CN117897493A

CN117897493A - Retinal disorders

Info

Publication number: CN117897493A
Application number: CN202280055858.0A
Authority: CN
Inventors: P·威多森; K·宾利
Original assignee: Icarovik Ltd
Current assignee: Icarovik Ltd
Priority date: 2021-06-18
Filing date: 2022-06-17
Publication date: 2024-04-16
Also published as: GB202204231D0

Abstract

The present invention relates to retinal disorders and to genetic constructs and recombinant vectors comprising such constructs, and their use in gene therapy methods for treating, preventing or ameliorating a variety of retinal disorders. The constructs and vectors are particularly useful for the treatment of Geographic Atrophy (GA) and dry age-related macular degeneration (dry AMD). The invention also relates to the use of the constructs and vectors for reducing complement activation and retinal cell damage and loss. The invention also relates to the pharmaceutical compositions themselves, and their use in the treatment, prevention or amelioration of retinal disorders and reduction of complement activation and retinal cell damage and loss.

Description

Retinal disorders

The present invention relates to retinal disorders and to genetic constructs and recombinant vectors comprising such constructs, and their use in gene therapy methods for treating, preventing or ameliorating a variety of retinal disorders. The constructs and vectors are particularly, but not exclusively, useful for the treatment of geographic atrophy (geographic atrophy, GA) and dry age-related macular degeneration (dry AMD). The invention also relates to the use of said constructs and vectors for reducing complement activation and retinal cell damage and loss. The invention also relates to the pharmaceutical compositions themselves, and their use in the treatment, prevention or amelioration of retinal disorders and reduction of complement activation and retinal cell damage and loss.

It is estimated that approximately 500 tens of thousands of people worldwide have Geographic Atrophy (GA) 1, and more than 100 tens of thousands of people in the United states alone [2], corresponding to an average of 1 in 29 people over 75 years of age. GA is a chronic progressive macular degeneration, which is part of advanced age-related macular degeneration (AMD). The disease is characterized by local atrophy of retinal tissue and choroidal capillaries, leading to central blind spots (central scotomas) and permanent vision loss. Age and family history of AMD are the most widely recognized GA risk factors [3]. Smoking, either in the past or in the present, also significantly increases the risk of individuals suffering from GA [3,4], and to date no studies have found any sex differences in the prevalence of GA disease [3,5]. This study of age-related eye disease found that thyroid hormone or antacid users had an increased risk of developing GA, and other studies showed that patients with coronary heart disease, lens clouding, or past cataract surgery had a greater risk of developing GA [6]. However, the pathogenesis of GA is still unclear.

The natural course of AMD begins in the early stages and is characterized by pigment translocation and the presence of drusen, a yellow deposit formed between the Retinal Pigment Epithelium (RPE) and Bruch's membrane [7,8]. Reticular pseudodrusen are particularly relevant to the development of GA [9, 10]. Advanced stages of AMD are characterized by choroidal neovascularization or GA. GA is considered a well-defined region of the posterior pole, accompanied by atrophy of the RPE, overlying photoreceptors and choroidal capillaries.

One of the major components of drusen is the accumulated autofluorescent membrane-bound lipofuscin [10]. Lipofuscin is typically present in the aged retina and is significantly elevated within the RPE cell layer of GA patients [11-13]. Lipofuscin is a major contributor to intrinsic fluorescence of the human fundus, and as such is more intense in GA patients on normally aged retinas [14,15]. Around age 90, lipofuscin particles account for about 20% of the cellular area of the macular RPE [12]. As the region of atrophy expands, visual function also declines [10,12,13,16]. Clinically, exudative and non-exudative AMD differ greatly, but neither is the advanced stage of AMD unique. Individuals with GA are at a higher risk of choroidal neovascularization and patients with exudative AMD are at a higher risk of developing atrophic areas.

Lipofuscin contains a complex mixture of pigments, but the main component has been shown to be A2E (N-retinylidene-N-retinylethanolamine; and iso-A2E, which is readily interconvertible), formed from two molecules of all-trans retinol or 11-cis retinol and ethanolamine found in the photoreceptor outer segment membrane (photoreceptor outer segment membrane) [17-22]. Lipofuscin levels have been shown to be directly related to the histopathological lesions of GA [12, 13, 22], suggesting that lipofuscin is one of the major factors of GA. Not only does A2E act as a detergent, thereby interfering with normal RPE lysosomal activity, A2E also interferes with cholesterol metabolism [23] and causes oxidative damage.

Several unsuccessful approaches have been attempted to reduce the level of A2E and drusen accumulation using drugs that slow the biochemical vision cycle. For example, retinol (Fenretinide, reVision Therapeutics) is an oral retinol binding protein (RBP 4) -transthyretin complex inhibitor that failed to slow GA progression in phase 2 clinical studies. Furthermore, the RPE65 isomerase inhibitor ACU-4426 (emixustat hydrochloride, acucela), while reducing A2E accumulation in the ABCA4/RDH8 double knockout mouse model [24, 25], failed to prevent GA progression in phase 2b/3 clinical studies [26].

The Complement System (CS) is part of the innate immune system, serving to protect against foreign pathogens such as microorganisms [27-29], and may play a role in reducing drusen. For example, complement factor H variant Y402H and ARMS2 are associated with increased risk of GA development [30,31], and drusen have been shown to contain multiple complement components in addition to lipofuscin [27-33]. This suggests that complement system mediated local inflammation may be an important factor in causing AMD.

CS consists of three biochemical pathways: classical Pathway (CP), lectin Pathway (LP) and Alternative Pathway (AP), each with a different trigger mechanism [27-29]. Although each pathway may be activated by a different component, these pathways all converge on a key protein component called complement factor 3 (C3).

The Classical Pathway (CP) is activated by antigen-antibody complexes, in particular by interactions 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 bind to form a C4b2a complex, a C3 convertase. The C4b2a complex further binds to C3b to form a C5 convertase.

Lectin Pathway (LP) is usually activated by binding to mannose residues on the surface of microorganisms, by binding to lectin or fibronectin (ficolin), similar to C1q, C1r and C1s, to form mannans binding lectin associated serine proteases (MASPs). Three MASP have been characterized to date; MASP-1, MASP-2 and MASP-3.MASP-2 has similar catalytic specificity to C1 and is able to cleave both C2 and C4.

The Alternative Pathway (AP) is closely related to the pathogenesis of AMD. In AP, complement factors B, P (properdin) and D participate in activation of C3 by forming a C3 convertase complex with C3B and activator B (Bb). C3 is then split into C3a and C3b. C3b can bind to C3a receptors on immune cells, causing inflammation, thereby allowing immune cells to permeate to further neutralize the pathogen. The C3b component further enhances the activity of the C3 convertase and also forms a complex with C4b and C2a (C4 b-C2a-C3 b) or Bb (C3 b-Bb-C3 b), both of which act as C5 convertases to generate C5a and C5b from the C5 protein. C5a can then bind to the C5b receptor on immune cells, similar to C3a, while C5b forms a complex with C6, C7, C8 and C9 (S5 b-9), forming a Membrane Attack Complex (MAC) or Terminal Complement Complex (TCC). TCC is able to destroy invading microorganisms by cell swelling and eventual lysis.

Complement Factor I (CFI) modulates AP activity [34,35], which is also known as a C3b/C4b deactivator, because it cleaves cell-bound or liquid phase C3b and C4b. Another regulatory factor is CD55 or Decay Accelerating Factor (DAF) [36-39], a membrane-bound protein that protects cells from complement-mediated lysis. The main function of CD55 is to inactivate the C3 convertase by cleavage of the C3 convertase into its constituent proteins [36-39]. Another regulatory protein, termed complement factor H-related protein-1 (CFHR 1), is a splice variant of complement factor H [40-43], also involved in reducing complement activation. The main mediators are complement factor H and its splice product FHL-1, which modulate complement activity on the fluid phase and surface by decay acceleration and cofactor activity. Factor H and FHL-1 target and attenuate the C3 convertase consisting of C3B and factor B. Another factor that reduces complement activation is known as membrane cofactor protein or CD46[44,46], which is membrane bound. Once bound to C3B, factor H and CFHL-1 occupy the factor B binding site in C3B, accelerating the decay and preventing the formation of new C3 convertases. In addition, factors H, CFHL-1 and CD46 act as cofactors for proteinase factor I, cleaving C3b to iC3b, and in the case of CD46, as cofactors for C4b. CFHR-1 does not mediate decay acceleration or factor I cofactor activity. As with factor H, CFHR-1 binds to C3b, recognizes its surface by binding to glycosaminoglycans, and inhibits the formation of C5 convertase and terminal complement complex [43].

Although there is evidence that the complement system plays a key role in mediating GA, assays of agents that modulate the complement system show different results. For example, monoclonal antibodies eculizumab (Aexion) and lanpalizumab (campalizumab) [44] (Genentech/Roche) have not been successful in slowing GA progression clinically. Furthermore, gyroscope Therapeutics incorporates complement factor I gene therapy into the clinical trial of GA (GT 005) [47]. However, while enhancing CFI in low or no CFI patients theoretically reduces complement activation, all three pathways are affected by this treatment, including CP and LP, which are responsible for antimicrobial activity. Thus, a systematic decrease in all complement system pathways may predispose the eye to bacterial infection, or may require longer periods of time for the infected eye to recover. In addition, early clinical trials of anti-complement monotherapy (e.g., avacincaptad pegol and complement factor I gene therapy) have witnessed that some GA patients develop wet AMD, which requires that they need to be injected monthly with anti-VEGF ranibizumab (ranibizumab) or aflibercept (aflibercept) to prevent rapid loss of vision.

PEDF is a 50kDa protein released in large amounts from the apical side of RPE cells [48,49], and shows retinal neuroprotective properties by interaction with PEDF receptors [50,51]. PEDF has multiple functions in the retina, namely anti-angiogenesis, and exhibits anti-neoplastic and neurotrophic properties [50-56], in part by inhibiting endothelial cell migration [54]. The presence of high density PEDF receptors in the RPE cell layer [57] suggests that it has an autocrine and paracrine neuroprotective effect in the retina. Furthermore, PEDF has been demonstrated to protect human RPE cells from oxidative stress (a phenomenon that occurs in GA patient retinas due to lipofuscin accumulation) by up-regulating the coupled protein-2 (UCP-2) [55, 58]. Thus, the inventors speculate that a decrease in retinal PEDF concentration in GA patients will make retinal pigment epithelium and photoreceptors extremely vulnerable to oxidative stress and complement attack.

Thus, there is a strong need for an improved therapy for the treatment of retinal disorders such as Geographic Atrophy (GA) and dry age-related macular degeneration (dry AMD) that activates PEDF receptors and neutralizes or reduces the complement system.

With significant creative effort, the inventors carefully designed and constructed a novel gene construct that encodes PEDF receptor agonists and anticomplementary proteins under the control of a single promoter, i.e., it is bicistronic. The promoters of this construct can be used to ensure maximum expression of both PEDF receptor agonists and anticomplementary proteins to reduce retinal cell damage and loss through two separate pathophysiological pathways, namely, increased activity of PEDF receptor and decreased complement activation.

Thus, according to a first aspect of the present invention there is provided a genetic construct comprising a promoter operably linked to a first coding sequence encoding a PEDF receptor agonist and a second coding sequence encoding an anti-complement protein.

PEDF receptor agonists restore PEDF concentration, thereby reducing inflammation, lowering the levels of toxic lipofuscin components, and protecting RPE and photoreceptor cells. In addition, anti-complement proteins are capable of neutralizing or attenuating the alternative complement pathway, thereby preventing further loss of RPE cells. Advantageously, the genetic constructs of the invention target the AP pathway, which means that the classical and lectin pathways of the complement system are preserved, so that the antimicrobial defense system, which can facilitate the destruction of invading pathogens, is maintained. Importantly, binding of anticomplements to PEDF receptor activation will limit release of retinal VEGF that would otherwise potentially convert dry AMD to a much more aggressive wet AMD pathophysiology. In addition, the promoters of the constructs can be used to ensure maximum expression of both PEDF receptor agonists and anti-complement proteins, to restore PEDF concentration and reduce inflammation, and to neutralize the complement pathway to prevent further loss of RPE cells.

The inventors surprisingly demonstrate in the examples that it is possible to combine Open Reading Frames (ORFs) encoding PEDF receptor agonists and anticomplementary proteins in a single gene construct. This is particularly challenging given the large size of PEDF receptor agonists and anti-complement proteins. It was not predicted that it would be possible to co-express physiologically useful concentrations of these two large proteins from a single expression cassette under the control of a single promoter, and that the expression cassette could be accommodated by an AAV vector (e.g., a rAAV-2 vector). Advantageously, with the construct of the invention, there is no need to inject recombinant proteins as described in the prior art. Furthermore, in the prior art, there is still a need to regularly inject proteins into the eye, which is clearly disadvantageous, whereas the constructs of the present invention surprisingly require only a single administration to achieve a long-term therapeutic effect, thereby providing significant benefits to the patient.

Preferably, the genetic construct of the present invention comprises an expression cassette, one embodiment of which is shown in FIG. 2. In one embodiment, as shown in fig. 2, the construct comprises a promoter, a first nucleotide sequence encoding an agonist of the PEDF receptor, and a second nucleotide sequence encoding an anticomplementary protein. Thus, preferably, the gene construct and expression cassette may be referred to as a bicistronic.

As shown in fig. 2, the first and second coding sequences encoding PEDF receptor agonists and anti-complement proteins may be placed in any order from 5 'to 3'. For example, in one embodiment, the coding sequence for a PEDF receptor agonist is placed 5' to the coding sequence for an anti-complement protein, preferably with a spacer sequence (spacer sequence) between the two. Alternatively, in another embodiment, the coding sequence for the anti-complement protein may be placed 5' to the coding sequence for the PEDF receptor agonist, preferably with a spacer sequence in between.

The promoter in the genetic construct of the first aspect may be any nucleotide sequence capable of inducing the binding and transcription of the first and second coding sequences by an RNA polymerase.

Promoters may be constitutive or tissue specific.

