence between the terminal glycine-proline of the viral-2A peptide, which would generate two proteins, the PEDF receptor agonist and the anti-complement protein (see FIG. 3).

[0042] 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.

[0043] 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:25, or a fragment or variant thereof, as follows: ATNFSLLKQAGDVEENPGP [SEQ ID NO:25] 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:25.

[0044] In this first embodiment, the P2A peptide spacer sequence comprises the nucleotide sequence designated herein as SEQ ID NO:26 (57 bp), or a fragment or variant thereof, as follows: GCCACCAACTTCAGCCTGCTGAAGCAGGCCGGCGACGTGGAGGAGAACCCCGGCCCC [SEQ ID NO:26] 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:27, or a fragment or variant thereof, as follows: QCTNYALLKLAGDVESNPGP [SEQ ID NO:27] 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:27.

[0045] In this second embodiment, the E2A peptide spacer sequence comprises the nucleotide sequence designated herein as SEQ ID NO:28 (60 bp), or a fragment or variant thereof, as follows: CAGTGCACCAACTACGCCCTGCTGAAGCTGGCCGGCGACGTGGAGAGCAACCCCGGCCCC [SEQ ID NO:28] 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:29, or a fragment or variant thereof, as follows: EGRGSLLTCGDVEENPGP [SEQ ID NO:29] 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:29.

[0046] In this third embodiment, the T2A peptide spacer sequence comprises the nucleotide sequence designated herein as SEQ ID NO:30 (54 bp), or a fragment or variant thereof, as follows: GAGGGCCGCGGCAGCCTGCTGACCTGCGGCGACGTGGAGGAGAACCCCGGCCCC [SEQ ID NO:30] 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:31, or a fragment or variant thereof, as follows: VKQTLNFDLLKLAGDVESNPGP [SEQ ID NO:31] 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:31.

[0047] In this fourth embodiment, the F2A peptide spacer sequence comprises the nucleotide sequence referred to herein as SEQ ID NO:32 (66 bp), or a fragment or variant thereof, as follows: GTGAAGCAGACCCTGAACTTCGACCTGCTGAAGCTGGCCGGCGACGTGGAGAGCAACCCCGGCCCC [SEQ ID NO:32] Thus, in a preferred embodiment, the peptide spacer sequence comprises a nucleotide sequence substantially as set out in any one of SEQ ID NOs: 26, 28, 30 or 32, or a fragment or variant thereof. Preferably, the peptide spacer sequence encodes an amino acid sequence substantially as set out in SEQ ID NOs: 25, 27, 29 or 31, or a fragment or variant thereof.

[0048] After translation skipping, the viral-2A peptide sequence remains fused to the C-terminus of the upstream protein (such as the PEDF receptor agonist), while the proline remains fused to the N-terminus of the downstream protein (such as the anti-complement protein). This may impose an immunogenic risk and potentially interfere with the intracellular signaling ability of the PEDF receptor. Therefore, we introduced an enzyme cleavage coding sequence directly upstream of the viral-2A peptide sequence, so that the remaining viral-2A peptide sequence is removed from both the encoded proteins (i.e., the PEDF receptor agonist and the anti-complement protein). The introduction of the enzyme cleavage site has the effect of removing the viral-2A peptide either in the cell 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 site for matrix metalloprotein-2 (MMP-2) or renin.

[0049] 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).

[0050] 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.

[0051] 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.

[0052] Thus, in a preferred embodiment, the genetic construct comprises a viral-2A excision sequence encoding an amino acid sequence designated herein as SEQ ID NO: 33, or a fragment or variant thereof, as follows, where B=basic amino acid, X=hydrophilic amino acid and S=serine: BB(X)BBS [SEQ ID NO:33] 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: 33, or a fragment or variant thereof.

[0053] In one embodiment, the viral-2A excision sequence encodes the amino acid sequence referred to herein as SEQ ID NO:34, or a fragment or variant thereof, as follows: RRSKRSGSG [SEQ ID NO:34] In this first embodiment, the viral-2A removal sequence comprises the nucleotide sequence referred to herein as SEQ ID NO:35, or a fragment or variant thereof, as follows: CGCCGCAGCAAGCGCAGCGGCAGCGGC [SEQ ID NO:35] In a second embodiment, the viral-2A excision sequence encodes the amino acid sequence referred to herein as SEQ ID NO:36, or a fragment or variant thereof, as follows: RRTKRSGSG [SEQ ID NO:36] In this second embodiment, the viral-2A excision sequence comprises the nucleotide sequence referred to herein as SEQ ID NO:37, or a fragment or variant thereof, as follows: CGCCGCACCAAGCGCAGCGGCAGCGGC [SEQ ID NO:37] Thus, in one embodiment, the viral-2A deletion sequence comprises a nucleotide sequence substantially as set forth in either SEQ ID NO: 35 or 37, or a fragment or variant thereof. Preferably, the viral-2A deletion sequence encodes an amino acid sequence substantially as set forth in SEQ ID NO: 34 or 36, or a fragment or variant thereof.

[0054] 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: 74], GPLGIAGA [SEQ ID NO: 75] or GPQGLLGQ [SEQ ID NO: 76], or a fragment or variant thereof.Cleavage preferably occurs after the second glycine residue.

[0055] The present inventors have generated a preferred amino acid sequence, designated herein as SEQ ID NO:38, which contains the gelatinase MMP-2 recognition sequence and the tripeptide GSG linker sequence.

[0056] Thus, in one embodiment, the viral-2A excision sequence encodes the amino acid sequence referred to herein as SEQ ID NO:38, or a fragment or variant thereof, as follows: GPQGIAGQGSG [SEQ ID NO:38] In this embodiment, the viral-2A removal sequence comprises a nucleotide sequence designated herein as SEQ ID NO:39, or a fragment or variant thereof, as follows: GGCCCCCTGGGCATCGCCGGCCAGGGGCAGCGGC [SEQ ID NO:39] Thus, in one embodiment, the viral-2A deletion sequence comprises a nucleotide sequence substantially as set forth in SEQ ID NO: 39, or a fragment or variant thereof. Preferably, the viral-2A deletion sequence encodes an amino acid sequence substantially as set forth in SEQ ID NO: 38, or a fragment or variant thereof.

[0057] 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: 77] or HPFHLLVYS [SEQ ID NO: 78], or a fragment or variant thereof.Cleavage preferably occurs after the leucine residue(s).

[0058] The present inventors have generated a preferred amino acid sequence, designated herein as SEQ ID NO:40, which contains a renin recognition sequence and a tripeptide GSG linker sequence.

[0059] Thus, in one embodiment, the viral-2A excision sequence encodes the amino acid sequence referred to herein as SEQ ID NO:40, or a fragment or variant thereof, as follows: HPFHLLVYSGSG [SEQ ID NO: 40] In this embodiment, the viral-2A removal sequence comprises a nucleotide sequence designated herein as SEQ ID NO:41, or a fragment or variant thereof, as follows: CGCCCCTTCCACCTGCTGGTCATCCACGGCAGCGGC [SEQ ID NO:41] Thus, in one embodiment, the viral-2A removal sequence comprises a nucleotide sequence substantially as set forth in SEQ ID NO: 41, 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: 40, or a fragment or variant thereof.

[0060] 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 the expression of the transgene, i.e., the PEDF receptor agonist and the anti-complement protein. Preferably, the WPRE coding sequence is located 3' of the transgene coding sequence.

[0061] 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:42, as follows: AATCAACCTCTGGATTACAAAATTTGTGAAAGATTGACTGGTATTCTTAACTATGTTGCTCCTTTTACGCTATGTGGATACGCTGCTTTAATGCCTTTGTATCATGCTATTGCTTCCCGTATGGCTTTCATTTTCTCCTCCTTGTATA AATCCTGGTTGCTGTCTCTTTATGAGGAGTTGTGGCCCGTTGTCAGGCAACGTGGCGTGGTGTGCACTGTGTTTGCTGACGCAACCCCCACTGGTTGGGGCATTGCCACCACCTGTCAGCTCCTTTCCGGGACTTTCGCTTTCCCCCT CCCTATTGCCACGGCGGAACTCATCGCCGCCTGCCTTGCCCGCTGCTGGACAGGGGCTCGGCTGTTGGGCACTGACAATTCCGTGGTGTTGTCGGGGAAGCTGACGTCCTTTCCATGGCTGCTCGCCTGTGTTGCCACCTGGATTCTG CGCGGGACGTCCTTCTGCTACGTCCCTTCGGCCCTCAATCCAGCGGACCTTCCTTCCCGCGGCCTGCTGCCGGCTCTGCGGCCTCTTCCGCGTCTTCGCCTTCGCCCTCAGACGAGTCGGATCTCCCTTTGGGCCGCCTCCCCGCCTG [SEQ ID NO:42] Preferably, the WPRE comprises a nucleic acid sequence substantially as set forth in SEQ ID NO: 42, or a fragment or variant thereof.

[0062] 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:43, as follows: AATCAACCTCTGGATTACAAAATTTGTGAAAGATTGACTGGTATTCTTAACTATGTTGCTCCTTTTACGCTATGTGGATACGCTGCTTTAATGCCTTTGTATCATGCTATTGCTTCCCGTATG GCTTTCATTTTCTCCTCCTTGTATAAATCCTGGTTAGTTCTTGCCACGGCGGAACTCATCGCCGCCTGCCTTGCCCGCTGCTGGACAGGGGCTCGGCTGTTGGGCACTGACAATTCCGTGGTGT [SEQ ID NO:43] Preferably, therefore, the truncated WPRE comprises a nucleic acid sequence substantially as set out in SEQ ID NO: 43, or a fragment or variant thereof.

[0063] 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: 43, or a fragment or variant thereof.

[0064] 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.