A suitable constitutive promoter may be a cytomegalovirus promoter. One embodiment of the nucleotide sequence encoding the Cytomegalovirus (CMV) promoter (508 bp) is referred to herein as SEQ ID No. 1, as shown below:

CGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACC

CCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAG

GGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCAC

TTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTC

AATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATG

GGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCAT

GGTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGACT

CACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTT

TGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAACTCCGCCC

CATTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGC

AGAGCT

[SEQ ID No:1]

in another embodiment, the promoter is preferably a truncated form of the CMV promoter. One embodiment (60 bp) of the nucleotide sequence encoding the truncated form of the CMV promoter is referred to herein as SEQ ID No. 2, as shown below:

AGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTGGTTTAGTG

AACCGTCAGAT

[SEQ ID No:2]

In another embodiment, the promoter is a fusion of a Cytomegalovirus (CMV) early enhancer element and a first intron of the chicken β -actin gene (CAG). One embodiment (583 bp) of the nucleotide sequence encoding the cytomegalovirus early enhancer element and the first intron of the chicken beta-actin gene is referred to herein as SEQ ID No:3, as shown below: CGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGACTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTCGAGGTGAGCCCCACGTTCTGCTTCACTCTCCCCATCTCCCCCCCCTCCCCACCCCCAATTTTGTATTTATTTATTTTTTAATTATTTTGTGCAGCGATGGGGGCGGGGGGGGGGGGGGGGCGCGCGCCAGGCGGGGCGGGGCGGGGCGAGGGGCGGGGCGGGGCGAGGCGGAGAGGTGCGGCGGCAGCCAATCAGAGCGGCGCGCTCCGAAAGTTTCCTTTTATGGCGAGGCGGCGGCGGCGGCGGCCCTATAAAAAGCGAAGCGCGCGGCGGGCG

[SEQ ID No:3]

A suitable tissue-specific promoter may be the vitelline-like macular dystrophin-2 (VMD 2) promoter (sometimes referred to as betatropin-1). Advantageously, the promoter limits transgene expression in RPE cells. One embodiment of the nucleotide sequence encoding the VMD2 promoter (2039 bp) is referred to herein as SEQ ID No. 4, as follows:

AATTCTGTCATTTTACTAGGGTGATGAAATTCCCAAGCAACACCATCCTTTTCAGATAAGGGCACTGAGGCTGAGAGAGGAGCTGAAACCTACCCGGCGTCACCACACACAGGTGGCAAGGCTGGGACCAGAAACCAGGACTGTTGACTGCAGCCCGGTATTCATTCTTTCCATAGCCCACAGGGCTGTCAAAGACCCCAGGGCCTAGTCAGAGGCTCCTCCTTCCTGGAGAGTTCCTGGCACAGAAGTTGAAGCTCAGCACAGCCCCCTAACCCCCAACTCTCTCTGCAAGGCC

TCAGGGGTCAGAACACTGGTGGAGCAGATCCTTTAGCCTCTGGATTTTA

GGGCCATGGTAGAGGGGGTGTTGCCCTAAATTCCAGCCCTGGTCTCAGC

CCAACACCCTCCAAGAAGAAATTAGAGGGGCCATGGCCAGGCTGTGCTA

GCCGTTGCTTCTGAGCAGATTACAAGAAGGGACTAAGACAAGGACTCCT

TTGTGGAGGTCCTGGCTTAGGGAGTCAAGTGACGGCGGCTCAGCACTCA

CGTGGGCAGTGCCAGCCTCTAAGAGTGGGCAGGGGCACTGGCCACAGA

GTCCCAGGGAGTCCCACCAGCCTAGTCGCCAGACCTTCTGTGGGATCAT

CGGACCCACCTGGAACCCCACCTGTGAGTACAAGGTGCCCCAGGTGGA

CTGGGCTGGGGCTTTGAGGCCTTCAGGGTTGGATGGCCATCTTGCGTATT

TGTGTGGGATATGCACACACAGGCAGCACATGCGCAGGTGTGTGGGCAC

CTGTGTGTCTGTGCAAATGCCCTGAGGTGGGAATGAGCTTGGTGTGCATC

AGGCACAGCCAGCCAGTGTGGCTGCAGCAAAACACACAGGGAAAGAAT

GGAGGGGGCATCAATCACTGCTTCAGTAAATTTTTATTGAGCGCCTTCTA

CGAGAACACAAGAGGAGCTTCCATTCTGAGGAGGAAACAGGCAGGAAA

CAGGCAGATATCCTGTATAATTTCAAGTAGTGATAAGTGCTCTCTAGAAAT

ATCAAGCAAGGTGAGGAGACACAGAGCACCGGTGGCAGTGGGGCTCTA

TTTCCAGGTTGGATGGTTGGGAACATCCTTTCTAAAGGGAACCTGGAGT

GGGAAGGAACCATGCAGGTATCTCAGGAAGAGCTTCCTCCAGGCAGGA

AGATCAGCAGGTGGAAAGGCCCTGGAGCCACCATTCAGTAAACATCATT

TGAGCATCTCTACCAGCTAGGTTCCATTATGGGAATGGGAATATGGTGGT

GGACAGGGCTGCCTGGTCCCTTCCATACTTCTCACACTAGGGTGGTTGA

GAGAGCTTGGGAGCTAACGAACAAGATGGGCTGAGAACACTGCCTAGC

CCAGAGGACCTGAGCTTAGTGTGTAGACATTGCTGCTGTTACTGCCTTTG

TCATTGTATTATTTATTTATTTATTTATTTATTTTTAGACAGAGTTTTGCTCTT

CTTACCCAGGCTGGAGTGCAATGGCGTGATCTCAGCTCACTGCAACCTC

CACCTCCTGGGTTCAAGCGATTCTCCTGCCTCAGCCTCCTGAGTAGCTGG

GATTACAGGCACCCGCACCACGCCTGGATAATTTTTTTGTATTTTTAGTAG

AGACAGGGTTTCACCATGTTGGCCAGGCTGGTCTCGAACTCCTGACCTT

AGGTGATCCACCTGCCTCGACTTCCCAAAGTGCTGGGATTATAGGCATGA

GCCACTGCGCCCAGTGATTATAGAAAGTTAAAGGCACATGGCAATGCAC

ACGCCTATCTACGTCTTCCCTGCCAAAGCAAAGGGCAGCCTCTGGGCTC

ACTTTCTTGCGTTTCTACTTCCAAAAGGCAGTCAGAACTGGCAGGGCCT

TGGAGACCACTTCATCCACCTCCTAGGGTCCCTATGGGAGAGTTGAGGT

CCAGAGCAGGGAAGGGTCCTGACAGGCTCTGACCAGGGCCTCTGATCC

CTACAAACCCCCAATCGGTGTCCCTCTCTACCAGGACCCAAGCCCACCT

GCTGCAGCCCACTGCCTGGCC

[SEQ ID No:4]

In yet a further preferred embodiment, the promoter is a truncated form of the VMD2 promoter. One embodiment of the nucleotide sequence encoding the truncated version of the VMD2 promoter (623 bp) is referred to herein as SEQ ID No. 5, as shown below:

AATTCTGTCATTTTACTAGGGTGATGAAATTCCCAAGCAACACCATCCTTT

TCAGATAAGGGCACTGAGGCTGAGAGAGGAGCTGAAACCTACCCGGCG

TCACCACACACAGGTGGCAAGGCTGGGACCAGAAACCAGGACTGTTGA

CTGCAGCCCGGTATTCATTCTTTCCATAGCCCACAGGGCTGTCAAAGACC

CCAGGGCCTAGTCAGAGGCTCCTCCTTCCTGGAGAGTTCCTGGCACAGA

AGTTGAAGCTCAGCACAGCCCCCTAACCCCCAACTCTCTCTGCAAGGCC

TCAGGGGTCAGAACACTGGTGGAGCAGATCCTTTAGCCTCTGGATTTTA

GGGCCATGGTAGAGGGGGTGTTGCCCTAAATTCCAGCCCTGGTCTCAGC

CCAACACCCTCCAAGAAGAAATTAGAGGGGCCATGGCCAGGCTGTGCTA

GCCGTTGCTTCTGAGCAGATTACAAGAAGGGACTAAGACAAGGACTCCT

TTGTGGAGGTCCTGGCTTAGGGAGTCAAGTGACGGCGGCTCAGCACTCA

CGTGGGCAGTGCCAGCCTCTAAGAGTGGGCAGGGGCACTGGCCACAGA

GTCCCAGGGAGTCCCACCAGCCTAGTCGCCAGACC

[SEQ ID No:5]

in yet another preferred embodiment, the nucleotide sequence (462 bp) encoding the truncated form of the VMD2 promoter is referred to herein as SEQ ID No. 6, as set forth below: TCATTCTTTCCATAGCCCACAGGGCTGTCAAAGACCCCAGGGCCTAGTCAGAGGCTCCTCCTTCCTGGAGAGTTCCTGGCACAGAAGTTGAAGCTCAGCACAGCCCCCTAACCCCCAACTCTCTCTGCAAGGCCTCAGGGGTCAGAACACTGGTGGAGCAGATCCTTTAGCCTCTGGATTTTAGGGCCATGGTAGAGGGGGTGTTGCCCTAAATTCCAGCCCTGGTCTCAGCCCAACACCCTCCAAGAAGAAATTAGAGGGGCCATGGCCAGGCTGTGCTAGCCGTTGCTTCTGAGCAGATTACAAGAAGGGACTAAGACAAGGACTCCTTTGTGGAGGTCCTGGCTTAGGGAGTCAAGTGACGGCGGCTCAGCACTCACGTGGGCAGTGCCAGCCTCTAAGAGTGGGCAGGGGCACTGGCCACAGAGTCCCAGGGAGTCCCACCAGCCTAGTCGCCAGACC

[SEQ ID No:6]

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

GGGTAGGGGAGGCGCTTTTCCCAAGGCAGTCTGGAGCATGCGCTTTAGCAGCCCCGCTGGGCACTTGGCGCTACACAAGTGGCCTCTGGCCTCGCACACATTCCACATCCACCGGTAGGCGCCAACCGGCTCCGTTCTTTGGTGGCCCCTTCGCGCCACCTTCTACTCCTCCCCTAGTCAGGAAGTTCCCCCCCGCCCCGCAGCTCGCGTCGTGCAGGACGTGACAAATGGAAGTAGCACGTCTCACTAGTCTCGTGCAGATGGACAGCACCGCTGAGCAATGGAAGCGGGTAGGCCTTTGGGGCAGCGGCCAATAGCAGCTTTGCTCCTTCGCTTTCTGGGCTCAGAGGCTGGGAAGGGGTGGGTCCGGGGGCGGGCTCAGGGGCGGGCTCAGGGGCGGGGCGGGCGCCCGAAGGTCCTCCGGAGGCCCGGCATTCTGCACGCTTCAAAAGCGCACGTCTGCCGCGCTGTTCTCCTCTTCCTCATCTCCGGGCCTTTCG

[SEQ ID No:7]

In a further embodiment, the promoter is EF1a derived from the human EEF1A1 gene expressing the α subunit of eukaryotic elongation factor 1. One embodiment of the nucleotide sequence encoding the EF 1. Alpha. Promoter (1182 bp) is referred to herein as SEQ ID No. 8, as set forth below:

GCTCCGGTGCCCGTCAGTGGGCAGAGCGCACATCGCCCACAGTCCCCGAGAAGTTGGGGGGAGGGGTCGGCAATTGAACCGGTGCCTAGAGAAGGTGGCGCGGGGTAAACTGGGAAAGTGATGTCGTGTACTGGCTCCGCCTTTTTCCCGAGGGTGGGGGAGAACCGTATATAAGTGCAGTAGTCGCCGTGAACGTTCTTTTTCGCAACGGGTTTGCCGCCAGAACACAGGTAAGTGCCGTGTGTGGTTCCCGCGGGCCTGGCCTCTTTACGGGTTATGGCCCTTGCGTGCCTTGAATTACTTCCACGCCCCTGGCTGCAGTACGTGATTCTTGATCCCGAGCTTCGGGTTGGAAGTGGGTGGGAGAGTTCGAGGCCTTGCGCTTAAGGAGCCCCTTCGCCTCGTGCTTGAGTTGAGGCCTGGCTTGGGCGCTGGGGCCGCCGCGTGCGAATCTGGTGGCACCTTCGCGCCTGTCTCGCTGCTTTCGATAAGTCTCTAGCCATTTAAAATTTTTGATGACCTGCTGCGACGCTTTTTTTCTGGCAAGATAGTCTTGTAAATGCGGGCCAAGATCTGCACACTGGTATTTCGGTTTTTGGGGCCGCGGGCGGCGACGGGGCCCGTGCGTCCCAGCGCACATGTTCGGCGAGGCGGGGCCTGCGAGCGCGGCCACCGAGAATCGGACGGGGGTAGTCTCAAGCTGGCCGGCCTGCTCTGGTGCCTGGCCTCGCGCCGCCGTGTATCGCCCCGCCCTGGGCGGCAAGGCTGGCCCGGTCGGCACCAGTTGCGTGAGCGGAAAGATGGCCGCTTCCCGGCCCTGCTGCAGGGAGCTCAAAATGGAGGACGCGGCGCTCGGGAGAGCGGGCGGGTGAGTCACCCACACAAAGGAAAAGGGCCTTTCCGTCCTCAGCCGTCGCTTCATGTGACTCCACGGAGTACCGGGCGCCGTCCAGGCACCTCGATTAGTTCTCGAGCTTTTGGAGTACGTCGTCTTTAGGTTGGGGGGAGGGGTTTTATGCGATGGAGTTTCCCCACACTGAGTGGGTGGAGACTGAAGTTAGGCCAGCTTGGCACTTGATGTAATTCTCCTTGGAATTTGCCCTTTTTGAGTTTGGATCTTGGTTCATTCTCAAGCCTCAGACAGTGGTTCAAAGTTTTTTTCTTCCATTTCAGGTGTCGTGA

[SEQ ID No:8]

in a further embodiment, the promoter is EF1a without a large intron. One embodiment (230 bp) of the nucleotide sequence encoding the EF 1. Alpha. Promoter is referred to herein as SEQ ID No. 9, as follows:

GCTCCGGTGCCCGTCAGTGGGCAGAGCGCACATCGCCCACAGTCCCCGA

GAAGTTGGGGGGAGGGGTCGGCAATTGAACCGGTGCCTAGAGAAGGTG

GCGCGGGGTAAACTGGGAAAGTGATGTCGTGTACTGGCTCCGCCTTTTT

CCCGAGGGTGGGGGAGAACCGTATATAAGTGCAGTAGTCGCCGTGAACG

TTCTTTTTCGCAACGGGTTTGCCGCCAGAACACAG

[SEQ ID No:9]

thus, preferably, the promoter comprises a 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.

To ensure survival of damaged and fragile RPE cells in GA patients, the inventors incorporated PEDF receptor agonists into the gene constructs of the invention. The inventors carefully consider the sequence of the PEDF receptor agonist and create several preferred embodiments of the protein that can be encoded by the first coding sequence in the genetic construct of the first aspect.

In one embodiment, the first coding sequence comprises a nucleotide sequence encoding 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 shown below in MQALVLLLCIGALLGHSSCQNPASPPEEGSPDPDSTGALVEEEDPFFKVPVNKLAAAVSNFGYDLYRVRSSTSPTTNVLLSPLSVATALSALSLGAEQRTESIIHRALYYDLISSPDIHGTYKELLDTVTAPQKNLKSASRIVFEKKLRIKSSFVAPLEKSYGTRPRVLTGNPRLDLQEINNWVQAQMKGKLARSTKEIPDEISILLLGVAHFKGQWVTKFDSRKTSLEDFYLDEERTVRVPMMSDPKAVLRYGLDSDLSCKIAQLPLTGSMSIIFFLPLKVTQNLTLIEESLTSEFIHDIDRELKTVQAVLTVPKLKLSYEGEVTKSLQEMKLQSLFDSPDFSKITGKPIKLTQVEHRAGFEWNEDGAGTTPSPGLQPAHLTFPLDYHLNQPFIFVLRDTDTGALLFIGKILDPRGP

[SEQ ID No:10]

Preferably, in this embodiment, the first coding sequence comprises the nucleotide sequence (1254 bp) referred to herein as SEQ ID No:11, as set forth below:

ATGCAGGCCCTGGTGCTACTCCTCTGCATTGGAGCCCTCCTCGGGCACAG

CAGCTGCCAGAACCCTGCCAGCCCCCCGGAGGAGGGCTCCCCAGACCC

CGACAGCACAGGGGCGCTGGTGGAGGAGGAGGATCCTTTCTTCAAAGT

CCCCGTGAACAAGCTGGCAGCGGCTGTCTCCAACTTCGGCTATGACCTG

TACCGGGTGCGATCCAGCACGAGCCCCACGACCAACGTGCTCCTGTCTC

CTCTCAGTGTGGCCACGGCCCTCTCGGCCCTCTCGCTGGGAGCGGAGCA

GCGAACAGAATCCATCATTCACCGGGCTCTCTACTATGACTTGATCAGCA

GCCCAGACATCCATGGTACCTATAAGGAGCTCCTTGACACGGTCACTGCC

CCCCAGAAGAACCTCAAGAGTGCCTCCCGGATCGTCTTTGAGAAGAAGC

TGCGCATAAAATCCAGCTTTGTGGCACCTCTGGAAAAGTCATATGGGACC

AGGCCCAGAGTCCTGACGGGCAACCCTCGCTTGGACCTGCAAGAGATC

AACAACTGGGTGCAGGCGCAGATGAAAGGGAAGCTCGCCAGGTCCACA

AAGGAAATTCCCGATGAGATCAGCATTCTCCTTCTCGGTGTGGCGCACTT

CAAGGGGCAGTGGGTAACAAAGTTTGACTCCAGAAAGACTTCCCTCGA

GGATTTCTACTTGGATGAAGAGAGGACCGTGAGGGTCCCCATGATGTCG

GACCCTAAGGCTGTTTTACGCTATGGCTTGGATTCAGATCTCAGCTGCAA

GATTGCCCAGCTGCCCTTGACCGGAAGCATGAGTATCATCTTCTTCCTGC

CCCTGAAAGTGACCCAGAATTTGACCTTGATAGAGGAGAGCCTCACCTC

CGAGTTCATTCATGACATAGACCGAGAACTGAAGACCGTGCAGGCGGTC

CTCACTGTCCCCAAGCTGAAGCTGAGTTATGAAGGCGAAGTCACCAAGT

CCCTGCAGGAGATGAAGCTGCAATCCTTGTTTGATTCACCAGACTTTAGC

AAGATCACAGGCAAACCCATCAAGCTGACTCAGGTGGAACACCGGGCT

GGCTTTGAGTGGAACGAGGATGGGGCGGGAACCACCCCCAGCCCAGGG

CTGCAGCCTGCCCACCTCACCTTCCCGCTGGACTATCACCTTAACCAGCC

TTTCATCTTCGTACTGAGGGACACAGACACAGGGGCCCTTCTCTTCATTG

GCAAGATTCTGGACCCCAGGGGCCCC

[SEQ ID No:11]

codon analysis of the endogenous PEDF 1254 nucleotide sequence (SEQ ID No: 11) using online rare codon analysis tools (www.jcat.de and https:// www.genscript.com/tools/cart-codon-analysis) showed that this sequence showed poor Codon Adaptation Index (CAI) (JCAT CAI=0.40 and Genscript CAI=0.78). High expression transgenes should ideally exhibit a CAI of between 0.80 and 1.00 to achieve efficient gene expression. Thus, rare codons contained in endogenous PEDF transgenes were identified and replaced with codons more commonly used in the expression host (i.e., human eye) to generate a codon optimized 1254 sequence (JCAT cai= 0.96;Genscript CAI =1.00).