[0065] 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:44, as follows: AGCAGACATGATAAGATACATTGATGAGTTTGGACAAACCACAACTAGAATGCAGTGAAAAAAATGCTTTATTTGTGAAATTTGTGATGCTATTGCTTTATTTGTAACCATTATAAGCTGCAATAAACAAGTTAACAACAACAATTGCATTCATTTTATGTTTCAGGTTCAGGGGGAGGTGTGGGAGGTTTTTTAAAGCAAGTAAAACCTCTACAAATGTGGTA [SEQ ID NO:44] In another embodiment, the polyA tail comprises a 169 nucleotide sequence polyA component, referred to herein as SEQ ID NO:45, as follows: TTCGAGCAACTTGTTATTGCAGCTTATAATGGTTACAAATAAAGCAATAGCATCACAAATTTCACAAATAAAGCATTTTTTTCACTGCATTCTAGTTGTGGTTTGTCCAAACTCATCAATGTATCTTATCATGTCTGGATCGTCTAGCATCGAAGATCCCCCGATCTG [SEQ ID NO:45] 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:84, as follows: CTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTTCCTAATAAAATGAGGAAATTGCATCGCATTGTCTGAGTAGGTGTCATTCTATTCTGGGGGTGGGTGGGGCAGGACAGCAAGGGGGAGGATTGGGAAGACAATAGCAGGCATGCTGGGGATGCGGTGGGCTCTATGG [SEQ ID NO:84] Preferably, therefore, the poly A tail comprises a nucleic acid sequence substantially as set out in SEQ ID NO: 44, 45 or 84, or a fragment or variant thereof.

[0066] 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.

[0067] 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:46, as follows: CGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCAAAGCCCGGGCGTCGGGCGACCTTTGGTCGCCCGGCCTCAGTGAGCGAGCGAGCGCGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCTTG [SEQ ID NO:46] 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:47, as follows: AGGAACCCCTAGTGATGGAGTTGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCCCGGGCTTTGCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGC [SEQ ID NO:47] Preferably, the left and / or right inverted terminal repeat comprises a nucleic acid sequence substantially as set out in SEQ ID NO: 46 or 47, or a fragment or variant thereof.

[0068] 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 [59, 60]. 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.

[0069] 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.

[0070] In one embodiment, the non-coding intron is the Minute Virus of Mice (MVM) small (121 bp) intron

[61] , referred to herein as SEQ ID NO: 48, as follows: AGGTACGATGGCGCCTCCAGCTAAAAGAGCTAAAAGAGGTAAGGGTTTAAGGGATGGTTGGTTGGTGGGGTATTAATGTTTAATTACCTGTTTTACAGGCCTGAAATCACTTGGTTTTAGG [SEQ ID NO:48] 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:49, as follows: GTAAGTATCAAGGTTACAAGACAGGTTTAAGGAGACCAATAGAAACTGGGCTTGTCGAGACAGAGAAGACTCTTGCGTTTCTGATAGGCACCTATTGGTCTTACTGACATCCACTTTGCCTTTCTCTCCACAG [SEQ ID NO:49] 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:50, as follows: GTGAGCGGGCGGGACGGCCCTTCTCCCGGGCTGTAATTAGCGCTTGGTTTAATGACGGCTTGTTTCTTTTCTGTGGCTGCGTGAAAGCCTTGAGGGGCTCCGGGAGGCTAGAGCCTCTGCTAACCATGTTCATGCCTTCTTCTTTTTCCTACAGCTCCTGGCAACGTGCTGGTTATTGTGCTGTCTCATCATTTTGGCAAAGAATTC [SEQ ID NO:50] Thus, preferably the non-coding intron comprises a nucleic acid sequence substantially as set out in SEQ ID NO: 48, 49 or 50, or a fragment or variant thereof.

[0071] To allow for correct folding of the polypeptides encoded by the genetic constructs, intracellular transport and secretion of the PEDF receptor agonist and anticomplement 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 for cleavage of the signal peptide from the final mature secreted protein.

[0072] 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.

[0073] In one embodiment, the inventors optimized the signal peptide using the online program Signal-P 5.0 (http: / / www.cbs.dtu.dk / services / SignalP / data.php) to increase the level of enzymatic cleavage by signal peptidase, thereby enhancing the cellular secretion of mature PEDF. The N-terminal glutamine [Q] and alanine [A] amino acids of the endogenous signal peptide sequence MQALVLLLCIGALLGHSSC [SEQ ID NO: 79] were replaced with basic amino acids such as arginine [R] or lysine [K] residues, and the terminal three amino acids [SSC] were replaced with [VFC].

[0074] Thus, in one embodiment, the signal peptide coding sequence encodes the amino acid sequence referred to herein as SEQ ID NO:51, or a fragment or variant thereof, as follows: MRRLVLLLCIGALLGHVFC [SEQ ID NO:51] Preferably, in this embodiment, the signal peptide coding sequence comprises the nucleotide sequence referred to herein as SEQ ID NO:52, or a fragment or variant thereof, as follows: ATGCGCCGCCTGGTGCTGCTGCTGTGCATCGGCGCCCTGCTGGGCCACGTGTTCTGC [SEQ ID NO:52] For the anti-complement proteins (anti-C3b, anti-Bb or anti-C5 single chain variable fragment, CD55, sCD46, CFHL1 or CFHR1) to be released from producer cells, the coding sequence is preceded by a modified form of the signal peptide derived from human brain-derived neurotrophic factor (BDNF) in which the N-terminal amino acid is replaced by arginine [R] or lysine [K].

[0075] Thus, in one embodiment, the signal peptide coding sequence encodes the amino acid sequence referred to herein as SEQ ID NO:53, or a fragment or variant thereof, as follows: MRRFLTVISFLLYFGCAFA [SEQ ID NO:53] Preferably, in this embodiment, the signal peptide coding sequence comprises the nucleotide sequence referred to herein as SEQ ID NO:54, or a fragment or variant thereof, as follows: ATGCGCCGCTTCCTGACCGTGATCAGCTTCCTGCTGTACTTCGGCTGCGCCTTCGCC [SEQ ID NO:54] Thus, preferably, the signal peptide coding sequence comprises a nucleotide sequence substantially as set out in SEQ ID NO: 52 or 54, or a fragment or variant thereof. Preferably, the signal peptide coding sequence encodes an amino acid sequence substantially as set out in SEQ ID NO: 51 or 53, or a fragment or variant thereof.

[0076] In a preferred embodiment, the genetic construct can include, in this designated order, a 5' promoter; a first coding sequence that encodes a PEDF receptor agonist; and a second coding sequence that encodes an anti-complement protein at 3'.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, those skilled in the art will understand that the first and second coding sequences that encode a PEDF receptor agonist and an anti-complement protein can be arranged in any 5' to 3' order.

[0077] In certain embodiments, the genetic construct can include, in this specified order: a 5' promoter; a first coding sequence encoding a PEDF receptor agonist; a spacer sequence; and a 3' second coding sequence encoding an anti-complement protein.

[0078] In certain embodiments, the genetic construct can include, in this specified order: a 5' promoter; a first coding sequence encoding a PEDF receptor agonist; a viral-2A excision sequence; a spacer sequence; and a 3' second coding sequence encoding an anti-complement protein.

[0079] In certain embodiments, the genetic construct may include, in this specified order: a 5'ITR; a promoter; a first coding sequence encoding a PEDF receptor agonist; a viral-2A excision sequence; a spacer sequence; a second coding sequence encoding an anti-complement protein; a sequence encoding a WPRE; a sequence encoding a polyA tail; and a 3'ITR.

[0080] 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 a PEDF receptor agonist; a viral-2A excision sequence; a spacer sequence; a second coding sequence encoding an anti-complement protein; a sequence encoding a WPRE; a sequence encoding a polyA tail; and a 3'ITR.

[0081] 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 a PEDF receptor agonist; a viral-2A excision sequence; a spacer sequence; a second signal peptide coding sequence; a second coding sequence encoding an anti-complement protein; a sequence encoding a WPRE; a sequence encoding a polyA tail; and a 3'ITR.