Thus, in another embodiment, the first coding sequence comprises the nucleotide sequence (1254 bp) referred to herein as SEQ ID No. 12, or a fragment or variant thereof, as set forth below: ATGCAGGCCCTGGTGCTGCTGCTGTGCATCGGCGCCCTGCTGGGCCACAGCAGCTGCCAGAACCCCGCCAGCCCCCCCGAGGAGGGCAGCCCCGACCCCGACAGCACCGGCGCCCTGGTGGAGGAGGAGGACCCCTTCTTCAAGGTGCCCGTGAACAAGCTGGCCGCCGCCGTGAGCAACTTCGGCTACGACCTGTACCGCGTGCGCAGCAGCACCAGCCCCACCACCAACGTGCTGCTGAGCCCCCTGAGCGTGGCCACCGCCCTGAGCGCCCTGAGCCTGGGCGCCGAGCAGCGCACCGAGAGCATCATCCACCGCGCCCTGTACTACGACCTGATCAGCAGCCCCGACATCCACGGCACCTACAAGGAGCTGCTGGACACCGTGACCGCCCCCCAGAAGAACCTGAAGAGCGCCAGCCGCATCGTGTTCGAGAAGAAGCTGCGCATCAAGAGCAGCTTCGTGGCCCCCCTGGAGAAGAGCTACGGCACCCGCCCCCGCGTGCTGACCGGCAACCCCCGCCTGGACCTGCAGGAGATCAACAACTGGGTGCAGGCCCAGATGAAGGGCAAGCTGGCCCGCAGCACCAAGGAGATCCCCGACGAGATCAGCATCCTGCTGCTGGGCGTGGCCCACTTCAAGGGCCAGTGGGTGACCAAGTTCGACAGCCGCAAGACCAGCCTGGAGGACTTCTACCTGGACGAGGAGCGCACCGTGCGCGTGCCCATGATGAGCGACCCCAAGGCCGTGCTGCGCTACGGCCTGGACAGCGACCTGAGCTGCAAGATCGCCCAGCTGCCCCTGACCGGCAGCATGAGCATCATCTTCTTCCTGCCCCTGAAGGTGACCCAGAACCTGACCCTGATCGAGGAGAGCCTGACCAGCGAGTTCATCCACGACATCGACCGCGAGCTGAAGACCGTGCAGGCCGTGCTGACCGTGCCCAAGCTGAAGCTGAGCTACGAGGGCGAGGTGACCAAGAGCCTGCAGGAGATGAAGCTGCAGAGCCTGTTCGACAGCCCCGACTTCAGCAAGATCACCGGCAAGCCCATCAAGCTGACCCAGGTGGAGCACCGCGCCGGCTTCGAGTGGAACGAGGACGGCGCCGGCACCACCCCCAGCCCCGGCCTGCAGCCCGCCCACCTGACCTTCCCCCTGGACTACCACCTGAACCAGCCCTTCATCTTCGTGCTGCGCGACACCGACACCGGCGCCCTGCTGTTCATCGGCAAGATCCTGGACCCCCGCGGCCCC

[SEQ ID No:12]

In yet another preferred embodiment, the first coding sequence comprises a nucleotide sequence (1254 bp) comprising a modified signal peptide sequence, referred to herein as SEQ ID No. 13, or a fragment or variant thereof, as set forth below:

ATGCGCCGCCTGGTGCTGCTGCTGTGCATCGGCGCCCTGCTGGGCCACG

TGTTCTGCCAGAACCCCGCCAGCCCCCCCGAGGAGGGCAGCCCCGACC

CCGACAGCACCGGCGCCCTGGTGGAGGAGGAGGACCCCTTCTTCAAGG

TGCCCGTGAACAAGCTGGCCGCCGCCGTGAGCAACTTCGGCTACGACCT

GTACCGCGTGCGCAGCAGCACCAGCCCCACCACCAACGTGCTGCTGAGC

CCCCTGAGCGTGGCCACCGCCCTGAGCGCCCTGAGCCTGGGCGCCGAG

CAGCGCACCGAGAGCATCATCCACCGCGCCCTGTACTACGACCTGATCA

GCAGCCCCGACATCCACGGCACCTACAAGGAGCTGCTGGACACCGTGAC

CGCCCCCCAGAAGAACCTGAAGAGCGCCAGCCGCATCGTGTTCGAGAA

GAAGCTGCGCATCAAGAGCAGCTTCGTGGCCCCCCTGGAGAAGAGCTAC

GGCACCCGCCCCCGCGTGCTGACCGGCAACCCCCGCCTGGACCTGCAG

GAGATCAACAACTGGGTGCAGGCCCAGATGAAGGGCAAGCTGGCCCGC

AGCACCAAGGAGATCCCCGACGAGATCAGCATCCTGCTGCTGGGCGTGG

CCCACTTCAAGGGCCAGTGGGTGACCAAGTTCGACAGCCGCAAGACCA

GCCTGGAGGACTTCTACCTGGACGAGGAGCGCACCGTGCGCGTGCCCAT

GATGAGCGACCCCAAGGCCGTGCTGCGCTACGGCCTGGACAGCGACCTG

AGCTGCAAGATCGCCCAGCTGCCCCTGACCGGCAGCATGAGCATCATCT

TCTTCCTGCCCCTGAAGGTGACCCAGAACCTGACCCTGATCGAGGAGAG

CCTGACCAGCGAGTTCATCCACGACATCGACCGCGAGCTGAAGACCGTG

CAGGCCGTGCTGACCGTGCCCAAGCTGAAGCTGAGCTACGAGGGCGAG

GTGACCAAGAGCCTGCAGGAGATGAAGCTGCAGAGCCTGTTCGACAGC

CCCGACTTCAGCAAGATCACCGGCAAGCCCATCAAGCTGACCCAGGTGG

AGCACCGCGCCGGCTTCGAGTGGAACGAGGACGGCGCCGGCACCACCC

CCAGCCCCGGCCTGCAGCCCGCCCACCTGACCTTCCCCCTGGACTACCA

CCTGAACCAGCCCTTCATCTTCGTGCTGCGCGACACCGACACCGGCGCC

CTGCTGTTCATCGGCAAGATCCTGGACCCCCGCGGCCCC

[SEQ ID No:13]

thus, in a preferred embodiment, the first coding sequence comprises a sequence substantially as set forth in SEQ ID No: 11. 12 or 13 or a fragment or variant thereof. Preferably, the PEDF receptor agonist comprises a sequence substantially as set forth in SEQ ID No:10 or a fragment or variant thereof.

It is understood that the second coding sequence encodes an anticomplement 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 anticomplementary protein minimally affects the Classical Pathway (CP) and/or Lectin Pathway (LP) of the complement system. Preferably, the anti-complement proteins do not target the Classical Pathway (CP) and/or Lectin Pathway (LP) of the complement system.

Preferably, the anti-complement proteins are capable of neutralizing complement factors C3b, bb and/or C5. Thus, in this embodiment, the anti-complement protein is an anti-C3 b, anti-Bb and/or anti-C5 antibody or antigen-binding fragment thereof.

The antigen binding fragment thereof may comprise or consist of any fragment selected from the group consisting of: VH, VL, fd, fv, fab, fab ', scFv, F (ab') ₂ And an Fc fragment. The antigen binding fragment may comprise Complementarity Determining Regions (CDRs) that bind to C3b and/or Bb epitopes.

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-C3 b single-chain variable fragment, an anti-Bb single-chain variable fragment, or an anti-C5 single-chain variable fragment.

Alternatively, in another preferred embodiment, the anti-complement protein is a soluble form of normal membrane bound CD55 (sCD 55) (sometimes also referred to as decay accelerating factor; DAF). Preferably, the anti-complement protein is non-membrane-attached CD55 (sCD 55). Soluble CD55 (DAF) destabilizes the complement protein complex, thereby reducing the activity of this biochemical pathway.

In another preferred embodiment, the anti-complement protein is complement factor H-related protein-1 (CFHR 1). Preferably, CFHR1 reduces the cascade of complement system activity.

In another preferred embodiment, the anti-complement protein is a soluble form of normal membrane bound CD46 (sCD 46). Preferably, in this embodiment, the anti-complement protein is soluble (non-membrane bound) human complement regulatory protein CD46 (sCD 46).

In another preferred embodiment, the anti-complement protein is complement factor H-like protein 1 (CFHL 1). CFHL1 is a splice variant of factor H that includes a regulatory domain and inhibits complement activation at the level of central complement component C3 and higher.

In a preferred embodiment, the amino acid sequence of the anti-C3 b single chain variable fragment is referred to herein as SEQ ID No. 14, or a fragment or variant thereof, as set forth below: DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYATLPTFEQGTKVEIKRGGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFSFTSSSVSPGKGLEWVGLIYPYNGFNYYADSVKGRFTISADTSLQMNSLRAEDTAVYYCARNALYGSGGYYAMDYWGQGTLVTVSS

[SEQ ID No:14]

In a preferred embodiment, the nucleic acid sequence encoding the anti-C3 b single-stranded variable fragment (726 bp) is referred to herein as SEQ ID No. 15, or a fragment or variant thereof, as set forth below: GACATCCAGATGACCCAGAGCCCCAGCAGCCTGAGCGCCAGCGTGGGCGACCGCGTGACCATCACCTGCCGCGCCAGCCAGGACGTAAGCACCGCCGTGGCCTGGTACCAGCAGAAGCCCGGCAAGGCCCCCAAGCTGCTGATCTACAGCGCCAGCTTCCTGTACAGCGGCGTGCCCAGCCGCTTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGACCATCAGCAGCCTGCAGCCCGAGGACTTCGCCACCTACTACTGCCAGCAGAGCTACGCCACCCTGCCCACCTTCGAGCAGGGCACCAAGGTGGAGATCAAGCGCGGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGAGGTGCAGCTGGTGGAGAGCGGCGGCGGCCTGGTGCAGCCCGGCGGCAGCCTGCGCCTGAGCTGCGCCGCCAGCGGCTTCAGCTTCACCAGCAGCAGCGTGAGCCCCGGCAAGGGCCTGGAGTGGGTGGGCCTGATCTACCCCTACAACGGCTTCAACTACTACGCCGACAGCGTGAAGGGCCGCTTCACCATCAGCGCCGACACCAGCCTGCAGATGAACAGCCTGCGCGCCGAGGACACCGCCGTGTACTACTGCGCCCGCAACGCCCTGTACGGCAGCGGCGGCTACTACGCCATGGACTACTGGGGCCAGGGCACCCTGGTGACCGTGAGCAGC

[SEQ ID No:15]

In another embodiment, the amino acid sequence of the anti-Bb single-chain variable fragment is referred to herein as SEQ ID No. 16, or a fragment or variant thereof, as set forth below:

DVQITQSPSYLAASPGETITINCRASKSISKYLAWYQDKPGKTNKLLIYSGSTLQSGIPSRFSGSGSGTDFTLTISSLEPEDFAMYYCQQHDEYPWTFGGGTKLEIKRGGGGGSGGGGSGGGGSGGGGSQVQLQQSGAELAKPGASVRMSCKASGYTFTNYWIHWVKQRPGQGLEWIGYINPNTGYNDYNQKFKDKATLTADKSSSTVYMQLSSLTSEDSAVYYCARGGQLGLRRAMDYWGQGTSVTVSS

[SEQ ID No:16]

in a preferred embodiment, the nucleic acid sequence encoding the anti-Bb single-stranded variable fragment (750 bp) is referred to herein as SEQ ID No. 17, or a fragment or variant thereof, as shown below:

GACGTGCAGATCACCCAGAGCCCCAGCTACCTGGCCGCCAGCCCCGGCGAGACCATCACCATCAACTGCCGCGCCAGCAAGAGCATCAGCAAGTACCTGGCCTGGTACCAGGACAAGCCCGGCAAGACCAACAAGCTGCTGATCTACAGCGGCAGCACCCTGCAGAGCGGCATCCCCAGCCGCTTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGACCATCAGCAGCCTGGAGCCCGAGGACTTCGCCATGTACTACTGCCAGCAGCACGACGAGTACCCCTGGACCTTCGGCGGCGGCACCAAGCTGGAGATCAAGCGCGGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCCAGGTGCAGCTGCAGCAGAGCGGCGCCGAGCTGGCCAAGCCCGGCGCCAGCGTGCGCATGAGCTGCAAGGCCAGCGGCTACACCTTCACCAACTACTGGATCCACTGGGTGAAGCAGCGCCCCGGCCAGGGCCTGGAGTGGATCGGCTACATCAACCCCAACACCGGCTACAACGACTACAACCAGAAGTTCAAGGACAAGGCCACCCTGACCGCCGACAAGAGCAGCAGCACCGTGTACATGCAGCTGAGCAGCCTGACCAGCGAGGACAGCGCCGTGTACTACTGCGCCCGCGGCGGCCAGCTGGGCCTGCGCCGCGCCATGGACTACTGGGGCCAGGGCACCAGCGTGACCGTGAGCAGC

[SEQ ID No:17]

in another embodiment, the amino acid sequence of the soluble form of normal plasma membrane bound CD55 (sCD 55, sometimes also referred to as decay accelerating factor; DAF) is referred to herein as SEQ ID No. 18, or a fragment or variant thereof, as shown below:

DCGLPPDVPNAQPALEGRTSFPEDTVITYKCEESFVKIPGEKDSVICLKGSQWSDIEEFCNRSCEVPTRLNSASLKQPYITQNYFPVGTVVEYECRPGYRREPSLSPKLTCLQNLKWSTAVEFCKKKSCPNPGEIRNGQIDVPGGILFGATISFSCNTGYKLFGSTSSFCLISGSSVQWSDPLPECREIYCPAPPQIDNGIIQGERDHYGYRQSVTYACNKGFTMIGEHSIYCTVNNDEGEWSGPPPECRGKSLTSKVPPTVQKPTTVNVPTTEVSPTSQKTTTKTTTPNAQATRSTPVSRTTKHFHETTPNKGSGTTSG