[0082] From the foregoing, the skilled artisan will recognize the nucleotide sequence of an 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 PEDF-Furin-P2A-Anti-C3b SCVF is referred to herein as SEQ ID NO: 55, as follows: MRRLVLLLCIGALLGHVFCQNPASPPEEGSPDPDSTGALVEEEDPFFKVPVNKLAAAVSNFGYDLYRVRSSTSPTTNVLLSPLSVATALSALSLGAEQRTESIIHRALYYDLISSPDIHGTYKELLDTVTAPQKNLKSASRIVFEKKLRIKSSFVAPLEKSYGTRPRVLTGNPRLD LQEINNWVQAQMKGKLARSTKEIPDEISILLLGVAHFKGQWVTKFDSRKTSLEDFYLDEERTVRVPMMSDPKAVLRYGLDSDLSCKIAQLPLTGSMSIIFFLPLKVTQNLTLIEESLTSEFIHDIDRELKTVQAVLTVPKLKLSYEGEVTKSLQEMKLQSLFDSPDFSKITGKPIKL TQVEHRAGFEWNEDGAGTTPSPGLQPAHLTFPLDYHLNQPFIFVLRDTTDTGALLFIGKILDPRGPRRSKRSGSGATNFSLLKQAGDVEENPGPMRRLLTFISILALVGAFADIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGS GSGTDFTLTISSLQPEDFATYYCQQSYATLPTFEQGTKVEIKRGGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFSFTSSSVSPGKGLEWVGLIYPYNGFNYYADSVKGRFTISADTSLQMNSLRAEDTAVYYCARNALYGSGGYYAMDYWGQGTLVTVSS [SEQ ID NO:55] Preferably, in this embodiment, the construct comprises the 2121 nucleotide sequence designated herein as SEQ ID NO:56 (contained within plasmid IKC157P), or a fragment or variant thereof, as follows: [SEQ ID NO:56] In another embodiment, the amino acid sequence of PEDF-furin-P2A-sCD55 is as follows, referred to herein as SEQ ID NO:57, or a fragment or variant thereof: MRRLVLLLCIGALLGHVFCQNPASPPEEGSPDPDSTGALVEEEDPFFKVPVNKLAAAVSNFGYDLYRVRSSTSPTTNVLLSPLSVATALSALSLGAEQRTESIIHRALYYDLISSPDIHGTYKELLDTVTAPQKNLKSASRIVFEKKLRIKSSFVAPLEKSYGTRPRVLTGNPRLDLQEINNWVQAQMKGKLARST KEIPDEISILLLGVAHFKGQWVTKFDSRKTSLEDFYLDEERTVRVPMMSDPKAVLRYGLDSDLSCKIAQLPLTGSMSIIFFLPLKVTQNLTLIEESLTSEFIHDIDRELKTVQAVLTVPKLKLSYEGEVTKSLQEMKLQSLFDSPDFSKITGKPIKLTQVEHRAGFEWNEDGAGTTPSPGLQPAHLTFPLDYHLNQ PFIFVLRDTTDTGALLFIGKILDPRGPRRSKRSGSGATNFSLLKQAGDVEENPGPMRRFLTVISFLLYFGCAFADCGLPPDVPNAQPALEGRTSFPEDTVITYKCEESFVKIPGEKDSVICLKGSQWSDIEEFCNRSCEVPTRLNSASLKQPYITQNYFPVGTVVEYECRPGYRREPSLSPKLTCLQNLKWSTAVEF CKKKSCPNPGEIRNGQIDVPGGILFGATISFSCNTGYKLFGSTSSFCLISGSSVQWSDPLPECREIYCPAPPQIDNGIIQGERDHYGYRQSVTYACNKGFTMIGEHSIYCTVNNDEGEWSGPPPECRGKSLTSKVPPTVQKPTTVNVPTTEVSPTSQKTTTKTTTPNAQATRSTPVSRTTKHFHETTPNKGSGTTSG [SEQ ID NO:57] Preferably, in this embodiment, the construct comprises the 2358 nucleotide sequence designated herein as SEQ ID NO:58 (as contained in plasmid IKC158P), or a fragment or variant thereof, as follows: [SEQ ID NO:58] In another embodiment, the amino acid sequence of sCD55-Furin-P2A-PEDF is as follows, referred to herein as SEQ ID NO:59, or a fragment or variant thereof: MRRFLTVISFLLYFGCAFADCGLPPDVPNAQPALEGRTSFPEDTVITYKEESFVKIPGEKDSVICLKGSQWSDIEEFCNRSCEVPTRLNSASLKQPYITQNYFPVGTVVEYECRPGYRREPSLSPKLTCLQNLKWSTAVEFCKKKSCPNPGEIRNGQIDVPGGILFGATISFSCNTGYKLFGSTSSFCLISGSSV QWSDPLPECREIYCPAPPQIDNGIIQGERDHYGYRQSVTYACNKGFTMIGEHSIYCTVNNDEGEWSGPPPECRGKSLTSKVPPTVQKPTTVNVPTTEVSPTSQKTTTKTTTPNAQATRSTPVSRTTKHFHETTPNKGSGTTSGRRSKRSGSGATNFSLLKQAGDVEENPGPMRRLVLLLCIGALLGHVFCQNPASP PEEGSDPDSTGALVEEEDPFFKVPVNKLAAAVSNFGYDLYRVRSSTSPTTNVLLSPLSVATALSALSLGAEQRTESIIHRALYYDLISSPDIHGTYKELLDTVTAPQKNLKSASRIVFEKKLRIKSFVAPLEKSYGTRPRVLTGNPRLDLQEINNWVQAQMKGKLARSTKEIPDEISILLLGVAHFKGQWVTKF DSRKTSLEDFYLDEERTVRVPMMSDPKAVLRYGLDSDLSCKIAQLPLTGSMSIIFFLPLKVTQNLTLIEESLTSEFIHDIDRELKTVQAVLTVPKLKLSYEGEVTKSLQEMKLQSLFDSPDFSKITGKPIKLTQVEHRAGFEWNEDGAGTTPSPGLQPAHLTFPLDYHLNQPFIFVLRDTDTGALLFIGKILDPRGP [SEQ ID NO:59] Preferably, in this embodiment, the construct comprises the 2358 nucleotide sequence designated herein as SEQ ID NO:60 (as contained in plasmid IKC126P), or a fragment or variant thereof, as follows: [SEQ ID NO:60] In another embodiment, the amino acid sequence of PEDF-Furin-P2A-CFHR1 is as follows, referred to herein as SEQ ID NO:61, or a fragment or variant thereof: MRRLVLLLCIGALLGHVFCQNPASPPEEGSPDPDSTGALVEEEDPFFKVPVNKLAAAVSNFGYDLYRVRSSTSPTTNVLLSPLSVATALSALSLGAEQRTESIIHRALYYDLISSPDIHGTYKELLDTVTAPQKNLKSASRIVFEKKLRIKSSFVAPLEKSYGTRPRVLTGNPRLDLQEINNWVQAQMKGKLAR STKEIPDEISILLLGVAHFKGQWVTKFDSRKTSLEDFYLDEERTVRVPMMSDPKAVLRYGLDSDLSCKIAQLPLTGSMSIIFFLPLKVTQNLTLIEESLTSEFIHDIDRELKTVQAVLTVPKLKLSYEGEVTKSLQEMKLQSLFDSPDFSKITGKPIKLTQVEHRAGFEWNEDGAGTTPSPGLQPAHLTFPLDY HLNQPFIFVLRDTTDTGALLFIGKILDPRGPRRSKRSGSGATNFSLLKQAGDVEENPGPMRRFLTVISFLLYFGCAFAEATFCDFPKINHGILYDEEKYKPFSQVPTGEVFYYSCEYNFVSPSKSFWTRITCTEEGWSPTPKCLRLCFFPFVENGHSESSGQTHLEGDTVQIICNTGYRLQNNENNISCVERGWS TPPKCRSTDTSCVNPPTVQNAHILSRQMSKYPSGERVRYECRSPYEMFGDEEVMCLNGNWTEPPQCKDSTGKCGPPPPIDNGDITSFPLSVYAPASSVEYQCQNLYQLEGNKRITCRNGQWSEPPKCLHPCVISREIMENYNIALRWTAKQKLYLRTGESAEFVCKRGYRLSSRSHTLRTTCWDGKLEYPTCAKR [SEQ ID NO:61] Preferably, in this embodiment, the construct comprises the 2334 nucleotide sequence designated herein as SEQ ID NO:62 (as contained in plasmid IKC127P), or a fragment or variant thereof, as follows: [SEQ ID NO:62] In another embodiment, the amino acid sequence of CFHR1-Furin-P2A-PEDF is as follows, referred to herein as SEQ ID NO:63, or a fragment or variant thereof: MRRFLTVISFLLYFGCAFAEATFCDFPKINNHGILYDEEKYKPFSQVPTGEVFYYSCEYNFVSPSKSFWTRITCTEEGWSPTPKCLRLCFFPFVENGHSESSGQTHLEGDTVQIICNTGYRLQNNENNISCVERGWSTPPKCRSTDTSCVNPPTVQNAHILSRQMSKYPSGERVRYECRSPYEMFGDEEVMCLN GNWTEPPQCKDSTGKCGPPPPIDNGDITSFPLSVYAPASSVEYQCQNLYQLEGNKRITCRNGQWSEPPKCLHPCVISREIMENYNIALRWTAKQKLYLRTGESAEFVCKRGYRLSSRSHTLRTTCWDGKLEYPTCAKRSKRSGSATNFSLLKQAGDVEENPGPMRRLVLLLCIGALLGHVFCQNPASPPEEGSP DPDSTGALVEEEDPFFKVPVNKLAAAVSNFGYDLYRVRSSTSPTTNVLLSPLSVATALSALSLGAEQRTESIIHRALYYDLISSPDIHGTYKELLDTVTAPQKNLKSASRIVFEKKLRIKSSFVAPLEKSYGTRPRVLTGNPRLDLQEINNWVQAQMKGKLARSTKEIPDEISILLLGVAHFKGQWVTKFDSR KTSLEDFYLDEERTVRVPMMSDPKAVLRYGLDSDLSCKIAQLPLTGSMSIIFFLPLKVTQNLTLIEESLTSEFIHDIDRELKTVQAVLTVPKLKLSYEGEVTKSLQEMKLQSLFDSPDFSKITGKPIKLTQVEHRAGFEWNEDGAGTTPSPGLQPAHLTFPLDYHLNQPFIFVLRDTDTGALLFIGKILDPRGP [SEQ ID NO:63] Preferably, in this embodiment, the construct comprises the 2325 nucleotide sequence designated herein as SEQ ID NO:64 (as contained in plasmid IKC128P), or a fragment or variant thereof, as follows: [SEQ ID NO:64] In another embodiment, the amino acid sequence of PEDF-Furin-P2A-Anti-C5 SCVF is as follows, referred to herein as SEQ ID NO:85, or a fragment or variant thereof: MRRLVLLLCIGALLGHSSCQNPASPPEEGSPDPDSTGALVEEEDPFFKVPVNKLAAAVSNFGYDLYRVRSSTSPTTNVLLSPLSVATALSALSLGAEQRTESIIHRALYYDLISSPDIHGTYKELLDTVTAPQKNLKSASRIVFEKKLRIKSSFVAPLEKSYGTRPRVLTGNPRLDL QEINNWVQAQMKGKLARSTKEIPDEISILLLGVAHFKGQWVTKFDSRKTSLEDFYLDEERTVRVPMMSDPKAVLRYGLDSDLSCKIAQLPLTGSMSIIFFLPLKVTQNLTLIEESLTSEFIHDIDRELKTVQAVLTVPKLKLSYEGEVTKSLQEMKLQSLFDSPDFSKITGKPIKLTQ VEHRAGFEWNEDGAGTTPSPGLQPAHLTFPLDYHLNQPFIFVLRDTTDTGALLFIGKILDPRGPRVRRGSGATNFSLLKQAGDVEENPGPMRRLLILALVGAAVADIQMTQSPSSLSASVGDRVTITCQASQSINNQLSWYQQKPGKAPKLLIYYASTLASGYPSRFSGSGSGTDFTLT ISSLQPEDFATYYCQGSYYSGGWDYGFGQGTKVEIKRGGGGGGSGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFSFSGRYWIQWVRQAPGKGLEWVASVWPGITGTNYANWAKGRFTISRDDSKNTLYLQMNSLRAEDTAVYYCAREPVAWGGGLDLWGQGTLVTVSS [SEQ ID NO:85] Preferably, in this embodiment, the construct comprises the 2136 nucleotide sequence designated herein as SEQ ID NO:86 (as contained in plasmid IKC094P), or a fragment or variant thereof, as follows: [SEQ ID NO:86] In another embodiment, the amino acid sequence of PEDF-Furin-P2A-Anti-Bb SCVF is as follows, referred to herein as SEQ ID NO:65, or a fragment or variant thereof: MRRLVLLLCIGALLGHSSCQNPASPPEEGSPDPDSTGALVEEEDPFFKVPVNKLAAAVSNFGYDLYRVRSSTSPTTNVLLSPLSVATALSALSLGAEQRTESIIHRALYYDLISSPDIHGTYKELLDTVTAPQKNLKSASRIVFEKKLRIKSSFVAPLEKSYGTRPRVLTGNPRLDL QEINNWVQAQMKGKLARSTKEIPDEISILLLGVAHFKGQWVTKFDSRKTSLEDFYLDEERTVRVPMMSDPKAVLRYGLDSDLSCKIAQLPLTGSMSIIFFLPLKVTQNLTLIEESLTSEFIHDIDRELKTVQAVLTVPKLKLSYEGEVTKSLQEMKLQSLFDSPDFSKITGKPIKLT QVEHRAGFEWNEDGAGTTPSPGLQPAHLTFPLDYHLNQPFIFVLRDTTDTGALLFIGKILDPRGPRVRRGSGATNFSLLKQAGDVEENPGPMRRLLILALVGAAVADVQITQSPSYLAASPGETITINCRASKSISKYLAWYQDKPGKTNKLLIYSGSTLQSGIPSRFSGSGSGTDFT LTISSLEPEDFAMYYCQQHDEYPWTFGGGTKLEIKRGGGGGSGGGGSGGGGSGGGGSQVQLQQSGAELAKPGASVRMSCKASGYTFTNYWIHWVKQRPGQGLEWIGYINPNTGYNDYNQKFKDKATLTADKSSSTVYMQLSSLTSEDSAVYYCARGGQLGLRRAMDYWGQGTSVTVSS [SEQ ID NO:65] Preferably, in this embodiment, the construct comprises the 2130 nucleotide sequence designated herein as SEQ ID NO:66 (as contained in plasmid IKC093P), or a fragment or variant thereof, as follows: [SEQ ID NO:66] In another embodiment, the amino acid sequence of PEDF-Furin-P2A-Anti-Bb SCVF is as follows, referred to herein as SEQ ID NO:67, or a fragment or variant thereof: MRRLVLLLCIGALLGHSSCQNPASPPEEGSPDPDSTGALVEEEDPFFKVPVNKLAAAVSNFGYDLYRVRSSTSPTTNVLLSPLSVATALSALSLGAEQRTESIIHRALYYDLISSPDIHGTYKELLDTVTAPQKNLKSASRIVFEKKLRIKSSFVAPLEKSYGTRPRVLTGNPRLDL QEINNWVQAQMKGKLARSTKEIPDEISILLLGVAHFKGQWVTKFDSRKTSLEDFYLDEERTVRVPMMSDPKAVLRYGLDSDLSCKIAQLPLTGSMSIIFFLPLKVTQNLTLIEESLTSEFIHDIDRELKTVQAVLTVPKLKLSYEGEVTKSLQEMKLQSLFDSPDFSKITGKPIKLTQ VEHRAGFEWNEDGAGTTPSPGLQPAHLTFPLDYHLNQPFIFVLRDTTDTGALLFIGKILDPRGPRRSKRSGSGATNFSLLKQAGDVEENPGPMRRLLILALVGAAVADVQITQSPSYLAASPGETITINCRASKSISKYLAWYQDKPGKTNKLLIYSGSTLQSGIPSRFSGSGSGTDFT LTISSLEPEDFAMYYCQQHDEYPWTFGGGTKLEIKRGGGGGSGGGGSGGGGSGGGGSQVQLQQSGAELAKPGASVRMSCKASGYTFTNYWIHWVKQRPGQGLEWIGYINPNTGYNDYNQKFKDKATLTADKSSSTVYMQLSSLTSEDSAVYYCARGGQLGLRRAMDYWGQGTSVTVSS [SEQ ID NO:67] Preferably, in this embodiment, the construct comprises the 2136 nucleotide sequence designated herein as SEQ ID NO:68 (as contained in plasmid IKC129P), or a fragment or variant thereof, as follows: [SEQ ID NO:68] In another embodiment, the amino acid sequence of PEDF-furin-P2A-sCD46 is as follows, referred to herein as SEQ ID NO:69, or a fragment or variant thereof: MRRLVLLLCIGALLGHVFCQNPASPPEEGSPDPDSTGALVEEEDPFFKVPVNKLAAAVSNFGYDLYRVRSSTSPTTNVLLSPLSVATALSALSLGAEQRTESIIHRALYYDLISSPDIHGTYKELLDTVTAPQKNLKSASRIVFEKKLRIKSSFVAPLEKSYGTRPRVLTGNPRLDLQEINNWVQAQMKGKLA RSTKEIPDEISILLLGVAHFKGQWVTKFDSRKTSLEDFYLDEERTVRVPMMSDPKAVLRYGLDSDLSCKIAQLPLTGSMSIIFFLPLKVTQNLTLIEESLTSEFIHDIDRELKTVQAVLTVPKLKLSYEGEVTKSLQEMKLQSLFDSPDFSKITGKPIKLTQVEHRAGFEWNEDGAGTTPSPGLQPAHLTFPLD YHLNQPFIFVLRDTTDTGALLFIGKILDPRGPRRSKRSGSGATNFSLLKQAGDVEENPGPMRRFLTVISFLLYFGCAFACEEPPTFEAMELIGKPKPYYEIGERVDYKCKKGYFYIPPLATHTICDRNHTWLPVSDDACYRETCPYIRDPLNGQAVPANGTYEFGYQMHFICNEGYYLIGEEILYCELKGSVAIW SGKPPICEKVLCTPPPKIKNGKHTFSEVEVFEYLDAVTYSCDPAPGPDPFSLIGESTIYCGDNSVWSRAAPECKVVKCRFPVVENGKQISGFGKKFYYKATVMFECDKGFYLDGSDTIVCDSNSTWDPPVPKCLKVLPPSSTKPPALSHSVSTSSTTKSPASSASGPRPTYKPVSNYPGYPKPEEGILDSLDV [SEQ ID NO:69] Preferably, in this embodiment, the construct comprises the 2328 nucleotide sequence designated herein as SEQ ID NO:70 (as contained in plasmid IKC159P), or a fragment or variant thereof, as follows: [SEQ ID NO:70] In another embodiment, the amino acid sequence of sCD46-Furin-P2A-PEDF is as follows, referred to herein as SEQ ID NO:71, or a fragment or variant thereof: MRRFLTVISFLLYFGCAFACEEPPTFEAMELIGKPKPYYEIGERVDYKCKKGYFYIPPLATHTICDRNHTWLPVSDDACYRETCPYIRDPLNGQAVPANGTYEFGYQMHFICNEGYYLIGEEILYCELKGSVAIWSGKPPICEKVLCTPPPKIKNGKHTFSEVEVFEYLDAVTYSCDPAPGPDPFSLIGESTI YCGDNSVWSRAAPECKVVKCRFPVVENGKQISGFGKKFYYKATVMFECDKGFYLDGSDTIVCDSNSTWDPPVPKCLKVLPPSSTKPPALSHSVSTSSTTKSPASSASGPRPTYKPVSNYPGYPKPEEGILDSLDVRRSKRSGSGATNFSLLKQAGDVEENPGPMRRLVLLLCIGALLGHVFCQNPASPPEEGS PDPDSTGALVEEEDPFFKVPVNKLAAAVSNFGYDLYRVRSSTSPTTNVLLSPLSVATALSALSLGAEQRTESIIHRALYYDLISSPDIHGTYKELLDTVTAPQKNLKSASRIVFEKKLRIKSFVAPLEKSYGTRPRVLTGNPRLDLQEINNWVQAQMKGKLARSTKEIPDEISILLLGVAHFKGQWVTKFDSR KTSLEDFYLDEERTVRVPMMSDPKAVLRYGLDSDLSCKIAQLPLTGSMSIIFFLPLKVTQNLTLIEESLTSEFIHDIDRELKTVQAVLTVPKLKLSYEGEVTKSLQEMKLQSLFDSPDFSKITGKPIKLTQVEHRAGFEWNEDGAGTTPSPGLQPAHLTFPLDYHLNQPFIFVLRDTDTGALLFIGKILDPRGP [SEQ ID NO:71] Preferably, in this embodiment, the construct comprises the 2328 nucleotide sequence designated herein as SEQ ID NO:72 (as contained in plasmid IKC145P), or a fragment or variant thereof, as follows: [SEQ ID NO:72] In another embodiment, the amino acid sequence of [PEDF-Furin-P2A-CFHL1] is as follows, referred to herein as SEQ ID NO:87, or a fragment or variant thereof: MRRLVLLLCIGALLGHVFCQNPASPPEEGSPDPDSTGALVEEEDPFFKVPVNKLAAAVSNFGYDLYRVRSSTSPTTNVLLSPLSVATALSALSLGAEQRTESIIHRALYYD LISSPDIHGTYKELLDTVTAPQKNLKSASRIVFEKKLRIKSSFVAPLEKSYGTRPRVLTGNPRLDLQEINNWVQAQMKGKLARSTKEIPDEISILLLGVAHFKGQWVTKFD SRKTSLEDFYLDEERTVRVPMMSDPKAVLRYGLDSDLSCKIAQLPLTGSMSIIFFLPLKVTQNLTLIEESLTSEFIHDIDRELKTVQAVLTVPKLKLSYEGEVTKSLQEMK LQSLFDSPDFSKITGKPIKLTQVEHRAGFEWNEDGAGTTPSPGLQPAHLTFPLDYHLNQPFIFVLRDTTDTGALLFIGKILDPRGPRRSKRSGSGATNFSLLKQAGDVEENPG PMRRLLTFISILALVGAFAEDCNELPPRRNTEILTGSWSDQTYPEGTQAIYKCRPGYRSLGNVIMVCRKGEWVALNPLRKCQKRPCGHPGDTPFGTFTLTGGNVFEYGVKA VYTCNEGYQLLGEINYRECDTDGWTNDIPICEVVKCLPVTAPENGKIVSSAMEPDREYHFGQAVRFVCNSGYKIEGDEEMHCSDDGFWSKEKPKCVEISCKSPDVINGSPI SQKIIYKENERFQYKCNMGYEYSERGDAVCTESGWRPLPSCEEKSCDNPYIPNGDYSPLRIKHRTGDEITYQCRNGFYPATRGNTAKCTSTGWIPAPRCTLKPCDYPDIKH GGLYHENMRRPYFPVAVGKYYSYYCDEHFETPSGSYWDHIHCTQDGWSPAVPCLRKCYFPYLENGYNQNYGRKFVQGKSIDVACHPGYALPKAQTTVTCMENGWSPTPRCIR [SEQ ID NO:87] Preferably, in this embodiment, the construct comprises the 2670 nucleotide sequence designated herein as SEQ ID NO:88 (as contained in plasmid IKC161P), or a fragment or variant thereof, as follows: [SEQ ID NO:88] In another embodiment, the amino acid sequence of [CFHL1-Furin-P2A-PEDF] is referred to herein as SEQ ID NO:89, or a fragment or variant thereof, as follows: MRRLLTFISILALVGAFAEDCNELPPRRNTEILTGSWSDQTYPEGTQAIYKCRPGYRSLGNVIMVCRKGEWVALNPLRKCQKRPCGHPGDTPFGTFTLTGGNVFEYGVKAV YTCNEGYQLLGEINYRECDTDGWTNDIPICEVVKCLPVTAPENGKIVSSAMEPDREYHFGQAVRFVCNSGYKIEGDEEMHCSDDGFWSKEKPKCVEISCKSPDVINGSPIS QKIIYKENERFQYKCNMGYEYSERGDAVCTESGWRPLPSCEEKSCDNPYIPNGDYSPLRIKHRTGDEITYQCRNGFYPATRGNTAKCTSTGWIPAPRCTLKPCDYPDIKHG GLYHENMRRPYFPVAVGKYYSYYCDEHFETPSGSYWDHIHCTQDGWSPAVPCLRKCYFPYLENGYNQNYGRKFVQGKSIDVACHPGYALPKAQTTVTCMENGWSPTPRCIRR RSKRSGSGATNFSLLKQAGDVEENPGPMRRLVLLLCIGALLGHVFCQNPASPPEEGSPDPDSTGALVEEEDPFFKVPVNKLAAAVSNFGYDLYRVRSSTSPTTNVLLSPLS VATALSALSLGAEQRTESIIHRALYYDLISSPDIHGTYKELLDTVTAPQKNLKSASRIVFEKKLRIKSFVAPLEKSYGTRPRVLTGNPRLDLQEINNWVQAQMKGKLARS TKEIPDEISILLLGVAHFKGQWVTKFDSRKTSLEDFYLDEERTVRVPMMSDPKAVLRYGLDSDLSCKIAQLPLTGSMSIIFFLPLKVTQNLTLIEESLTSEFIHDIDRELK TVQAVLTVPKLKLSYEGEVTKSLQEMKLQSLFDSPDFSKITGKPIKLTQVEHRAGFEWNEDGAGTTPSPGLQPAHLTFPLDYHLNQPFIFVLRDTTDTGALLFIGKILDPRGP [SEQ ID NO:89] Preferably, in this embodiment, the construct comprises the 2670 nucleotide sequence designated herein as SEQ ID NO:90 (as contained in plasmid IKC174P), or a fragment or variant thereof, as follows: [SEQ ID NO: 90] Thus, in a preferred embodiment the construct encodes an amino acid sequence substantially as set out in SEQ ID NO: 55, 57, 59, 61, 63, 65, 67, 69, 71, 85, 87 or 89, or a fragment or variant thereof.