[SEQ ID No:18]

in a preferred embodiment, the nucleic acid sequence (960 bp) encoding a soluble form of normal plasma membrane-bound CD55 (sCD 55, sometimes also referred to as decay accelerating factor; DAF) is referred to herein as SEQ ID No. 19, or a fragment or variant thereof, as shown below:

GACTGCGGCCTGCCCCCCGACGTGCCCAACGCCCAGCCCGCCCTGGAGGGCCGCACCAGCTTCCCCGAGGACACCGTGATCACCTACAAGTGCGAGGAGAGCTTCGTGAAGATCCCCGGCGAGAAGGACAGCGTGATCTGCCTGAAGGGCAGCCAGTGGAGCGACATCGAGGAGTTCTGCAACCGCAGCTGCGAGGTGCCCACCCGCCTGAACAGCGCCAGCCTGAAGCAGCCCTACATCACCCAGAACTACTTCCCCGTGGGCACCGTGGTGGAGTACGAGTGCCGCCCCGGCTACCGCCGCGAGCCCAGCCTGAGCCCCAAGCTGACCTGCCTGCAGAACCTGAAGTGGAGCACCGCCGTGGAGTTCTGCAAGAAGAAGAGCTGCCCCAACCCCGGCGAGATCCGCAACGGCCAGATCGACGTGCCCGGCGGCATCCTGTTCGGCGCCACCATCAGCTTCAGCTGCAACACCGGCTACAAGCTGTTCGGCAGCACCAGCAGCTTCTGCCTGATCAGCGGCAGCAGCGTGCAGTGGAGCGACCCCCTGCCCGAGTGCCGCGAGATCTACTGCCCCGCCCCCCCCCAGATCGACAACGGCATCATCCAGGGCGAGCGCGACCACTACGGCTAC

CGCCAGAGCGTGACCTACGCCTGCAACAAGGGCTTCACCATGATCGGCG

AGCACAGCATCTACTGCACCGTGAACAACGACGAGGGCGAGTGGAGCG

GCCCCCCCCCCGAGTGCCGAGGCAAGAGCCTGACCAGCAAGGTGCCCC

CCACCGTGCAGAAGCCCACCACCGTGAACGTGCCCACCACCGAGGTGA

GCCCCACCAGCCAGAAGACCACCACCAAGACCACCACCCCCAACGCCC

AGGCCACCCGCAGCACCCCCGTGAGCCGCACCACCAAGCACTTCCACG

AGACCACCCCCAACAAGGGCAGCGGCACCACCAGCGGC

[SEQ ID No:19]

in a further embodiment, the amino acid sequence of human complement factor H-related protein-1 (CFHR 1) is referred to herein as SEQ ID No. 20, or a fragment or variant thereof, as shown below: EATFCDFPKINHGILYDEEKYKPFSQVPTGEVFYYSCEYNFVSPSKSFWTRITCTEEGWSPTPKCLRLCFFPFVENGHSESSGQTHLEGDTVQIICNTGYRLQNNENNISCVERGWSTPPKCRSTDTSCVNPPTVQNAHILSRQMSKYPSGERVRYECRSPYEMFGDEEVMCLNGNWTEPPQCKDSTGKCGPPPPIDNGDITSFPLSVYAPASSVEYQCQNLYQLEGNKRITCRNGQWSEPPKCLHPCVISREIMENYNIALRWTAKQKLYLRTGESAEFVCKRGYRLSSRSHTLRTTCWDGKLEYPTCAKR

[SEQ ID No:20]

In a preferred embodiment, the nucleic acid sequence encoding human complement factor H-related protein-1 (CFHR 1) (936 bp) is referred to herein as SEQ ID No. 21, or a fragment or variant thereof, as shown below: GAAGCAACATTTTGTGATTTTCCAAAAATAAACCATGGAATTCTATATGATGAAGAAAAATATAAGCCATTTTCCCAGGTTCCTACAGGGGAAGTTTTCTATTACTCCTGTGAATATAATTTTGTGTCTCCTTCAAAATCATTTTGGACTCGCATAACATGCACAGAAGAAGGATGGTCACCAACACCAAAGTGTCTCAGACTGTGTTTCTTTCCTTTTGTGGAAAATGGTCATTCTGAATCTTCAGGACAAACACATCTGGAAGGTGATACTGTGCAAATTATTTGCAACACAGGATACAGACTTCAAAACAATGAGAACAACATTTCATGTGTAGAACGGGGCTGGTCCACCCCTCCCAAATGCAGGTCCACTGACACTTCCTGTGTGAATCCGCCCACAGTACAAAATGCTCATATACTGTCGAGACAGATGAGTAAATATCCATCTGGTGAGAGAGTACGTTATGAATGTAGGAGCCCTTATGAAATGTTTGGGGATGAAGAAGTGATGTGTTTAAATGGAAACTGGACAGAACCACCTCAATGCAAAGATTCTACGGGAAAATGTGGGCCCCCTCCACCTATTGACAATGGGGACATTACTTCATTCCCGTTGTCAGTATATGCTCCAGCTTCATCAGTTGAGTACCAATGCCAGAACTTGTATCAACTTGAGGGTAACAAGCGAATAACATGTAGAAATGGACAATGGTCAGAACCACCAAAATGCTTACATCCGTGTGTAATATCCCGAGAAATTATGGAAAATTATAACATAGCATTAAGGTGGACAGCCAAACAGAAGCTTTATTTGAGAACAGGTGAATCAGCTGAATTTGTGTGTAAACGGGGATATCGTCTTTCATCACGTTCTCACACATTGCGAACAACATGTTGGGATGGGAAACTGGAGTATCCAACTTGTGCAAAAAGA

[SEQ ID No:21]

In another embodiment, the codon-optimized nucleic acid sequence (936 bp) encoding human complement factor H-related protein-1 (CFHR 1) is referred to herein as SEQ ID No. 22, or a fragment or variant thereof, as shown below:

GAGGCCACCTTCTGCGACTTCCCCAAGATCAACCACGGCATCCTGTACG

ACGAGGAGAAGTACAAGCCCTTCAGCCAGGTGCCCACCGGCGAGGTGT

TCTACTACAGCTGCGAGTACAACTTCGTGAGCCCCAGCAAGAGCTTCTG

GACCCGCATCACCTGCACCGAGGAGGGCTGGAGCCCCACCCCCAAGTG

CCTGCGCCTGTGCTTCTTCCCCTTCGTGGAGAACGGCCACAGCGAGAGC

AGCGGCCAGACCCACCTGGAGGGCGACACCGTGCAGATCATCTGCAAC

ACCGGCTACCGCCTGCAGAACAACGAGAACAACATCAGCTGCGTGGAG

CGCGGCTGGAGCACCCCCCCCAAGTGCCGCAGCACCGACACCAGCTGC

GTGAACCCCCCCACCGTGCAGAACGCCCACATCCTGAGCCGCCAGATGA

GCAAGTACCCCAGCGGCGAGCGCGTGCGCTACGAGTGCCGCAGCCCCTA

CGAGATGTTCGGCGACGAGGAGGTGATGTGCCTGAACGGCAACTGGAC

CGAGCCCCCCCAGTGCAAGGACAGCACCGGCAAGTGCGGCCCCCCCCC

CCCCATCGACAACGGCGACATCACCAGCTTCCCCCTGAGCGTGTACGCC

CCCGCCAGCAGCGTGGAGTACCAGTGCCAGAACCTGTACCAGCTGGAG

GGCAACAAGCGCATCACCTGCCGCAACGGCCAGTGGAGCGAGCCCCCC

AAGTGCCTGCACCCCTGCGTGATCAGCCGCGAGATCATGGAGAACTACA

ACATCGCCCTGCGCTGGACCGCCAAGCAGAAGCTGTACCTGCGCACCGG

CGAGAGCGCCGAGTTCGTGTGCAAGCGCGGCTACCGCCTGAGCAGCCG

CAGCCACACCCTGCGCACCACCTGCTGGGACGGCAAGCTGGAGTACCCC

ACCTGCGCCAAGCGC

[SEQ ID No:22]

in a further embodiment, the amino acid sequence of normal plasma membrane-bound CD46 (sCD 46) in soluble form is referred to herein as SEQ ID No. 23, or a fragment or variant thereof, as shown below: CEEPPTFEAMELIGKPKPYYEIGERVDYKCKKGYFYIPPLATHTICDRNHTWLPVSDDACYRETCPYIRDPLNGQAVPANGTYEFGYQMHFICNEGYYLIGEEILYCELKGSVAIWSGKPPICEKVLCTPPPKIKNGKHTFSEVEVFEYLDAVTYSCDPAPGPDPFSLIGESTIYCGDNSVWSRAAPECKVVKCRFPVVENGKQISGFGKKFYYKATVMFECDKGFYLDGSDTIVCDSNSTWDPPVPKCLKVLPPSSTKPPALSHSVSTSSTTKSPASSASGPRPTYKPPVSNYPGYPKPEEGILDSLDV

[SEQ ID No:23]

In a preferred embodiment, the nucleic acid sequence (930 bp) encoding the soluble form of normal plasma membrane-bound CD46 (sCD 46) is referred to herein as SEQ ID No. 24, or a fragment or variant thereof, as shown below:

TGCGAGGAGCCCCCCACCTTCGAGGCCATGGAGCTGATCGGCAAGCCCA

AGCCCTACTACGAGATCGGCGAGCGCGTGGACTACAAGTGCAAGAAGG

GCTACTTCTACATCCCCCCCCTGGCCACCCACACCATCTGCGACCGCAAC

CACACCTGGCTGCCCGTGAGCGACGACGCCTGCTACCGCGAGACCTGCC

CCTACATCCGCGACCCCCTGAACGGCCAGGCCGTGCCCGCCAACGGCAC

CTACGAGTTCGGCTACCAGATGCACTTCATCTGCAACGAGGGCTACTACC

TGATCGGCGAGGAGATCCTGTACTGCGAGCTGAAGGGCAGCGTGGCCAT

CTGGAGCGGCAAGCCCCCCATCTGCGAGAAGGTGCTGTGCACCCCCCCC

CCCAAGATCAAGAACGGCAAGCACACCTTCAGCGAGGTGGAGGTGTTCGAGTACCTGGACGCCGTGACCTACAGCTGCGACCCCGCCCCCGGCCCCGACCCCTTCAGCCTGATCGGCGAGAGCACCATCTACTGCGGCGACAACAGCGTGTGGAGCCGCGCCGCCCCCGAGTGCAAGGTGGTGAAGTGCCGCTTCCCCGTGGTGGAGAACGGCAAGCAGATCAGCGGCTTCGGCAAGAAGTTCTACTACAAGGCCACCGTGATGTTCGAGTGCGACAAGGGCTTCTACCTGGACGGCAGCGACACCATCGTGTGCGACAGCAACAGCACCTGGGACCCCCCCGTGCCCAAGTGCCTGAAGGTGCTGCCCCCCAGCAGCACCAAGCCCCCCGCCCTGAGCCACAGCGTGAGCACCAGCAGCACCACCAAGAGCCCCGCCAGCAGCGCCAGCGGCCCCCGCCCCACCTACAAGCCCCCCGTGAGCAACTACCCCGGCTACCCCAAGCCCGAGGAGGGCATCCTGGACAGCCTGGACGTG

[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 set forth below:

EDCNELPPRRNTEILTGSWSDQTYPEGTQAIYKCRPGYRSLGNVIMVCRKGEWVALNPLRKCQKRPCGHPGDTPFGTFTLTGGNVFEYGVKAVYTCNEGYQLLGEINYRECDTDGWTNDIPICEVVKCLPVTAPENGKIVSSAMEPDREYHFGQAVRFVCNSGYKIEGDEEMHCSDDGFWSKEKPKCVEISCKSPDVINGSPISQKIIYKENERFQYKCNMGYEYSERGDAVCTESGWRPLPSCEEKSCDNPYIPNGDYSPLRIKHRTGDEITYQCRNGFYPATRGNTAKCTSTGWIPAPRCTLKPCDYPDIKHGGLYHENMRRPYFPVAVGKYYSYYCDEHFETPSGSYWDHIHCTQDGWSPAVPCLRKCYFPYLENGYNQNYGRKFVQGKSIDVACHPGYALPKAQTTVTCMENGWSPTPRCIR

[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 set out below:

GAGGACTGCAACGAGCTGCCCCCCCGCCGCAACACCGAGATCCTGACCGGCAGCTGGAGCGACCAGACCTACCCCGAGGGCACCCAGGCCATCTAC

AAGTGCCGCCCCGGCTACCGCAGCCTGGGCAACGTGATCATGGTGTGCC

GCAAGGGCGAGTGGGTGGCCCTGAACCCCCTGCGCAAGTGCCAGAAGC

GCCCCTGCGGCCACCCCGGCGACACCCCCTTCGGCACCTTCACCCTGAC

CGGCGGCAACGTGTTCGAGTACGGCGTGAAGGCCGTGTACACCTGCAAC

GAGGGCTACCAGCTGCTGGGCGAGATCAACTACCGCGAGTGCGACACC

GACGGCTGGACCAACGACATCCCCATCTGCGAGGTGGTGAAGTGCCTGC

CCGTGACCGCCCCCGAGAACGGCAAGATCGTGAGCAGCGCCATGGAGC

CCGACCGCGAGTACCACTTCGGCCAGGCCGTGCGCTTCGTGTGCAACAG

CGGCTACAAGATCGAGGGCGACGAGGAGATGCACTGCAGCGACGACGG

CTTCTGGAGCAAGGAGAAGCCCAAGTGCGTGGAGATCAGCTGCAAGAG

CCCCGACGTGATCAACGGCAGCCCCATCAGCCAGAAGATCATCTACAAG

GAGAACGAGCGCTTCCAGTACAAGTGCAACATGGGCTACGAGTACAGCG

AGCGCGGCGACGCCGTGTGCACCGAGAGCGGCTGGCGCCCCCTGCCCA

GCTGCGAGGAGAAGAGCTGCGACAACCCCTACATCCCCAACGGCGACT

ACAGCCCCCTGCGCATCAAGCACCGCACCGGCGACGAGATCACCTACCA

GTGCCGCAACGGCTTCTACCCCGCCACCCGGGGCAACACCGCCAAGTGC

ACCAGCACCGGCTGGATCCCCGCCCCCCGCTGCACCCTGAAGCCCTGCG

ACTACCCCGACATCAAGCACGGCGGCCTGTACCACGAGAACATGCGCCG

CCCCTACTTCCCCGTGGCCGTGGGCAAGTACTACAGCTACTACTGCGACG

AGCACTTCGAGACCCCCAGCGGCAGCTACTGGGACCACATCCACTGCAC

CCAGGACGGCTGGAGCCCCGCCGTGCCCTGCCTGCGCAAGTGCTACTTC

CCCTACCTGGAGAACGGCTACAACCAGAACTACGGCCGCAAGTTCGTGC

AGGGCAAGAGCATCGACGTGGCCTGCCACCCCGGCTACGCCCTGCCCAA

GGCCCAGACCACCGTGACCTGCATGGAGAACGGCTGGAGCCCCACCCC

CCGCTGCATCCGC

[SEQ ID No:81]

In a preferred embodiment, the amino acid sequence of the anti-C5 single chain variable fragment is referred to herein as SEQ ID No. 82, or a fragment or variant thereof, as set forth below:

DIQMTQSPSSLSASVGDRVTITCQASQSINNQLSWYQQKPGKAPKLLIYYAS

TLASGYPSRFSGSGSGTDFTLTISSLQPEDFATYYCQGSYYSGGWDYGFGQG

TKVEIKRGGGGGSGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCA

ASGFSFSGRYWIQWVRQAPGKGLEWVASVWPGITGTNYANWAKGRFTISR

DDSKNTLYLQMNSLRAEDTAVYYCAREPVAWGGGLDLWGQGTLVTVSS

[SEQ ID No:82]

in a preferred embodiment, the nucleic acid sequence encoding the anti-C5 single-stranded variable fragment (759 bp) is referred to herein as SEQ ID No. 83, or a fragment or variant thereof, as shown below: GACATCCAGATGACCCAGAGCCCCAGCAGCCTGAGCGCCAGCGTGGGCGACCGCGTGACCATCACCTGCCAGGCCAGCCAGAGCATCAACAACCAGCTGAGCTGGTACCAGCAGAAGCCCGGCAAGGCCCCCAAGCTGCTGATCTACTACGCCAGCACCCTGGCCAGCGGCTACCCCAGCCGCTTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGACCATCAGCAGCCTGCAGCCCGAGGACTTCGCCACCTACTACTGCCAGGGCAGCTACTACAGCGGCGGCTGGGACTACGGCTTCGGCCAGGGCACCAAGGTGGAGATCAAGCGCGGCGGCGGCGGCGGCAGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGAGGTGCAGCTGGTGGAGAGCGGCGGCGGCCTGGTGCAGCCCGGCGGCAGCCTGCGCCTGAGCTGCGCCGCCAGCGGCTTCAGCTTCAGCGGCCGCTACTGGATCCAGTGGGTGCGCCAGGCCCCCGGCAAGGGCCTGGAGTGGGTGGCCAGCGTGTGGCCCGGCATCACCGGCACCAACTACGCCAACTGGGCCAAGGGCCGCTTCACCATCAGCCGCGACGACAGCAAGAACACCCTGTACCTGCAGATGAACAGCCTGCGCGCCGAGGACACCGCCGTGTACTACTGCGCCCGCGAGCCCGTGGCCTGGGGCGGCGGCCTGGACCTGTGGGGCCAGGGCACCCTGGTGACCGTGAGCAGCTGA

[SEQ ID No:83]

Thus, in a preferred embodiment, the second coding sequence comprises a sequence substantially as set forth in SEQ ID No: 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 shown in SEQ ID No. 14, 16, 18, 20, 23, 80 or 82, or a fragment or variant thereof.