[0083] Preferably, the construct comprises a nucleotide sequence substantially as set out in SEQ ID NO: 56, 58, 60, 62, 64, 66, 68, 70, 72, 86, 88 or 90, or a fragment or variant thereof.

[0084] The present inventors have created a series of recombinant expression vectors which contain the constructs of the present invention.

[0085] Thus, in a second aspect there is provided a recombinant vector comprising a genetic construct according to the first aspect.

[0086] In one embodiment, the recombinant vector (for example, known as “IKC0121V”) comprises a nucleotide sequence referred to herein as SEQ ID NO:73, or a fragment or variant thereof, as follows: [SEQ ID NO:73] Thus, in one embodiment the recombinant vector comprises a nucleotide sequence substantially as set out in SEQ ID NO: 73, or a fragment or variant thereof.

[0087] 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.

[0088] 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.

[0089] The term "recombinant AAV (rAAV) vector" can refer to a recombinant AAV-derived nucleic acid, which can contain at least one terminal repeat sequence.

[0090] The capsid coat of AAV and recombinant vectors is known to be composed of three capsid proteins, termed VP1, VP2 and VP3, all of which contain a significant amount of overlapping amino acids between them but unique N-terminal sequences. The AAV virus contains 60 subunits each of the VP1, VP2 and VP3 capsid proteins in a 1:1:10 ratio that 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 were identified by screening DNA variants from libraries of capsid coding sequences

[63] . 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.

[0091] 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, AA7, 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.

[0092] 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.

[0093] The constructs and expression vectors described herein can be used to treat retinal disorders, in particular dry age-related macular degeneration and geographic atrophy, and more generally to reduce complement activation and retinal cell damage and loss.

[0094] 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.

[0095] 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 complement activation and retinal cell damage and loss.

[0096] In a fifth aspect, there is provided a method for treating, preventing or ameliorating a retinal disorder or reducing complement activation and retinal cell damage and loss 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.

[0097] Preferably, the genetic construct or recombinant vector of the invention is used in gene therapy techniques.The PEDF receptor agonist encoded by the construct or vector activates PEDF receptor, thereby preserving RPE and photoreceptor cells, reducing VEGF release, and preventing dry AMD from transforming into wet AMD.The anti-complement protein encoded by the construct or vector neutralizes complement factors, thereby reducing complement activation and reducing GA area and retinal cell loss.

[0098] In one embodiment, the retinal disorder to be treated can be dry age-related macular degeneration or geographic atrophy. In addition, the retinal disorder to be treated can be any pathophysiological condition involving retinal damage due to complement activation.

[0099] In another embodiment, the constructs and vectors can be used to reduce complement activation and retinal cell damage and loss.The constructs and vectors can be used to treat retinal cell damage and loss associated with the following conditions: retinitis pigmentosa, Stargardt's disease, diabetic macular degeneration, age-related macular degeneration and Leber's congenital amaurosis.In another embodiment, the constructs and vectors can be used to treat retinal cell damage and loss associated with glaucoma.

[0100] 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 complement activation and retinal cell damage and loss. 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 complement activation and retinal cell damage and loss.

[0101] 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.

[0102] 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.

[0103] 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.

[0104] 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.

[0105] 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).

[0106] 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).

[0107] 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.

[0108] Generally, between 0.001 μg / kg (body weight) and 10 mg / kg (body weight) of DNA plasmid or 1 × 10 8 GC / mL ~ 1 × 1013 A daily dose of the viral vector of the present invention between GC / mL can be used to treat, ameliorate or prevent retinal disorders, depending on the genetic construct or recombinant vector used.

[0109] The genetic construct or recombinant vector may be administered before, during or after the onset of retinal damage or retinal capillary dysfunction. 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.

[0110] 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 and frequency of administration of drugs) of the genetic constructs or recombinant vectors of the present invention. The inventors believe themselves to be the first to suggest a bicistronic genetic construct encoding a promoter operably linked to a coding sequence that would boost PEDF concentrations and attenuate the complement cascade, thereby improving retinal survival and reducing GA disease progression.

[0111] 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.

[0112] 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.

[0113] 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.

[0114] 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.

[0115] 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.

[0116] 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.

[0117] In some embodiments, the viral titer of the pharmaceutical composition of the invention is 5×10 per milliliter or less. 10 ~5×10 13The 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 that described in

[64] .

[0118] 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.

[0119] 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.

[0120] 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.

[0121] 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.

[0122] 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.

[0123] 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).

[0124] 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: 1-90, etc.

[0125] 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.

[0126] 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.

[0127] 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.

[0128] It will therefore be appreciated that accurate alignment of protein or DNA sequences is a complex process. The popular multiple alignment program ClustalW [65, 66] 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.

[0129] 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.

[0130] 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.

[0131] 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.

[0132] 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.

[0133] 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]

[0134] [Figure 1] FIG. 1 illustrates one embodiment of viral vectors of the present invention (top of figure) expressing various transgene proteins, namely, a PEDF receptor agonist and an anti-complement protein, and their biological effects in reducing pathophysiology associated with retinal disorders such as geographic atrophy and dry AMD. [Diagram 2] 2 shows a schematic diagram of one embodiment of a genetic construct according to the invention. The construct is a bicistronic cassette in which the coding sequence for the PEDF receptor agonist is preceded by a signal peptide directing cellular secretion; the coding sequence for the anti-complement protein is similarly preceded by a signal peptide directing cellular secretion; the two are linked in either orientation by an enzyme-cleavable viral-2A linker sequence / skipping site. [Diagram 3] Figure 3 illustrates the intracellular processing of genetic material from a gene therapy construct to generate two mature therapeutic proteins capable of protecting retinal cells and neutralizing or attenuating complement activation. Step 1 is the transcription of messenger RNA by a single promoter. 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 signal peptide, which removes the remaining proline amino acid from the N-terminus of the downstream component prior to secretion from the target retinal cell. [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]Figure 6 shows expression of PEDF protein in supernatants collected from either HEK293T or ARPE-19 cells 24 hours after transfection with a series of expression plasmids, and Western blots illustrating efficient furin cleavage in constructs with optimized sequences compared to the basic sequence, and release of the viral-2A linker from the C-terminus of the upstream protein (Figure 6A). IKC036P [null control], IKC030P [PEDF only], IKC093P [hPEDF-basic furin-viral P2A-anti-Bb SCVF], IKC094P [hPEDF-basic furin-viral P2A-anti-C5 SCVF], IKC104P [hPEDF-optimized furin-viral P2A-anti-C3b SCVF], IKC121P [hPEDF-optimized furin-viral P2A-anti-C3b SCVF], IKC122P [hPEDF-optimized furin-viral P2A-sCD55]. Figure 6B shows the percentage furin cleavage in HEK293T supernatants, and Figure 6C shows the percentage furin cleavage in ARPE-19 supernatants. [Figure 7] FIG. 7 is a diagrammatic representation of an ELISA assay to illustrate PEDF concentrations released into cell culture medium from HEK293T cells 24 hours after transfection with a series of bicistronic or control plasmids; IKC036 [null control], IKC093P, IKC104P, IKC121P and IKC122P. [Figure 8] FIG. 8 is a Western blot illustrating intracellular processing and release of PEDF protein and non-membrane-bound anti-complement factors into the culture medium following transfection of HEK293T cells with plasmids IKC157P, IKC158P, IKC159P and IKC161P versus the null control IKC166 plasmid. [Figure 9]FIG. 9 shows HEK293T cells expressing PEDF and non-membrane-bound anti-complement proteins prior to secretion and released into the culture medium following transfection with plasmids IKC093P, IKC094P, IKC157P, IKC158P, IKC159P and IKC161P versus the null control IKC166P plasmid using immunocytochemistry (light staining). [Figure 10] FIG. 10A and FIG. 10B illustrate the production and release of soluble sCD55(DAF) from HEK293T or ARPE-19 cell culture medium, respectively, following transfection with IKC122P plasmid or control IKC036 null plasmid. [Figure 11] Figure 11 illustrates data showing neutralization and / or reduction of complement C3b in human serum after incubation of serum with cell growth medium from HEK293T cells transfected with a null control plasmid (IKC036P), or IKC087P, IKC104P, or IKC0121P, which secreted single chain variable fragments capable of binding and neutralizing human C3b. Note that IKC087P has a non-optimized expression cassette and that the ELISA antibody has 80% cross-reactivity with human C3 (constituting approximately 2 / 3 of the immunoreactivity, so a 30% reduction in the reading would equate to nearly 100% neutralization of C3b, since SCVF does not bind C3). [Figure 12] Figure 12A demonstrates the ability of supernatants collected from HEK293T cells transfected with the IKC122P plasmid construct producing sCD55 to reduce the generation of recombinant C3b (C3 convertase) from parental C3 in the presence of factors B and D, compared to a null control plasmid (IKC036P) and the IKC121P plasmid construct expressing a single chain variable fragment capable of binding and neutralizing human C3b. Figure 12B shows that the reduction in the percentage conversion of C3 to C3b in the presence of IKC121P is significant compared to the IKC036P control. [Figure 13]Figure 13A illustrates that supernatants collected from HEK293T cells transfected with constructs IKC139P and IKC143P, which generate factor I cofactors sCD46 and CFHL1, respectively, can facilitate recombinant C3b degradation into two iC3b fragments (68 and 43 kDa) in the presence of low concentrations of recombinant CFI. Note the absence of degradation of C3b produced by the null control plasmid IKC036P or the PBS control. Figure 13B shows the percentage of iC3b fragments in the presence of IKC139P and IKC143P compared to IKC036P and the PBS control, which showed no degradation of C3b to iC3b. [Figure 14] FIG. 14 shows an embodiment of a plasmid map of the "IKC121P" vector of the present invention. [Figure 15] FIG. 15 shows Western blot data of PEDF and anti-complement transgene expression expressed and secreted into the culture medium 48 hours after transduction of HEK293T cells with vectors IKC157V, IKC158V, IKC159V, IKC161V, IKC167V versus the null control vector IKC166V. [Figure 16] Figure 16 compares the effect of adding HEK293T transfected cells with plasmids expressing either soluble CD46 (IKC137P) or complement I (IKC139P) on complement factor C3b (39 nM) degradation by low concentrations of recombinant complement factor I (11 nM) and factor H (0.5 nM) compared to null control transfected cells (IKC036P). Note that supplementation of recombinant complement factor I with cell culture medium collected from HEK293T cells transfected with IKC137P (complement I) did not increase C3b degradation compared to the IKC036P null control. In contrast, supplementation with cell culture medium collected from IKC139P (soluble CD46)-transfected HEK293T cells significantly increased enzymatic C3b degradation, as evidenced by a decrease in the C3b alpha chain band and an increase in the iC3b (68 kDa and 43 kDa) bands. [Figure 17]Figure 17 illustrates that intravitreal injection of IKC159V rAAV2 vectors increases vitreous PEDF concentrations (A) and protects retinal ganglion cells (B and C) when challenged with the neurotoxin N-methyl-D-aspartate (NMDA) (challenging for 8 days 3 weeks after gene therapy delivery). In addition, the rAAV2 vectors are able to significantly degrade recombinant complement C3b (ex-vivo) and secrete sufficient soluble CD46 into the vitreous compared to vitreous isolated from animals treated with the null IKC166V vector (D). [Figure 18] Figure 18 shows the beneficial effect of IKC159V rAAV2 vector in preventing the transient decrease in ARPE-19 cell transepithelial resistance when challenged with mild oxidative stress (hydrogen peroxide) and complement attack (addition of human serum proteins). The data show the transient loss of transepithelial resistance at 2 hours and the percentage change from baseline when IKC159V or IKC166V (null vector) transduced cells were treated with both hydrogen peroxide and human serum. The comparison standard was ARPE-19 cells treated with hydrogen peroxide or human serum only. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0135] Working Example With reference to Figure 1 and Figure 2, the present inventors have designed and constructed a novel genetic construct that encodes (i) an agonist of PEDF receptor and (ii) an anti-complement protein under the control of a single promoter. As illustrated in Figure 3, the present inventors have also advantageously introduced a spacer sequence into the genetic construct (e.g., a viral-2A peptide spacer sequence), which allows the expression of all of the peptides encoded by the construct 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 a viral-2A removal sequence into the construct, such as a furin recognition sequence, with reference to Figure 6.

[0136] As illustrated in FIG. 3, the bicistronic expression cassette produces two mature therapeutic proteins, a PEDF receptor agonist and an anti-complement protein (FIGS. 7-11 and 15). The PEDF receptor agonist acts to protect the retinal pigment epithelium (RPE) and other retinal cells, such as photoreceptors, from cytotoxic biochemical insults and cell death. In dry AMD and geographic atrophy patients, the endogenous concentration of PEDF in the eye is significantly depleted by disease pathology, thereby reducing the ability of the retina to function normally. The genetic construct of the present invention will replenish retinal PEDF concentrations, thus restoring retinal defense mechanisms against oxidative damage and other pathophysiological factors operating in dry AMD. As illustrated in FIG. 17, boosting PEDF concentrations will result in prevention of further loss to RPE cells and overlying photoreceptors, thereby slowing or halting vision loss. In addition, anti-complement proteins can reduce complement system activation, which has also been shown to play a significant role in GA, as illustrated in Figures 12, 13, 16-18. By providing better protection from retinal cell loss via increased retinal PEDF concentrations coupled with reduced complement activation, the bicistronic gene construct can attenuate or halt further retinal damage and associated vision loss.

[0137] Next, the inventors introduced the genetic construct into a recombinant expression vector such as rAAV2 (see, e.g., FIG. 14). 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.

[0138] IKC036P is a null control. IKC030P contains PEDF only. IKC093P contains [hPEDF-basic furin-viral P2A-anti-Bb SCVF], IKC094P contains [hPEDF-basic furin-viral P2A-anti-C5 SCVF], IKC104P contains [hPEDF-optimized furin-viral P2A-anti-C3b SCVF], IKC121P contains [hPEDF-optimized furin-viral P2A-anti-C3b SCVF], IKC122P contains [hPEDF-optimized furin-viral P2A-sCD55], and IKC157P contains [hPEDF-optimized furin-viral P2A-anti-C3b SCVF], IKC158P contains [hPEDF-optimized furin-virus P2A-sCD55], IKC159P contains [hPEDF-optimized furin-virus P2A-sCD46], IKC161P contains [hPEDF-optimized furin-virus P2A-CFHL1], and IKC166P contains [null control]. Recombinant AAV vector production The DNA plasmids were used to produce recombinant AAV2 vectors. HEK293 cells (2.5 × 10 8 HEK293 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 HEK293 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).