Many of the gene constructs proposed in the scientific literature that express two or more genes have (i) a dual promoter to drive expression of two or more genes, respectively, or (ii) an Internal Ribosome Entry Site (IRES) to link genes, such as from an encephalomyocarditis virus (EMCV), to enable translation of genes from a single transcript within a recombinant viral vector driven by a single promoter. However, the efficiency of IRES-dependent translation varies significantly in different cells and tissues, and IRES-dependent translation may be significantly lower than cap-dependent translation, meaning that the expression of genes downstream of the IRES is typically lower compared to genes at position 1 (i.e., the 5' end) of the expression cassette. Furthermore, the limited coding capacity of rAAV vectors (typically <5 kb) prevents the incorporation of large genes/ORFs, such as PEDF receptor agonists and anti-complement protein coding sequences using dual promoters and/or IRES linkers (where EMCV IRES is 553 nucleotides in length).

Thus, in a preferred embodiment, the genetic construct comprises a spacer sequence disposed between the first and second coding sequences. See, for example, fig. 3, wherein a spacer sequence (v 2A) is placed between the first and second coding sequences. The spacer sequence encodes a peptide spacer configured to produce PEDF receptor agonist and anticomplementary proteins as separate molecules. This is possible because the spacer is configured to skip transcription of the linear ribosomal sequence to produce individual molecules or peptides. It is understood that the individual molecules are active.

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

Preferably, the viral-2A peptide sequence links the first coding sequence to the second coding sequence. This allows the construct to overcome the size limitations that occur when expressed in various vectors and allows all peptides encoded by the construct of the first aspect to be expressed as a single mRNA transcript under the control of a single promoter.

Thus, in one embodiment, after transcription of a single mRNA transcript encoding the PEDF receptor agonist, the viral-2A peptide and the anti-complement protein, a translational jump may occur at the viral-2A peptide sequence between the terminal glycine-prolines of the viral-2A peptide (translational skipping). This translational jump will thus result in two proteins, namely PEDF receptor agonist and anticomplementary protein (see fig. 3).

The inventors have generated four embodiments of the spacer sequence. For all embodiments described herein, one important part of the peptide spacer is the C-terminus.

In one embodiment, the peptide spacer is P2A. Preferably, the P2A peptide spacer encodes an amino acid sequence referred to herein as SEQ ID No. 25, or a fragment or variant thereof, as shown below:

ATNFSLLKQAGDVEENPGP

[SEQ ID No:25]

preferably, the digestion or cleavage site for the peptide spacer is placed between the terminal glycine and the terminal proline in SEQ ID No. 25.

In this first embodiment, the P2A peptide spacer sequence comprises the nucleotide sequence (57 bp) referred to herein as SEQ ID No. 26 or a fragment or variant thereof, as shown below:

GCCACCAACTTCAGCCTGCTGAAGCAGGCCGGCGACGTGGAGGAGAACCCCGGCCCC

[SEQ ID No:26]

in a second embodiment, the peptide spacer is E2A. Preferably, the E2A peptide spacer encodes an amino acid sequence referred to herein as SEQ ID No. 27, or a fragment or variant thereof, as shown below:

QCTNYALLKLAGDVESNPGP

[SEQ ID No:27]

preferably, the digestion or cleavage site for the peptide spacer is placed between the terminal glycine and the terminal proline in SEQ ID No. 27.

In this second embodiment, the E2A peptide spacer comprises the nucleotide sequence (60 bp) referred to herein as SEQ ID No. 28, or a fragment or variant thereof, as shown below:

CAGTGCACCAACTACGCCCTGCTGAAGCTGGCCGGCGACGTGGAGAGCAACCCCGGCCCC

[SEQ ID No:28]

In a third embodiment, the peptide spacer is T2A. Preferably, the T2A peptide spacer encodes an amino acid sequence referred to herein as SEQ ID No. 29, or a fragment or variant thereof, as shown below:

EGRGSLLTCGDVEENPGP

[SEQ ID No:29]

preferably, the digestion or cleavage site for the peptide spacer is placed between the terminal glycine and the terminal proline in SEQ ID No. 29.

In this third embodiment, the T2A peptide spacer comprises the nucleotide sequence (54 bp) referred to herein as SEQ ID No. 30, or a fragment or variant thereof, as shown below:

GAGGGCCGCGGCAGCCTGCTGACCTGCGGCGACGTGGAGGAGAACCCCGGCCCC

[SEQ ID No:30]

in a fourth preferred embodiment, the peptide spacer is F2A. Preferably, the F2A peptide spacer encodes an amino acid sequence referred to herein as SEQ ID No. 31, or a fragment or variant thereof, as shown below:

VKQTLNFDLLKLAGDVESNPGP

[SEQ ID No:31]

preferably, the digestion or cleavage site for the peptide spacer is placed between the terminal glycine and the terminal proline in SEQ ID No. 31.

In this fourth embodiment, the F2A peptide spacer comprises the nucleotide sequence (66 bp) referred to herein as SEQ ID No. 32, or a fragment or variant thereof, as shown below:

GTGAAGCAGACCCTGAACTTCGACCTGCTGAAGCTGGCCGGCGACGTGGAGAGCAACCCCGGCCCC

[SEQ ID No:32]

thus, in a preferred embodiment, the peptide spacer comprises a nucleotide sequence substantially as shown in any one of SEQ ID Nos. 26, 28, 30 or 32, or a fragment or variant thereof. Preferably, the peptide spacer encodes an amino acid sequence substantially as shown in SEQ ID No. 25, 27, 29 or 31, or a fragment or variant thereof.

Following a translational jump, the viral-2A peptide sequence remains fused to the C-terminus of the upstream protein (e.g., PEDF receptor agonist) and the proline remains fused to the N-terminus of the downstream protein (e.g., anticomplementary protein). This carries an immunogenicity risk and may interfere with intracellular signaling capacity at PEDF receptors. Thus, the inventors introduced an enzyme cleavage coding sequence immediately upstream (directly upstream) of the viral-2A peptide sequence, such that the remaining viral-2A peptide sequence from both coding proteins (i.e., PEDF receptor agonist and anticomplementary protein) was removed. The introduction of the enzyme cleavage site has the effect of removing the virus-2A peptide in the cell before releasing the secreted protein (in the case of furin) or immediately after secretion of the protein from the target (retina) cell (in the case of the enzyme recognition site of matrix metalloprotein-2 (MMP-2) or renin).

Thus, in one embodiment, the construct further comprises a viral-2A removal sequence. Preferably, the virus-2A removal sequence is placed 5' to the virus-2A sequence. Preferably, the virus-2A removal sequence is separated from the virus-2A sequence by a linker sequence comprising the tripeptide glycine-serine-glycine sequence (G-S-G).

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

Currently, furin recognition sequences are generally considered to comprise three or four basic amino acids (arginine or lysine), with an optional non-basic amino acid at position 2, and with cleavage by furin at a position after the last basic amino acid. However, using various plasmid constructs, the inventors determined that the basic furin recognition sequence does not always result in enzymatic activity and isolation of the viral-2A sequence. Thus, the inventors have generated preferred furin recognition sequences for use in the genetic constructs of the invention.

Thus, in a preferred embodiment, the genetic construct comprises a viral-2A removal sequence encoding the amino acid sequence referred to herein as SEQ ID No. 33, or a fragment or variant thereof, wherein: b=basic amino acid, x=hydrophilic amino acid, and s=serine, as shown below:

BB(X)BBS

[SEQ ID No:33]

preferably, the hydrophilic amino acid (X) is serine (S) or threonine (T). Thus, in one embodiment, the viral-2A removal sequence encodes an amino acid sequence substantially as shown in SEQ ID No. 33, or a fragment or variant thereof.

In one embodiment, the virus-2A removal sequence encodes an amino acid sequence referred to herein as SEQ ID No. 34, or a fragment or variant thereof, as shown below:

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 set forth below:

CGCCGCAGCAAGCGCAGCGGCAGCGGC

[SEQ ID No:35]

in a second embodiment, the viral-2A removal sequence encodes an amino acid sequence referred to herein as SEQ ID No:36, or a fragment or variant thereof, as shown below:

RRTKRSGSG

[SEQ ID No:36]

in this second embodiment, the viral-2A removal sequence comprises the nucleotide sequence referred to herein as SEQ ID No. 37, or a fragment or variant thereof, as set forth below:

CGCCGCACCAAGCGCAGCGGCAGCGGC

[SEQ ID No:37]

thus, in one embodiment, the viral-2A removal sequence comprises a nucleotide sequence substantially as shown in SEQ ID No. 35 or 37, or a fragment or variant thereof. Preferably, the viral-2A removal sequence encodes an amino acid sequence substantially as shown in SEQ ID No. 34 or 36, or a fragment or variant thereof.

Alternatively, in another embodiment, the viral-2A removal sequence is a gelatinase MMP-2 recognition sequence. Preferably, in this embodiment, the viral-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.

The inventors have generated a preferred amino acid sequence, referred to herein as SEQ ID No. 38, comprising a gelatinase MMP-2 recognition sequence and a tripeptide GSG linker sequence.

Thus, in one embodiment, the viral-2A removal sequence encodes an amino acid sequence referred to herein as SEQ ID No. 38, or a fragment or variant thereof, as shown below:

GPQGIAGQGSG

[SEQ ID No:38]

in this embodiment, the viral-2A removal sequence comprises the nucleotide sequence referred to herein as SEQ ID No:39, or a fragment or variant thereof, as shown below:

GGCCCCCTGGGCATCGCCGGCCAGGGCAGCGGC

[SEQ ID No:39]

thus, in one embodiment, the viral-2A removal sequence comprises a nucleotide sequence substantially as shown in SEQ ID No. 39 or a fragment or variant thereof. Preferably, the viral-2A removal sequence encodes an amino acid sequence substantially as shown in SEQ ID No. 38 or a fragment or variant thereof.

Alternatively, in another embodiment, the viral-2A removal sequence is a renin recognition sequence. Preferably, in this embodiment, the viral-2A removal 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.

The inventors have generated what is referred to herein as SEQ ID No:40 comprising a renin recognition sequence and a tripeptide GSG linker sequence.

Thus, in one embodiment, the viral-2A removal sequence encodes an amino acid sequence referred to herein as SEQ ID No. 40, or a fragment or variant thereof, as shown below:

HPFHLLVYSGSG

[SEQ ID No:40]

in this embodiment, the viral-2A removal sequence comprises the nucleotide sequence referred to herein as SEQ ID No. 41, or a fragment or variant thereof, as shown below:

CGCCCCTTCCACCTGCTGGTCATCCACGGCAGCGGC

[SEQ ID No:41]

thus, in one embodiment, the viral-2A removal sequence comprises a nucleotide sequence substantially as shown in SEQ ID No. 41 or a fragment or variant thereof. Preferably, the viral-2A removal sequence encodes an amino acid sequence substantially as shown in SEQ ID No. 40, or a fragment or variant thereof.

As shown in FIG. 2, the expression cassette further comprises sequences encoding a hepatitis virus post-transcriptional regulatory element (WPRE), a poly-A tail, and left-hand (left-hand) and right-hand (right-hand) Inverted Terminal Repeats (ITRs). Preferably, the genetic construct comprises a nucleotide sequence encoding a woodchuck hepatitis virus (WHP) post-transcriptional regulatory element (WPRE) that enhances expression of the transgene, namely PEDF receptor agonist and anti-complement proteins. Preferably, the WPRE coding sequence is placed 3' to the transgene coding sequence.

One embodiment of the woodchuck hepatitis virus posttranscriptional regulatory element (WPRE) is 592 nucleotides long, including a gamma-alpha-beta element, referred to herein as SEQ ID No. 42, as shown below:

AATCAACCTCTGGATTACAAAATTTGTGAAAGATTGACTGGTATTCTTAACTATGTTGCTCCTTTTACGCTATGTGGATACGCTGCTTTAATGCCTTTGTATCATGCTATTGCTTCCCGTATGGCTTTCATTTTCTCCTCCTTGTATAAATCCTGGTTGCTGTCTCTTTATGAGGAGTTGTGGCCCGTTGTCAGGCAACGTGGCGTGGTGTGCACTGTGTTTGCTGACGCAACCCCCACTGGTTGGGGCATTGCCACCACCTGTCAGCTCCTTTCCGGGACTTTCGCTTTCCCCCTCCCTATTGCCACGGCGGAACTCATCGCCGCCTGCCTTGCCCGCTGCTGGACAGGGGCTCGGCTGTTGGGCACTGACAATTCCGTGGTGTTGTCGGGGAAGCTGACGTCCTTTCCATGGCTGCTCGCCTGTGTTGCCACCTGGATTCTGCGCGGGACGTCCTTCTGCTACGTCCCTTCGGCCCTCAATCCAGCGGACCTTCCTTCCCGCGGCCTGCTGCCGGCTCTGCGGCCTCTTCCGCGTCTTCGCCTTCGCCCTCAGACGAGTCGGATCTCCCTTTGGGCCGCCTCCCCGCCTG

[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.

However, in a preferred embodiment, a truncated WPRE is used that is 247 nucleotides long due to the deletion of the β -element and is referred to herein as SEQ ID No. 43, as follows:

AATCAACCTCTGGATTACAAAATTTGTGAAAGATTGACTGGTATTCTTAACTATGTTGCTCCTTTTACGCTATGTGGATACGCTGCTTTAATGCCTTTGTATCATGCTATTGCTTCCCGTATGGCTTTCATTTTCTCCTCCTTGTATAAATCCTGGTTAGTTCTTGCCACGGCGGAACTCATCGCCGCCTGCCTTGCCCGCTGCTGGACAGGGGCTCGGCTGTTGGGCACTGACAATTCCGTGGTGT

[SEQ ID No:43]

thus, preferably, the truncated WPRE comprises a nucleic acid sequence substantially as set forth in SEQ ID No. 43 or a fragment or variant thereof.

Advantageously, the truncated WPRE sequences used in the constructs provide a total savings of about 300 nucleotides without adversely affecting transgene expression. Thus, preferably, the WPRE comprises a nucleic acid sequence substantially as set forth in SEQ ID No. 43 or a fragment or variant thereof.