[0139] Figure 4 illustrates enhanced green fluorescent protein (eGFP) reporter gene expression in HEK293 cells harvested 24 hours after transduction with rAAV2 vectors containing different promoter sequences: sCAG, sCAG-intron, CMV, hSYN1, and mPGK. As illustrated in Figure 4, both sCAG and CMV display high levels of eGFP transgene expression in HEK293 cells.

[0140] Moreover, Figure 5 shows both cross-section and flat mount images of mouse retina to illustrate the levels of eGFP expression 3 weeks after intravitreal injection with rAAV2 vectors containing different promoter sequences: sCAG, sCAG-intron, CMV, mPGK and hSYN1. As can be seen from 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

[0141] PEDF concentrations in HEK293T cells after cell transfection with various plasmid constructs Briefly, DNA plasmids were mixed with Opti-MEM (FisherSci; Loughborough, Leics., UK) and Lipofectamine 3000 (FisherSci) and added to HEK293T cells cultured at 80% confluency in 24-well plates such that each well received 0.5 μg of plasmid DNA and 0.75 μL of Lipofectamine. Cells were incubated for 24 hours at 37° C., 5% CO2. HEK293T cell incubation medium was collected and centrifuged to remove any cell debris, and the PEDF concentration produced by the cells was subsequently measured using a commercially available human PEDF ELISA kit (Abcam; Cambridge, UK; ab246535) or by Western blot (Abcam ab180711, 1:1000 dilution). The control null plasmids (IKC036P or IKC166P) were not shown to contribute any additional to the small amounts of PEDF produced by HEK293T cells.

[0142] Figures 6 and 7 demonstrate that the amount of PEDF secreted by HEK293T cells 24 hours after transfection with a series of plasmids was significantly greater than the null controls (IKC036P and IKC166P). EXAMPLES

[0143] Detection of furin activity and viral-2A peptide cleavage HEK293T cells were transfected with the plasmids described above. The molecular weight of the tested transgenes (PEDF and anticomplement protein) from the bicistronic construct was compared with the transgene construct that produces only a single transgene. To confirm the cleavage of the viral-2A peptide from the C-terminus, the 2A antibody (NBP2-59627) from Bio-Techne (Abingdon, Oxon, UK) was used to test for the presence of the viral-2A peptide.

[0144] Figure 6 illustrates the expression of PEDF protein (with and without viral-2A) in the supernatant collected from HEK293T and ARPE-19 cells 24 hours after transfection with a series of expression plasmids. Moreover, Figure 6B and Figure 6C illustrate the quantification of effective furin cleavage and release of the viral-2A linker from the C-terminus of PEDF in HEK293T and ARPE-19 cells transfected with a series of plasmids, showing that the optimized furin sequence in IKC104P, IKC121P and IKC122P plasmids is significantly more effective than the basic furin sequence in IKC093P and IKC094P plasmids. PEDF derived from HEK293T cells transfected with IKC030P plasmid was used as a control, since it does not have a C-terminal furin or viral 2A sequence. EXAMPLES

[0145] Detection of expressed anti-complement proteins Figures 8 and 9 demonstrate the production, correct processing and release of PEDF and downstream anti-complement proteins in HEK293T cells after transfection with the plasmids. In Figure 8, the release of both PEDF and anti-complement proteins into the culture medium from HEK293T cells transfected with either the control IKC166P (null) or IKC157P, IKC158P, IKC159P and IKC161P plasmids is shown by Western blot using the same antibodies used for immunocytochemistry (see below), all diluted 1:1000. The secondary antibody was goat-anti-rabbit (Abcam, ab6721) at 1:10,000 dilution.

[0146] Figure 9 shows PEDF and anti-complement proteins in transfected HEK293T cells grown on coverslips and stained by immunocytochemistry (anti-Bb, anti-C5 and anti-C3b SCVF stained with custom rabbit polyclonal antibodies generated by Genscript (Peptide 1); CD55 Abcam ab133684; CD46 Invitrogen PA535311; and CFH Abcam ab133536) prior to release. In Figure 15, release of both PEDF and anti-complement proteins into culture medium from HEK293T cells transduced with rAAV vectors, including control IKC166V (null vector) or IKC157V, IKC158V, IKC159V, IKC161V and IKC167V rAAV2 vectors, was assessed by Western blotting using the same antibodies as described above for Figures 8 and 9.

[0147] FIG. 10 illustrates the production and secretion of soluble / non-cell membrane bound forms of (DAF)sCD55 in both HEK293T and ARPE-19 cells 24 hours after transfection with plasmid IKC122P by Western blot using CD55 antibody (ab-133684 at 1:1000 dilution from Abcam, Cambridge, UK) and peroxidase-labeled goat anti-rabbit secondary antibody (1:10,000 dilution, abcam, ab6721). EXAMPLES

[0148] Demonstration of anti-complement protein activity The activity of anti-complement proteins is shown in Figures 11, 12, 13 and 16, whereby neutralization of C3b is shown in Figure 11, prevention of C3 convertase (C3bBb) generation is illustrated in Figure 12 and complement factor I (CFI)-mediated C3b cleavage assay is shown in Figures 13 and 16.

[0149] For the C3b neutralization assay shown in Figure 11, HEK293T cells were transfected with the plasmids described above. After 24 hours, the supernatant was collected and clarified by brief centrifugation. The supernatant (190 μL) was incubated with 10 μL of normal human serum (1:20,000 dilution) at room temperature for 30 minutes with gentle agitation. After incubation, samples were quantified using a human complement C3b ELISA kit (abcam, ab195461), which has 80% cross-reactivity with human C3 (constituting approximately 2 / 3 of the immunoreactivity, so a 30% reduction in the reading would equate to nearly 100% neutralization of C3b, since SCVF does not bind C3).

[0150] For the C3 convertase assay shown in Figure 12, recombinant proteins (C3, 0.2 μM; complement factor B, 0.2 μM and complement factor D, 0.02 μM, final concentration; Complement Technologies Inc., Tyler, TX75703, USA) were incubated in veronal buffer with HEK293T culture medium previously transfected with various plasmids in a total volume of 50 μL for 30 min at 37°C. After incubation, production of C3 convertase was measured by SDS-Page electrophoresis and staining of the gels with SimplyBlue Safe stain (Thermofisher).

[0151] For the C3b cleavage assay illustrated in Figure 13, HEK293T culture media previously transfected with various plasmids were incubated with C3b substrate (42 nM) and recombinant CFI (1.2 nM) (Complement Technologies Inc.) in a total volume of 60 μL for 60 min at 37°C. C3b degradation products (iC3b 64 kDa and 43 kDa) were examined by Western blot using a goat anti-human C3 antibody (AHP1752 diluted 1:2,000; BioRad) and a peroxidase-labeled donkey anti-goat secondary antibody (705-035-147, 1:10,000; Jackson ImmunoResearch Europe, Ely, UK).

[0152] For the C3b cleavage assay illustrated in Figure 16, medium from transfected HEK293T cells was combined with recombinant complement factors. The previously used concentrations of recombinant complement factors I and H were reduced to 11 nM and 0.5 nM, respectively, while the substrate C3b was maintained at around 39 nM. Note that the addition of more complement factor I from HEK293T culture medium (IKC137P) did not significantly increase C3b degradation, whereas the addition of HEK293T culture medium containing soluble CD46 cofactor could significantly boost C3b degradation (IKC139P). These data would suggest that C3b degradation is more sensitive to cofactor addition than to factor I supplementation. EXAMPLES

[0153] Demonstration of anti-complement protein and PEDF activity of bicistronic rAAV vectors in vivo The activity of both sCD46 and PEDF proteins following intravitreal delivery of IKC159V (a soluble CD46 vector) is shown in FIG.

[0154] Mice were intravitreally injected (2 μL) with IKC166V (null control) or IKC159V. After 21 days, mouse eyes were free dissected and vitreous samples (between 4-5 μL) were extracted and used to assess C3b degradation ex-vivo using the C3b cleavage assay method described above. Results showed significant degradation of C3b in the vitreous from IKC159V-treated eyes compared to the IKC166V (null) group.

[0155] Another set of mice receiving intravitreal injections (2 μL) of IKC166V (null control) or IKC159V was used in the NMDA study. 21 days after vector injection, mice received an additional intravitreal injection of NMDA (30 nmol / eye) or vehicle, and 8 days later, animals were terminated. Vitreous samples were obtained from vehicle-injected eyes and PEDF concentrations were measured using a commercially available PEDF ELISA kit (Abcam). Retinal flat mounts were prepared from all eyes and retinal ganglion cell counts were measured using RBPMS immunolabeling. Of note is the approximately 50% loss of retinal ganglion cells in the IKC166V (null) plus NMDA treatment group compared to the nearly complete protection in the IKC159V plus NMDA treatment group. EXAMPLES

[0156] Demonstration of the beneficial effect of a bicistronic rAAV2 vector (IKC159V) in preventing the loss of ARPE-19 cell transepithelial resistance For the ARPE-19 cell transepithelial resistance (TER) assay shown in Figure 18, ARPE-19 cells were grown in Transwell (24-well, Greiner) for 2 weeks with regular medium changes until a stable monolayer and TER were reached. The medium was then replaced with serum-free medium for another 2 weeks, followed by transduction of the monolayer with rAAV vectors for 48 hours. TER assay readings were obtained before and 1, 2, and 4 hours after exposure to 1 mM H2O2 and human serum. As illustrated in Figure 18, IKC159V rAAV2 vectors prevented the transient reduction of ARPE-19 cell transepithelial resistance when challenged with mild oxidative stress. Discussion and Conclusion As illustrated in the Examples, the inventors have surprisingly demonstrated that it is possible to combine open reading frames (ORFs) encoding a PEDF receptor agonist and an anti-complement protein in a single genetic construct.