Preferably, the genetic construct comprises a nucleotide sequence encoding a poly-A tail. Preferably, the poly-A tail coding sequence is placed 3 'to the transgene coding sequence, and preferably 3' to the WPRE coding sequence. polyA tails are important for nuclear export, translation and stability of mRNA. Over time, the poly tail becomes shorter and when it is short enough, the mRNA is enzymatically degraded.

Preferably, the poly-A tail comprises the simian virus-40 poly-A224 nucleotide sequence. One embodiment of the poly-A tail is referred to herein as SEQ ID No. 44, as follows: AGCAGACATGATAAGATACATTGATGAGTTTGGACAAACCACAACTAGAATGCAGTGAAAAAAATGCTTTATTTGTGAAATTTGTGATGCTATTGCTTTATTTGTAACCATTATAAGCTGCAATAAACAAGTTAACAACAACAATTGCATTCATTTTATGTTTCAGGTTCAGGGGGAGGTGTGGGAGGTTTTTTAAAGCAAGTAAAACCTCTACAAATGTGGTA

[SEQ ID No:44]

In another embodiment, the poly-A tail comprises a polyA component of nucleotide 169, which is referred to herein as SEQ ID No. 45, as set forth below:

TTCGAGCAACTTGTTTATTGCAGCTTATAATGGTTACAAATAAAGCAATAG

CATCACAAATTTCACAAATAAAGCATTTTTTTCACTGCATTCTAGTTGTGG

TTTGTCCAAACTCATCAATGTATCTTATCATGTCTGGATCGTCTAGCATCG

AAGATCCCCCGATCTG

[SEQ ID No:45]

in another embodiment, the poly-A tail comprises a bovine growth hormone 225 nucleotide sequence, which is referred to herein as SEQ ID No. 84, as set forth below:

CTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTT

CCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTTCCTAATAAAATGAG

GAAATTGCATCGCATTGTCTGAGTAGGTGTCATTCTATTCTGGGGGGTGG

GGTGGGGCAGGACAGCAAGGGGGAGGATTGGGAAGACAATAGCAGGCA

TGCTGGGGATGCGGTGGGCTCTATGG

[SEQ ID No:84]

thus, preferably, the poly-A tail comprises a nucleic acid sequence substantially as shown in SEQ ID No. 44, 45 or 84, or a fragment or variant thereof.

Preferably, the genetic construct comprises left and/or right Inverted Terminal Repeats (ITRs). Preferably, each ITR is placed at the 5 'and/or 3' end of the construct. The ITR can be specific for a viral (e.g., AAV or lentiviral) serotype, and can be any sequence, so long as it forms a hairpin loop in its secondary structure.

The DNA sequence of one embodiment of the ITR (the left ITR sequence is taken from a commercially available recombinant AAV genomic plasmid) is represented herein as SEQ ID No. 46, as follows: CGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCAAAGCCCGGGCGTCGGGCGACCTTTGGTCGCCCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCTTG

[SEQ ID No:46]

Another embodiment of the ITR (right ITR sequence taken from a commercially available recombinant AAV genomic plasmid) has the DNA sequence shown herein as SEQ ID No. 47, as follows: AGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCCCGGGCTTTGCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGC

[SEQ ID No:47]

Preferably, the left and/or right inverted terminal repeat comprises a nucleic acid sequence substantially as shown in SEQ ID No. 46 or 47, or a fragment or variant thereof.

Recently, it has been discovered that non-coding introns located between the promoter and the gene (near the 3 'end of the promoter and the 5' end of the gene) can promote gene expression in certain genomic sequences through mRNA accumulation [59, 60]. Thus, inclusion of an intron in the gene construct of the viral vector may facilitate more transgene expression and subsequent production of the mature protein when used in combination with a constitutive or regulated promoter.

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 placed 3 'of the promoter and 5' of the first coding sequence.

In one embodiment, the non-coding intron is the mouse adenovirus (minute virus of mice, MVM) small (121 bp) intron [61], referred to herein as SEQ ID No. 48, as shown below:

AGGTACGATGGCGCCTCCAGCTAAAAGAGCTAAAAGAGGTAAGGGTTTAAGGGATGGTTGGTTGGTGGGGTATTAATGTTTAATTACCTGTTTTACAGGCCTGAAATCACTTGGTTTTAGG

[SEQ ID No:48]

in another embodiment, the non-coding intron is the sequence (133 bp) of the 5 '-donor site and the branching from the first intron of the human β -globin gene and the 3' -acceptor site of the heavy chain variable region intron of the immunoglobulin gene, 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), referred to herein as SEQ ID No. 50, as shown below:

GTGAGCGGGCGGGACGGCCCTTCTCCTCCGGGCTGTAATTAGCGCTTGGTTTAATGACGGCTTGTTTCTTTTCTGTGGCTGCGTGAAAGCCTTGAGGGGCTCCGGGAGGCTAGAGCCTCTGCTAACCATGTTCATGCCTTCTTCTTTTTCCTACAGCTCCTGGGCAACGTGCTGGTTATTGTGCTGTCTCATCATTTTGGCAAAGAATTC

[SEQ ID NO.50]

thus, preferably, the non-coding intron comprises a nucleic acid sequence substantially as shown in SEQ ID No. 48, 49 or 50, or a fragment or variant thereof.

To allow for proper folding of the polypeptides encoded by the genetic constructs, intracellular trafficking of PEDF receptor agonists and anticomplementary proteins, and secretion from the cells of interest, the coding sequences for these proteins are preceded by a novel N-terminal minimal signal peptide coding sequence derived from known human secreted protein sequences. The secretory signal peptide comprises a methionine starting amino acid, a series of 2 or more basic amino acids (arginine or lysine), followed by a series of hydrophobic amino acids (leucine, isoleucine, valine or phenylalanine) and a final cleavage sequence to allow cleavage of the signal peptide from the final mature secretory protein.

Thus, in one embodiment, the genetic construct comprises a signal peptide coding sequence. Advantageously, the novel signal peptide coding sequences produced by the present inventors optimize intracellular cleavage and transport of secreted proteins within cells. Preferably, the genetic construct comprises a first signal peptide coding sequence placed before the first coding sequence and a second signal peptide coding sequence placed before the second coding sequence. Preferably, the first and second signal peptide coding sequences are placed 5' to the first and second coding sequences, respectively.

In one embodiment, the inventors used Signal peptide optimized at the thread sequence Signal-P5.0 (http:// www.cbs.dtu.dk/services/SignalP/data.php) to increase the cleavage level of Signal peptidase, thereby enhancing cell secretion of mature PEDF. The N-terminal glutamine [ Q ] and alanine [ A ] amino acids of endogenous signal peptide sequence MQALVLLLCIGALLGHSSC [ SEQ ID No. 79] have been substituted with basic amino acids such as arginine [ R ] or lysine [ K ] residues, and the terminal three amino acids [ SSC ] have been replaced with [ VFC ].

Thus, in one embodiment, the signal peptide coding sequence encodes an amino acid sequence referred to herein as SEQ ID No.51, or a fragment or variant thereof, as shown below:

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 set forth below:

ATGCGCCGCCTGGTGCTGCTGCTGTGCATCGGCGCCCTGCTGGGCCACGTGTTCTGC

[SEQ ID No:52]

in order to release the anticomplementary protein (anti-C3 b, anti-Bb or anti-C5 single chain variable fragment, CD55, sCD46, CFHL1 or CFHR 1) from the producer cell, the coding sequence is processed by a modified form of a signal peptide from human brain-derived neurotrophic factor (BDNF) in which the N-terminal amino acid has been replaced by arginine [ R ] or lysine [ K ].

Thus, in one embodiment, the signal peptide coding sequence encodes an amino acid sequence referred to herein as SEQ ID No:53, or a fragment or variant thereof, as shown below:

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 set forth below:

ATGCGCCGCTTCCTGACCGTGATCAGCTTCCTGCTGTACTTCGGCTGCGCCTTCGCC

[SEQ ID No:54]

thus, preferably, the signal peptide coding sequence comprises a nucleotide sequence substantially as shown 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 shown in SEQ ID No. 51 or 53, or a fragment or variant thereof.

In a preferred embodiment, the genetic construct may comprise a 5' promoter in the following specific order; a first coding sequence encoding a PEDF receptor agonist; and a 3' second coding sequence encoding an anticomplementary protein. The use of 5 'and 3' to denote that these features are either upstream or downstream does not mean that these features are necessarily terminal features. It will be appreciated by those skilled in the art that the first and second coding sequences encoding the PEDF receptor agonist and the anticomplementary protein may be placed in any 5 'to 3' order.

In particular embodiments, the genetic construct may comprise a 5' promoter in the following particular order; a first coding sequence encoding a PEDF receptor agonist; a spacer sequence; and a 3' second coding sequence encoding an anticomplementary protein.

In particular embodiments, the genetic construct may comprise a 5' promoter in the following particular order; a first coding sequence encoding a PEDF receptor agonist; virus-2A removal sequences; a spacer sequence; and a 3' second coding sequence encoding an anticomplementary protein.

In particular embodiments, the genetic construct may comprise 5' itrs in the following particular order; a promoter; a first coding sequence encoding a PEDF receptor agonist; virus-2A removal sequences; a spacer sequence; a second coding sequence encoding an anti-complement protein; a sequence encoding WPRE; a sequence encoding a poly a tail; and 3' ITR.

In particular embodiments, the genetic construct may comprise 5' itrs in the following particular order; a promoter; a non-coding intron; a first coding sequence encoding a PEDF receptor agonist; virus-2A removal sequences; a spacer sequence; a second coding sequence encoding an anti-complement protein; a sequence encoding WPRE; a sequence encoding a poly a tail; and 3' ITR.

In particular embodiments, the genetic construct may comprise 5' itrs in the following particular order; a promoter; a non-coding intron; a first signal peptide coding sequence; a first coding sequence encoding a PEDF receptor agonist; virus-2A removal sequences; a spacer sequence; a second signal peptide coding sequence; a second coding sequence encoding an anti-complement protein; a sequence encoding WPRE; a sequence encoding a poly a tail; and 3' ITR.

From the above, the person skilled in the art will know 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-C3 b SCVF is referred to herein as SEQ ID No:55, as shown below:

MRRLVLLLCIGALLGHVFCQNPASPPEEGSPDPDSTGALVEEEDPFFKVPVN

KLAAAVSNFGYDLYRVRSSTSPTTNVLLSPLSVATALSALSLGAEQRTESIIH

RALYYDLISSPDIHGTYKELLDTVTAPQKNLKSASRIVFEKKLRIKSSFVAPL

EKSYGTRPRVLTGNPRLDLQEINNWVQAQMKGKLARSTKEIPDEISILLLGV

AHFKGQWVTKFDSRKTSLEDFYLDEERTVRVPMMSDPKAVLRYGLDSDLS

CKIAQLPLTGSMSIIFFLPLKVTQNLTLIEESLTSEFIHDIDRELKTVQAVLTVP

KLKLSYEGEVTKSLQEMKLQSLFDSPDFSKITGKPIKLTQVEHRAGFEWNE

DGAGTTPSPGLQPAHLTFPLDYHLNQPFIFVLRDTDTGALLFIGKILDPRGPR

RSKRSGSGATNFSLLKQAGDVEENPGPMRRLLTFISILALVGAFADIQMTQS

PSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVP

SRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYATLPTFEQGTKVEIKRGGGG

GSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFSFTSSSV

SPGKGLEWVGLIYPYNGFNYYADSVKGRFTISADTSLQMNSLRAEDTAVYY

CARNALYGSGGYYAMDYWGQGTLVTVSS

[SEQ ID No:55]

preferably, in this embodiment, the construct comprises the 2121 nucleotide sequence referred to herein as SEQ ID No:56 (contained within plasmid IKC 157P) or a fragment or variant thereof as shown below: ATGCGCCGCCTGGTGCTGCTGCTGTGCATCGGCGCCCTGCTGGGCCACGTGTTCTGCCAGAACCCCGCCAGCCCCCCCGAGGAGGGCAGCCCCGACCCCGACAGCACCGGCGCCCTGGTGGAGGAGGAGGACCCCTTCTTCAAGGTGCCCGTGAACAAGCTGGCCGCCGCCGTGAGCAACTTCGGCTACGACCTGTACCGCGTGCGCAGCAGCACCAGCCCCACCACCAACGTGCTGCTGAGCCCCCTGAGCGTGGCCACCGCCCTGAGCGCCCTGAGCCTGGGCGCCGAGCAGCGCACCGAGAGCATCATCCACCGCGCCCTGTACTACGACCTGATCAGCAGCCCCGACATCCACGGCACCTACAAGGAGCTGCTGGACACCGTGACCGCCCCCCAGAAGAACCTGAAGAGCGCCAGCCGCATCGTGTTCGAGAAGAAGCTGCGCATCAAGAGCAGCTTCGTGGCCCCCCTGGAGAAGAGCTACGGCACCCGCCCCCGCGTGCTGACCGGCAACCCCCGCCTGGACCTGCAGGAGATCAACAACTGGGTGCAGGCCCAGATGAAGGGCAAGCTGGCCCGCAGCACCAAGGAGATCCCCGACGAGATCAGCATCCTGCTGCTGGGCGTGGCCCACTTCAAGGGCCAGTGGGTGACCAAGTTCGACAGCCGCAAGACCAGCCTGGAGGACTTCTACCTGGACGAGGAGCGCACCGTGCGCGTGCCCATGATGAGCGACCCCAAGGCCGTGCTGCGCTACGGCCTGGACAGCGACCTGAGCTGCAAGATCGCCCAGCTGCCCCTGACCGGCAGCATGAGCATCATCTTCTTCCTGCCCCTGAAGGTGACCCAGAACCTGACCCTGATCGAGGAGAGCCTGACCAGCGAGTTCATCCACGACATCGACCGCGAGCTGAAGACCGTGCAGGCCGTGCTGACCGTGCCCAAGCTGAAGCTGAGCTACGAGGGCGAGGTGACCAAGAGCCTGCAGGAGATGAAGCTGCAGAGCCTGTTCGACAGCCCCGACTTCAGCAAGATCACCGGCAAGCCCATCAAGCTGACCCAGGTGGAGCACCGCGCCGGCTTCGAGTGGAACGAGGACGGCGCCGGCACCACCCCCAGCCCCGGCCTGCAGCCCGCCCACCTGACCTTCCCCCTGGACTACCACCTGAACCAGCCCTTCATCTTCGTGCTGCGCGACACCGACACCGGCGCCCTGCTGTTCATCGGCAAGATCCTGGACCCCCGCGGCCCCCGCCGCAGCAAGCGCAGCGGCAGCGGCGCCACCAACTTCAGCCTGCTGAAGCAGGCCGGCGACGTGGAGGAGAACCCCGGCCCCATGCGCCGCCTGCTGACCTTCATCAGCATCCTGGCCCTGGTGGGCGCCTTCGCCGACATCCAGATGACCCAGAGCCCCAGCAGCCTGAGCGCCAGCGTGGGCGACCGCGTGACCATCACCTGCCGCGCCAGCCAGGACGTAAGCACCGCCGTGGCCTGGTACCAGCAGAAGCCCGGCAAGGCCCCCAAGCTGCTGATCTACAGCGCCAGCTTCCTGTACAGCGGCGTGCCCAGCCGCTTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGACCATCAGCAGCCTGCAGCCCGAGGACTTCGCCACCTACTACTGCCAGCAGAGCTACGCCACCCTGCCCACCTTCGAGCAGGGCACCAAGGTGGAGATCAAGCGCGGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGAGGTGCAGCTGGTGGAGAGCGGCGGCGGCCTGGTGCAGCCCGGCGGCAGCCTGCGCCTGAGCTGCGCCGCCAGCGGCTTCAGCTTCACCAGCAGCAGCGTGAGCCCCGGCAAGGGCCTGGAGTGGGTGGGCCTGATCTACCCCTACAACGGCTTCAACTACTACGCCGACAGCGTGAAGGGCCGCTTCACCATCAGCGCCGACACCAGCCTGCAGATGAACAGCCTGCGCGCCGAGGACACCGCCGTGTACTACTGCGCCCGCAACGCCCTGTACGGCAGCGGCGGCTACTACGCCATGGACTACTGGGGCCAGGGCACCCTGGTGACCGTGAGCAGCTGA