[0157] PEDF receptor agonists restore the concentration of PEDF, thereby reducing inflammation and preserving RPE and photoreceptor cells. Moreover, anti-complement proteins can neutralize or attenuate the alternative complement pathway, thereby preventing further RPE cell loss. Advantageously, the genetic constructs of the present invention target the AP pathway, which means that the classical and lectin pathways of the complement system are preserved, thereby maintaining an antimicrobial defense system that can facilitate the destruction of invading pathogens.

[0158] References 1. Tufail A, et al. Objective measurement of reading speed and correlation with patient-reported functional reading independence. Presented at the 15th EURETINA Congress, Nice, France, September 17-20, 2015. 2. The Eye Diseases Prevalence Research Group (2004). Prevalence of age-related macular degeneration in the United States. Arch Ophthalmol., vol.122, PP: 564-572 3. Age-Related Eye Disease Study Research Group. Risk factors associated with age-related macular degeneration. A case-control study in the age-related eye disease study: Age-Related Eye Disease Study Report Number 3. Ophthalmology. 2000, vol.107, PP: 2224-232. 4. Chakravarthy U, Augood C, Bentham GC, de Jong PT, Rahu M, Seland J, Soubrane G, Tomazolli L, Topouzis F, Vingerling JR, Vioque J, Young IS, Fletcher AE (2007). Cigarette smoking and age-related macular degeneration in the EUREYE Study. Ophthalmology vol. 114(6), PP: 1157-63. 5. Smith W, Assink J, Klein R, Mitchell P, Klaver CC, Klein BE et al. 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Claims

1. A genetic construct comprising a promoter operably linked to a first coding sequence encoding an agonist of the PEDF receptor and a second coding sequence encoding an anti-complement 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 degeneration protein-2 (VMD2) promoter, a human phosphoglycerate kinase-1 (PGK-1) promoter, or an EF1 alpha promoter, and the promoter may comprise 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. The genetic construct of claim 1 , wherein the first coding sequence comprises a nucleotide sequence encoding a PEDF protein.

4. 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, 12 or 13, or a fragment or variant thereof, and / or the first coding sequence encodes an amino acid sequence substantially as set forth in SEQ ID NO: 10, or a fragment or variant thereof.

5. The genetic construct of claim 1 , wherein the anti-complement protein is capable of neutralizing or attenuating complement activation.

6. 2. The genetic construct of claim 1, wherein the anti-complement protein is capable of targeting the alternative pathway (AP) of the complement system, and preferably wherein the anti-complement protein minimally affects the classical pathway (CP) and / or the lectin pathway (LP) of the complement system.

7. 2. The genetic construct of claim 1, wherein the anti-complement protein is an anti-C3b, anti-Bb or anti-C5 antibody, or an antigen-binding fragment thereof, and the anti-complement protein may be a single chain variable fragment (SCVF).

8. The genetic construct of claim 1 , wherein the anti-complement protein is CD55, preferably soluble CD55 (sCD55).

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

10. The genetic construct of claim 1 , wherein the anti-complement protein is CD46, preferably soluble CD46 (sCD46).

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

12. 8. The genetic construct of claim 7, wherein the second coding sequence comprises a nucleotide sequence substantially as set forth in SEQ ID NO: 15, 17 or 83, 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: 14, 16 or 82, or a fragment or variant thereof.

13. 9. The genetic construct of claim 8, wherein the second coding sequence comprises a nucleotide sequence substantially as set forth in SEQ ID NO: 19, 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: 18, or a fragment or variant thereof.

14. 10. The genetic construct of claim 9, wherein the second coding sequence comprises a nucleotide sequence substantially as set forth in SEQ ID NO:21 or 22, 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:20, or a fragment or variant thereof.

15. 11. The genetic construct of claim 10, wherein the second coding sequence comprises a nucleotide sequence substantially as set forth in SEQ ID NO:24, 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:23, or a fragment or variant thereof.

16. 12. The genetic construct of claim 11, wherein the second coding sequence comprises a nucleotide sequence substantially as set forth in SEQ ID NO:81, 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:80, or a fragment or variant thereof.

17. 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 PEDF receptor agonist and the anti-complement protein as separate molecules.

18. A genetic construct according to claim 17, wherein said spacer sequence comprises and encodes a viral peptide spacer sequence, most preferably a viral-2A peptide spacer sequence.

19. 20. The genetic construct of claim 18, wherein the viral-2A peptide spacer sequence comprises an F2A, E2A, T2A or P2A sequence.

20. 18. The genetic construct of claim 17, wherein the spacer sequence comprises a nucleotide sequence substantially as set forth in SEQ ID NO: 26, 28, 30 or 32, or a fragment or variant thereof, and / or the spacer sequence encodes an amino acid sequence substantially as set forth in SEQ ID NO: 25, 27, 29 or 31, or a fragment or variant thereof.

21. 18. The genetic construct of claim 17, wherein the genetic construct comprises a viral-2A excision sequence, the viral-2A excision sequence being optionally located 5' to the viral-2A sequence.

22. 22. The genetic construct of claim 21, 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).

23. 22. The genetic construct of claim 21, wherein the viral-2A removal sequence is a furin recognition sequence, and the viral-2A removal sequence may encode an amino acid sequence substantially as set forth in SEQ ID NO: 33, or a fragment or variant thereof.

24. 24. The genetic construct of claim 23, wherein the viral-2A removal sequence comprises a nucleotide sequence substantially as set forth in SEQ ID NO: 35 or 37, 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: 34 or 36, or a fragment or variant thereof.

25. 22. The genetic construct of claim 21, wherein the viral-2A removal sequence is a gelatinase MMP-2 recognition sequence, and the viral-2A removal sequence may comprise a nucleotide sequence substantially as set forth in SEQ ID NO: 39, or a fragment or variant thereof, and / or the viral-2A removal sequence may encode an amino acid sequence substantially as set forth in SEQ ID NO: 38, or a fragment or variant thereof.

26. 22. The genetic construct of claim 21, wherein the viral-2A removal sequence is a renin recognition sequence and may comprise a nucleotide sequence substantially as set forth in SEQ ID NO:41, or a fragment or variant thereof, and / or the viral-2A removal sequence may encode an amino acid sequence substantially as set forth in SEQ ID NO:40, or a fragment or variant thereof.

27. 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), the WPRE may comprise a nucleic acid sequence substantially as set forth in SEQ ID NO: 42 or 43, or a fragment or variant thereof.

28. 2. The genetic construct of claim 1, wherein the genetic construct comprises a nucleotide sequence encoding a polyA tail, the polyA tail may comprise a nucleic acid sequence substantially as set forth in SEQ ID NO: 44, 45 or 84, or a fragment or variant thereof.

29. 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), the left and / or right inverted terminal repeat may comprise a nucleic acid sequence substantially as set forth in SEQ ID NO: 46 or 47, or a fragment or variant thereof.

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

31. 31. The genetic construct of claim 30, wherein the non-coding intron comprises a nucleic acid sequence substantially as set forth in SEQ ID NO: 48, 49 or 50, or a fragment or variant thereof.

32. 2. The genetic construct of claim 1, wherein the genetic construct comprises a signal peptide coding sequence, which may comprise a nucleotide sequence substantially as set forth in any one of SEQ ID NOs: 52 or 54, or a fragment or variant thereof, and / or the signal peptide coding sequence may encode an amino acid sequence substantially as set forth in SEQ ID NOs: 51 or 53, or a fragment or variant thereof.

33. 2. The genetic construct of claim 1 , encoding an amino acid sequence substantially as set out in SEQ ID NO: 55, 57, 59, 61, 63, 65, 67, 69, 71, 85, 87 or 89, or a fragment or variant thereof, and / or comprising a nucleotide sequence substantially as set out in SEQ ID NO: 56, 58, 60, 62, 64, 66, 68, 70, 72, 86, 88 or 90, or a fragment or variant thereof.

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

35. 35. The recombinant vector of claim 34, which is a recombinant AAV (rAAV) vector.

36. The recombinant vector of claim 35, wherein the rAAV is 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 or AAV-11.

37. The recombinant vector of claim 36, wherein the rAAV is rAAV serotype-2.

38. 35. The recombinant vector of claim 34, comprising a nucleotide sequence substantially as set forth in SEQ ID NO:67, or a fragment or variant thereof.

39. 35. A genetic construct as claimed in claim 1 or a recombinant vector as claimed in claim 34 for use as a medicine or in therapy.

40. A genetic construct as described in claim 1 or a recombinant vector as described in claim 34 for use in the treatment, prevention or amelioration of retinal disorders or for reducing complement activation and retinal cell damage and loss.

41. 41. The genetic construct or recombinant vector of claim 40, wherein the retinal disorder to be treated is dry age-related macular degeneration, geographic atrophy, and / or any pathophysiological condition involving retinal damage due to complement activation.

42. 42. The genetic construct or recombinant vector of claim 41, wherein the retinal disorder is dry age-related macular degeneration.

43. 42. The genetic construct or recombinant vector for use according to claim 41, wherein the retinal disorder is geographic atrophy.

44. 41. The genetic construct or recombinant vector of claim 40 for use in reducing complement activation and retinal cell damage and loss associated with any one of the following conditions: retinitis pigmentosa, Stargardt's disease, diabetic macular degeneration, age-related macular degeneration and / or Leber's congenital amaurosis.

45. A pharmaceutical composition comprising the genetic construct of claim 1 or the recombinant vector of claim 34 and a pharma- ceutically acceptable vehicle.

46. A method for preparing a pharmaceutical composition according to claim 45, comprising the step of contacting the genetic construct according to claim 1 or the recombinant vector according to claim 34 with a pharma- ceutically acceptable medium.

47. 41. The genetic construct or recombinant vector of claim 40 for use in reducing complement activation and retinal cell damage and loss associated with glaucoma.

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