[SEQ ID No:56]

In another embodiment, the amino acid sequence of PEDF-furin-P2A-sCD 55 is referred to herein as SEQ ID No. 57, or a fragment or variant thereof, as shown below: MRRLVLLLCIGALLGHVFCQNPASPPEEGSPDPDSTGALVEEEDPFFKVPVNKLAAAVSNFGYDLYRVRSSTSPTTNVLLSPLSVATALSALSLGAEQRTESIIHRALYYDLISSPDIHGTYKELLDTVTAPQKNLKSASRIVFEKKLRIKSSFVAPLEKSYGTRPRVLTGNPRLDLQEINNWVQAQMKGKLARSTKEIPDEISILLLGVAHFKGQWVTKFDSRKTSLEDFYLDEERTVRVPMMSDPKAVLRYGLDSDLSCKIAQLPLTGSMSIIFFLPLKVTQNLTLIEESLTSEFIHDIDRELKTVQAVLTVPKLKLSYEGEVTKSLQEMKLQSLFDSPDFSKITGKPIKLTQVEHRAGFEWNEDGAGTTPSPGLQPAHLTFPLDYHLNQPFIFVLRDTDTGALLFIGKILDPRGPRRSKRSGSGATNFSLLKQAGDVEENPGPMRRFLTVISFLLYFGCAFADCGLPPDVPNAQPALEGRTSFPEDTVITYKCEESFVKIPGEKDSVICLKGSQWSDIEEFCNRSCEVPTRLNSASLKQPYITQNYFPVGTVVEYECRPGYRREPSLSPKLTCLQNLKWSTAVEFCKKKSCPNPGEIRNGQIDVPGGILFGATISFSCNTGYKLFGSTSSFCLISGSSVQWSDPLPECREIYCPAPPQIDNGIIQGERDHYGYRQSVTYACNKGFTMIGEHSIYCTVNNDEGEWSGPPPECRGKSLTSKVPPTVQKPTTVNVPTTEVSPTSQKTTTKTTTPNAQATRSTPVSRTTKHFHETTPNKGSGTTSG

[SEQ ID No:57]

Preferably, in this embodiment, the construct comprises the 2358 nucleotide sequence referred to herein as SEQ ID No:58 (as contained within plasmid IKC 158P) or a fragment or variant thereof, as shown below: ATGCGCCGCCTGGTGCTGCTGCTGTGCATCGGCGCCCTGCTGGGCCACGTGTTCTGCCAGAACCCCGCCAGCCCCCCCGAGGAGGGCAGCCCCGACCCCGACAGCACCGGCGCCCTGGTGGAGGAGGAGGACCCCTTCTTCAAGGTGCCCGTGAACAAGCTGGCCGCCGCCGTGAGCAACTTCGGCTACGACCTGTACCGCGTGCGCAGCAGCACCAGCCCCACCACCAACGTGCTGCTGAGCCCCCTGAGCGTGGCCACCGCCCTGAGCGCCCTGAGCCTGGGCGCCGAGCAGCGCACCGAGAGCATCATCCACCGCGCCCTGTACTACGACCTGATCAGCAGCCCCGACATCCACGGCACCTACAAGGAGCTGCTGGACACCGTGACCGCCCCCCAGAAGAACCTGAAGAGCGCCAGCCGCATCGTGTTCGAGAAGAAGCTGCGCATCAAGAGCAGCTTCGTGGCCCCCCTGGAGAAGAGCTACGGCACCCGCCCCCGCGTGCTGACCGGCAACCCCCGCCTGGACCTGCAGGAGATCAACAACTGGGTGCAGGCCCAGATGAAGGGCAAGCTGGCCCGCAGCACCAAGGAGATCCCCGACGAGATCAGCATCCTGCTGCTGGGCGTGGCCCACTTCAAGGGCCAGTGGGTGACCAAGTTCGACAGCCGCAAGACCAGCCTGGAGGACTTCTACCTGGACGAGGAGCGCACCGTGCGCGTGCCCATGATGAGCGACCCCAAGGCCGTGCTGCGCTACGGCCTGGACAGCGACCTGAGCTGCAAGATCGCCCAGCTGCCCCTGACCGGCAGCATGAGCATCATCT

TCTTCCTGCCCCTGAAGGTGACCCAGAACCTGACCCTGATCGAGGAGAG

CCTGACCAGCGAGTTCATCCACGACATCGACCGCGAGCTGAAGACCGTG

CAGGCCGTGCTGACCGTGCCCAAGCTGAAGCTGAGCTACGAGGGCGAG

GTGACCAAGAGCCTGCAGGAGATGAAGCTGCAGAGCCTGTTCGACAGC

CCCGACTTCAGCAAGATCACCGGCAAGCCCATCAAGCTGACCCAGGTGG

AGCACCGCGCCGGCTTCGAGTGGAACGAGGACGGCGCCGGCACCACCC

CCAGCCCCGGCCTGCAGCCCGCCCACCTGACCTTCCCCCTGGACTACCA

CCTGAACCAGCCCTTCATCTTCGTGCTGCGCGACACCGACACCGGCGCC

CTGCTGTTCATCGGCAAGATCCTGGACCCCCGCGGCCCCCGCCGCAGCA

AGCGCAGCGGCAGCGGCGCCACCAACTTCAGCCTGCTGAAGCAGGCCG

GCGACGTGGAGGAGAACCCCGGCCCCATGCGCCGCTTCCTGACCGTGAT

CAGCTTCCTGCTGTACTTCGGCTGCGCCTTCGCCGACTGCGGCCTGCCCC

CCGACGTGCCCAACGCCCAGCCCGCCCTGGAGGGCCGCACCAGCTTCCC

CGAGGACACCGTGATCACCTACAAGTGCGAGGAGAGCTTCGTGAAGATC

CCCGGCGAGAAGGACAGCGTGATCTGCCTGAAGGGCAGCCAGTGGAGC

GACATCGAGGAGTTCTGCAACCGCAGCTGCGAGGTGCCCACCCGCCTGA

ACAGCGCCAGCCTGAAGCAGCCCTACATCACCCAGAACTACTTCCCCGT

GGGCACCGTGGTGGAGTACGAGTGCCGCCCCGGCTACCGCCGCGAGCC

CAGCCTGAGCCCCAAGCTGACCTGCCTGCAGAACCTGAAGTGGAGCAC

CGCCGTGGAGTTCTGCAAGAAGAAGAGCTGCCCCAACCCCGGCGAGAT

CCGCAACGGCCAGATCGACGTGCCCGGCGGCATCCTGTTCGGCGCCACC

ATCAGCTTCAGCTGCAACACCGGCTACAAGCTGTTCGGCAGCACCAGCA

GCTTCTGCCTGATCAGCGGCAGCAGCGTGCAGTGGAGCGACCCCCTGCC

CGAGTGCCGCGAGATCTACTGCCCCGCCCCCCCCCAGATCGACAACGGC

ATCATCCAGGGCGAGCGCGACCACTACGGCTACCGCCAGAGCGTGACCT

ACGCCTGCAACAAGGGCTTCACCATGATCGGCGAGCACAGCATCTACTG

CACCGTGAACAACGACGAGGGCGAGTGGAGCGGCCCCCCCCCCGAGTG

CCGAGGCAAGAGCCTGACCAGCAAGGTGCCCCCCACCGTGCAGAAGCC

CACCACCGTGAACGTGCCCACCACCGAGGTGAGCCCCACCAGCCAGAA

GACCACCACCAAGACCACCACCCCCAACGCCCAGGCCACCCGCAGCAC

CCCCGTGAGCCGCACCACCAAGCACTTCCACGAGACCACCCCCAACAA

GGGCAGCGGCACCACCAGCGGCTAA

[SEQ ID No:58]

In another embodiment, the amino acid sequence of sCD 55-furin-P2A-PEDF is referred to herein as SEQ ID No. 59, or a fragment or variant thereof, as shown below: MRRFLTVISFLLYFGCAFADCGLPPDVPNAQPALEGRTSFPEDTVITYKCEESFVKIPGEKDSVICLKGSQWSDIEEFCNRSCEVPTRLNSASLKQPYITQNYFPVGTVVEYECRPGYRREPSLSPKLTCLQNLKWSTAVEFCKKKSCPNPGEIRNGQIDVPGGILFGATISFSCNTGYKLFGSTSSFCLISGSSVQWSDPLPECREIYCPAPPQIDNGIIQGERDHYGYRQSVTYACNKGFTMIGEHSIYCTVNNDEGEWSGPPPECRGKSLTSKVPPTVQKPTTVNVPTTEVSPTSQKTTTKTTTPNAQATRSTPVSRTTKHFHETTPNKGSGTTSGRRSKRSGSGATNFSLLKQAGDVEENPGPMRRLVLLLCIGALLGHVFCQNPASPPEEGSPDPDSTGALVEEEDPFFKVPVNKLAAAVSNFGYDLYRVRSSTSPTTNVLLSPLSVATALSALSLGAEQRTESIIHRALYYDLISSPDIHGTYKELLDTVTAPQKNLKSASRIVFEKKLRIKSSFVAPLEKSYGTRPRVLTGNPRLDLQEINNWVQAQMKGKLARSTKEIPDEISILLLGVAHFKGQWVTKFDSRKTSLEDFYLDEERTVRVPMMSDPKAVLRYGLDSDLSCKIAQLPLTGSMSIIFFLPLKVTQNLTLIEESLTSEFIHDIDRELKTVQAVLTVPKLKLSYEGEVTKSLQEMKLQSLFDSPDFSKITGKPIKLTQVEHRAGFEWNEDGAGTTPSPGLQPAHLTFPLDYHLNQPFIFVLRDTDTGALLFIGKILDPRGP

[SEQ ID No:59]

Preferably, in this embodiment, the construct comprises the 2358 nucleotide sequence referred to herein as SEQ ID No. 60 (as contained within plasmid IKC 126P) or a fragment or variant thereof as shown below: ATGCGCCGCTTCCTGACCGTGATCAGCTTCCTGCTGTACTTCGGCTGCGCCTTCGCCGACTGCGGCCTGCCCCCCGACGTGCCCAACGCCCAGCCCGCCCTGGAGGGCCGCACCAGCTTCCCCGAGGACACCGTGATCACCTACAAGTGCGAGGAGAGCTTCGTGAAGATCCCCGGCGAGAAGGACAGCGTGATCTGCCTGAAGGGCAGCCAGTGGAGCGACATCGAGGAGTTCTGCAACCGCA

GCTGCGAGGTGCCCACCCGCCTGAACAGCGCCAGCCTGAAGCAGCCCT

ACATCACCCAGAACTACTTCCCCGTGGGCACCGTGGTGGAGTACGAGTG

CCGCCCCGGCTACCGCCGCGAGCCCAGCCTGAGCCCCAAGCTGACCTGC

CTGCAGAACCTGAAGTGGAGCACCGCCGTGGAGTTCTGCAAGAAGAAG

AGCTGCCCCAACCCCGGCGAGATCCGCAACGGCCAGATCGACGTGCCCG

GCGGCATCCTGTTCGGCGCCACCATCAGCTTCAGCTGCAACACCGGCTA

CAAGCTGTTCGGCAGCACCAGCAGCTTCTGCCTGATCAGCGGCAGCAGC

GTGCAGTGGAGCGACCCCCTGCCCGAGTGCCGCGAGATCTACTGCCCCG

CCCCCCCCCAGATCGACAACGGCATCATCCAGGGCGAGCGCGACCACTA

CGGCTACCGCCAGAGCGTGACCTACGCCTGCAACAAGGGCTTCACCATG

ATCGGCGAGCACAGCATCTACTGCACCGTGAACAACGACGAGGGCGAG

TGGAGCGGCCCCCCCCCCGAGTGCCGAGGCAAGAGCCTGACCAGCAAG

GTGCCCCCCACCGTGCAGAAGCCCACCACCGTGAACGTGCCCACCACC

GAGGTGAGCCCCACCAGCCAGAAGACCACCACCAAGACCACCACCCCC

AACGCCCAGGCCACCCGCAGCACCCCCGTGAGCCGCACCACCAAGCAC

TTCCACGAGACCACCCCCAACAAGGGCAGCGGCACCACCAGCGGCCGC

CGCAGCAAGCGCAGCGGCAGCGGCGCCACCAACTTCAGCCTGCTGAAG

CAGGCCGGCGACGTGGAGGAGAACCCCGGCCCCATGCGCCGCCTGGTG

CTGCTGCTGTGCATCGGCGCCCTGCTGGGCCACGTGTTCTGCCAGAACC

CCGCCAGCCCCCCCGAGGAGGGCAGCCCCGACCCCGACAGCACCGGCG

CCCTGGTGGAGGAGGAGGACCCCTTCTTCAAGGTGCCCGTGAACAAGC

TGGCCGCCGCCGTGAGCAACTTCGGCTACGACCTGTACCGCGTGCGCAG

CAGCACCAGCCCCACCACCAACGTGCTGCTGAGCCCCCTGAGCGTGGCC

ACCGCCCTGAGCGCCCTGAGCCTGGGCGCCGAGCAGCGCACCGAGAGC

ATCATCCACCGCGCCCTGTACTACGACCTGATCAGCAGCCCCGACATCCA

CGGCACCTACAAGGAGCTGCTGGACACCGTGACCGCCCCCCAGAAGAA

CCTGAAGAGCGCCAGCCGCATCGTGTTCGAGAAGAAGCTGCGCATCAA

GAGCAGCTTCGTGGCCCCCCTGGAGAAGAGCTACGGCACCCGCCCCCGC

GTGCTGACCGGCAACCCCCGCCTGGACCTGCAGGAGATCAACAACTGG

GTGCAGGCCCAGATGAAGGGCAAGCTGGCCCGCAGCACCAAGGAGATC

CCCGACGAGATCAGCATCCTGCTGCTGGGCGTGGCCCACTTCAAGGGCC

AGTGGGTGACCAAGTTCGACAGCCGCAAGACCAGCCTGGAGGACTTCT

ACCTGGACGAGGAGCGCACCGTGCGCGTGCCCATGATGAGCGACCCCA

AGGCCGTGCTGCGCTACGGCCTGGACAGCGACCTGAGCTGCAAGATCGC

CCAGCTGCCCCTGACCGGCAGCATGAGCATCATCTTCTTCCTGCCCCTGA

AGGTGACCCAGAACCTGACCCTGATCGAGGAGAGCCTGACCAGCGAGT

TCATCCACGACATCGACCGCGAGCTGAAGACCGTGCAGGCCGTGCTGAC

CGTGCCCAAGCTGAAGCTGAGCTACGAGGGCGAGGTGACCAAGAGCCT

GCAGGAGATGAAGCTGCAGAGCCTGTTCGACAGCCCCGACTTCAGCAA

GATCACCGGCAAGCCCATCAAGCTGACCCAGGTGGAGCACCGCGCCGG

CTTCGAGTGGAACGAGGACGGCGCCGGCACCACCCCCAGCCCCGGCCT

GCAGCCCGCCCACCTGACCTTCCCCCTGGACTACCACCTGAACCAGCCC

TTCATCTTCGTGCTGCGCGACACCGACACCGGCGCCCTGCTGTTCATCGG

CAAGATCCTGGACCCCCGCGGCCCCTAA

[SEQ ID No:60]

In another embodiment, the amino acid sequence of PEDF-furin-P2A-CFHR 1 is referred to herein as SEQ ID No. 61, or a fragment or variant thereof, as shown below: MRRLVLLLCIGALLGHVFCQNPASPPEEGSPDPDSTGALVEEEDPFFKVPVNKLAAAVSNFGYDLYRVRSSTSPTTNVLLSPLSVATALSALSLGAEQRTESIIHRALYYDLISSPDIHGTYKELLDTVTAPQKNLKSASRIVFEKKLRIKSSFVAPLEKSYGTRPRVLTGNPRLDLQEINNWVQAQMKGKLARSTKEIPDEISILLLGVAHFKGQWVTKFDSRKTSLEDFYLDEERTVRVPMMSDPKAVLRYGLDSDLSCKIAQLPLTGSMSIIFFLPLKVTQNLTLIEESLTSEFIHDIDRELKTVQAVLTVPKLKLSYEGEVTKSLQEMKLQSLFDSPDFSKITGKPIKLTQVEHRAGFEWNEDGAGTTPSPGLQPAHLTFPLDYHLNQPFIFVLRDTDTGALLFIGKILDPRGPRRSKRSGSGATNFSLLKQAGDVEENPGPMRRFLTVISFLLYFGCAFAEATFCDFPKINHGILYDEEKYKPFSQVPTGEVFYYSCEYNFVSPSKSFWTRITCTEEGWSPTPKCLRLCFFPFVENGHSESSGQTHLEGDTVQIICNTGYRLQNNENNISCVERGWSTPPKCRSTDTSCVNPPTVQNAHILSRQMSKYPSGERVRYECRSPYEMFGDEEVMCLNGNWTEPPQCKDSTGKCGPPPPIDNGDITSFPLSVYAPASSVEYQCQNLYQLEGNKRITCRNGQWSEPPKCLHPCVISREIMENYNIALRWTAKQKLYLRTGESAEFVCKRGYRLSSRSHTLRTTCWDGKLEYPTCAKR

[SEQ ID No:61]

Preferably, in this embodiment, the construct comprises the 2334 nucleotide sequence referred to herein as SEQ ID No. 62 (as contained within plasmid IKC 127P) or a fragment or variant thereof, as shown below: ATGCGCCGCCTGGTGCTGCTGCTGTGCATCGGCGCCCTGCTGGGCCACGTGTTCTGCCAGAACCCCGCCAGCCCCCCCGAGGAGGGCAGCCCCGACCCCGACAGCACCGGCGCCCTGGTGGAGGAGGAGGACCCCTTCTTCAAGGTGCCCGTGAACAAGCTGGCCGCCGCCGTGAGCAACTTCGGCTACGACCTGTACCGCGTGCGCAGCAGCACCAGCCCCACCACCAACGTGCTGCTGAGCCCCCTGAGCGTGGCCACCGCCCTGAGCGCCCTGAGCCTGGGCGCCGAGCAGCGCACCGAGAGCATCATCCACCGCGCCCTGTACTACGACCTGATCAGCAGCCCCGACATCCACGGCACCTACAAGGAGCTGCTGGACACCGTGACCGCCCCCCAGAAGAACCTGAAGAGCGCCAGCCGCATCGTGTTCGAGAAGAAGCTGCGCATCAAGAGCAGCTTCGTGGCCCCCCTGGAGAAGAGCTACGGCACCCGCCCCCGCGTGCTGACCGGCAACCCCCGCCTGGACCTGCAGGAGATCAACAACTGGGTGCAGGCCCAGATGAAGGGCAAGCTGGCCCGCAGCACCAAGGAGATCCCCGACGAGATCAGCATCCTGCTGCTGGGCGTGGCCCACTTCAAGGGCCAGTGGGTGACCAAGTTCGACAGCCGCAAGACCAGCCTGGAGGACTTCTACCTGGACGAGGAGCGCACCGTGCGCGTGCCCATGATGAGCGACCCCAAGGCCGTGCTGCGCTACGGCCTGGACAGCGACCTGAGCTGCAAGATCGCCCAGCTGCCCCTGACCGGCAGCATGAGCATCATCTTCTTCCTGCCCCTGAAGGTGACCCAGAACCTGACCCTGATCGAGGAGAGCCTGACCAGCGAGTTCATCCACGACATCGACCGCGAGCTGAAGACCGTGCAGGCCGTGCTGACCGTGCCCAAGCTGAAGCTGAGCTACGAGGGCGAGGTGACCAAGAGCCTGCAGGAGATGAAGCTGCAGAGCCTGTTCGACAGCCCCGACTTCAGCAAGATCACCGGCAAGCCCATCAAGCTGACCCAGGTGGAGCACCGCGCCGGCTTCGAGTGGAACGAGGACGGCGCCGGCACCACCCCCAGCCCCGGCCTGCAGCCCGCCCACCTGACCTTCCCCCTGGACTACCACCTGAACCAGCCCTTCATCTTCGTGCTGCGCGACACCGACACCGGCGCCCTGCTGTTCATCGGCAAGATCCTGGACCCCCGCGGCCCCCGCCGCAGCAAGCGCAGCGGCAGCGGCGCCACCAACTTCAGCCTGCTGAAGCAGGCCGGCGACGTGGAGGAGAACCCCGGCCCCATGCGCCGCTTCCTGACCGTGATCAGCTTCCTGCTGTACTTCGGCTGCGCCTTCGCCGAGGCCACCTTCTGCGACTTCCCCAAGATCAACCACGGCATCCTGTACGACGAGGAGAAGTACAAGCCCTTCAGCCAGGTGCCCACCGGCGAGGTGTTCTACTACAGCTGCGAGTACAACTTCGTGAGCCCCAGCAAGAGCTTCTGGACCCGCATCACCTGCACCGAGGAGGGCTGGAGCCCCACCCCCAAGTGCCTGCGCCTGTGCTTCTTCCCCTTCGTGGAGAACGGCCACAGCGAGAGCAGCGGCCAGACCCACCTGGAGGGCGACACCGTGCAGATCATCTGCAACACCGGCTACCGCCTGCAGAACAACGAGAACAACATCAGCTGCGTGGAGCGCGGCTGGAGCACCCCCCCCAAGTGCCGCAGCACCGACACCAGCTGCGTGAACCCCCCCACCGTGCAGAACGCCCACATCCTGAGCCGCCAGATGAGCAAGTACCCCAGCGGCGAGCGCGTGCGCTACGAGTGCCGCAGCCCCTACGAGATGTTCGGCGACGAGGAGGTGATGTGCCTGAACGGCAACTGGACCGAGCCCCCCCAGTGCAAGGACAGCACCGGCAAGTGCGGCCCCCCCCCCCCCATCGACAACGGCGACATCACCAGCTTCCCCCTGAGCGTGTACGCCCCCGCCAGCAGCGTGGAGTACCAGTGCCAGAACCTGTACCAGCTGGAGGGCAACAAGCGCATCACCTGCCGCAACGGCCAGTGGAGCGAGCCCCCCAAGTGCCTGCACCCCTGCGTGATCAGCCGCGAGATCATGGAGAACTACAACATCGCCCTGCGCTGGACCGCCAAGCAGAAGCTGTACCTGCGCACCGGCGAGAGCGCCGAGTTCGTGTGCAAGCGCGGCTACCGCCTGAGCAGCCGCAGCCACACCCTGCGCACCACCTGCTGGGACGGCAAGCTGGAGTACCCCACCTGCGCCAAGCGCTAA

[SEQ ID No:62]

In another embodiment, the amino acid sequence of CFHR 1-furin-P2A-PEDF is referred to herein as SEQ ID No. 63, or a fragment or variant thereof, as shown below: MRRFLTVISFLLYFGCAFAEATFCDFPKINHGILYDEEKYKPFSQVPTGEVFYYSCEYNFVSPSKSFWTRITCTEEGWSPTPKCLRLCFFPFVENGHSESSGQTHLEGDTVQIICNTGYRLQNNENNISCVERGWSTPPKCRSTDTSCVNPPTVQNAHILSRQMSKYPSGERVRYECRSPYEMFGDEEVMCLNGNWTEPPQCKDSTGKCGPPPPIDNGDITSFPLSVYAPASSVEYQCQNLYQLEGNKRITCRNGQWSEPPKCLHPCVISREIMENYNIALRWTAKQKLYLRTGESAEFVCKRGYRLSSRSHTLRTTCWDGKLEYPTCAKRSKRSGSATNFSLLKQAGDVEENPGPMRRLVLLLCIGALLGHVFCQNPASPPEEGSPDPDSTGALVEEEDPFFKVPVNKLAAAVSNFGYDLYRVRSSTSPTTNVLLSPLSVATALSALSLGAEQRTESIIHRALYYDLISSPDIHGTYKELLDTVTAPQKNLKSASRIVFEKKLRIKSSFVAPLEKSYGTRPRVLTGNPRLDLQEINNWVQAQMKGKLARSTKEIPDEISILLLGVAHFKGQWVTKFDSRKTSLEDFYLDEERTVRVPMMSDPKAVLRYGLDSDLSCKIAQLPLTGSMSIIFFLPLKVTQNLTLIEESLTSEFIHDIDRELKTVQAVLTVPKLKLSYEGEVTKSLQEMKLQSLFDSPDFSKITGKPIKLTQVEHRAGFEWNEDGAGTTPSPGLQPAHLTFPLDYHLNQPFIFVLRDTDTGALLFIGKILDPRGP

[SEQ ID No:63]

Preferably, in this embodiment, the construct comprises the 2325 nucleotide sequence referred to herein as SEQ ID No. 64 (as contained within plasmid IKC 128P) or a fragment or variant thereof, as shown below: ATGCGCCGCCTGCTGACCTTCATCAGCATCCTGGCCCTGGTGGGCGCCTTCGCCGAGGCCACCTTCTGCGACTTCCCCAAGATCAACCACGGCATCCTGTACGACGAGGAGAAGTACAAGCCCTTCAGCCAGGTGCCCACCGGCGAGGTGTTCTACTACAGCTGCGAGTACAACTTCGTGAGCCCCAGCAAGAGCTTCTGGACCCGCATCACCTGCACCGAGGAGGGCTGGAGCCCCACCCCCAAGTGCCTGCGCCTGTGCTTCTTCCCCTTCGTGGAGAACGGCCACAGCGAGAGCAGCGGCCAGACCCACCTGGAGGGCGACACCGTGCAGATCATCTGCAACACCGGCTACCGCCTGCAGAACAACGAGAACAACATCAGCTGCGTGGAGCGCGGCTGGAGCACCCCCCCCAAGTGCCGCAGCACCGACACCAGCTGCGTGAACCCCCCCACCGTGCAGAACGCCCACATCCTGAGCCGCCAGATGAGCAAGTACCCCAGCGGCGAGCGCGTGCGCTACGAGTGCCGCAGCCCCTACGAGATGTTCGGCGACGAGGAGGTGATGTGCCTGAACGGCAACTG

GACCGAGCCCCCCCAGTGCAAGGACAGCACCGGCAAGTGCGGCCCCCC

CCCCCCCATCGACAACGGCGACATCACCAGCTTCCCCCTGAGCGTGTAC

GCCCCCGCCAGCAGCGTGGAGTACCAGTGCCAGAACCTGTACCAGCTGG

AGGGCAACAAGCGCATCACCTGCCGCAACGGCCAGTGGAGCGAGCCCC

CCAAGTGCCTGCACCCCTGCGTGATCAGCCGCGAGATCATGGAGAACTA

CAACATCGCCCTGCGCTGGACCGCCAAGCAGAAGCTGTACCTGCGCACC

GGCGAGAGCGCCGAGTTCGTGTGCAAGCGCGGCTACCGCCTGAGCAGC

CGCAGCCACACCCTGCGCACCACCTGCTGGGACGGCAAGCTGGAGTAC

CCCACCTGCGCCAAGCGCAGCAAGCGCAGCGGCAGCGGCGCCACCAAC

TTCAGCCTGCTGAAGCAGGCCGGCGACGTGGAGGAGAACCCCGGCCCC

ATGCGCCGCCTGGTGCTGCTGCTGTGCATCGGCGCCCTGCTGGGCCACG

TGTTCTGCCAGAACCCCGCCAGCCCCCCCGAGGAGGGCAGCCCCGACC

CCGACAGCACCGGCGCCCTGGTGGAGGAGGAGGACCCCTTCTTCAAGG

TGCCCGTGAACAAGCTGGCCGCCGCCGTGAGCAACTTCGGCTACGACCT

GTACCGCGTGCGCAGCAGCACCAGCCCCACCACCAACGTGCTGCTGAGC

CCCCTGAGCGTGGCCACCGCCCTGAGCGCCCTGAGCCTGGGCGCCGAG

CAGCGCACCGAGAGCATCATCCACCGCGCCCTGTACTACGACCTGATCA

GCAGCCCCGACATCCACGGCACCTACAAGGAGCTGCTGGACACCGTGAC

CGCCCCCCAGAAGAACCTGAAGAGCGCCAGCCGCATCGTGTTCGAGAA

GAAGCTGCGCATCAAGAGCAGCTTCGTGGCCCCCCTGGAGAAGAGCTAC

GGCACCCGCCCCCGCGTGCTGACCGGCAACCCCCGCCTGGACCTGCAG

GAGATCAACAACTGGGTGCAGGCCCAGATGAAGGGCAAGCTGGCCCGC

AGCACCAAGGAGATCCCCGACGAGATCAGCATCCTGCTGCTGGGCGTGG

CCCACTTCAAGGGCCAGTGGGTGACCAAGTTCGACAGCCGCAAGACCA

GCCTGGAGGACTTCTACCTGGACGAGGAGCGCACCGTGCGCGTGCCCAT

GATGAGCGACCCCAAGGCCGTGCTGCGCTACGGCCTGGACAGCGACCTG

AGCTGCAAGATCGCCCAGCTGCCCCTGACCGGCAGCATGAGCATCATCT

TCTTCCTGCCCCTGAAGGTGACCCAGAACCTGACCCTGATCGAGGAGAG

CCTGACCAGCGAGTTCATCCACGACATCGACCGCGAGCTGAAGACCGTG

CAGGCCGTGCTGACCGTGCCCAAGCTGAAGCTGAGCTACGAGGGCGAG

GTGACCAAGAGCCTGCAGGAGATGAAGCTGCAGAGCCTGTTCGACAGC

CCCGACTTCAGCAAGATCACCGGCAAGCCCATCAAGCTGACCCAGGTGG

AGCACCGCGCCGGCTTCGAGTGGAACGAGGACGGCGCCGGCACCACCC

CCAGCCCCGGCCTGCAGCCCGCCCACCTGACCTTCCCCCTGGACTACCA

CCTGAACCAGCCCTTCATCTTCGTGCTGCGCGACACCGACACCGGCGCC

CTGCTGTTCATCGGCAAGATCCTGGACCCCCGCGGCCCCTAA

[SEQ ID No:64]

In another embodiment, the amino acid sequence of PEDF-furin-P2A-anti-C5 SCVF is referred to herein as SEQ ID No. 85, or a fragment or variant thereof, as shown below: MRRLVLLLCIGALLGHSSCQNPASPPEEGSPDPDSTGALVEEEDPFFKVPVNKLAAAVSNFGYDLYRVRSSTSPTTNVLLSPLSVATALSALSLGAEQRTESIIHRALYYDLISSPDIHGTYKELLDTVTAPQKNLKSASRIVFEKKLRIKSSFVAPLEKSYGTRPRVLTGNPRLDLQEINNWVQAQMKGKLARSTKEIPDEISILLLGVAHFKGQWVTKFDSRKTSLEDFYLDEERTVRVPMMSDPKAVLRYGLDSDLSCKIAQLPLTGSMSIIFFLPLKVTQNLTLIEESLTSEFIHDIDRELKTVQAVLTVPKLKLSYEGEVTKSLQEMKLQSLFDSPDFSKITGKPIKLTQVEHRAGFEWNEDGAGTTPSPGLQPAHLTFPLDYHLNQPFIFVLRDTDTGALLFIGKILDPRGPRVRRGSGATNFSLLKQAGDVEENPGPMRRLLILALVGAAVADIQMTQSPSSLSASVGDRVTITCQASQSINNQLSWYQQKPGKAPKLLIYYASTLASGYPSRFSGSGSGTDFTLTISSLQPEDFATYYCQGSYYSGGWDYGFGQGTKVEIKRGGGGGSGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFSFSGRYWIQWVRQAPGKGLEWVASVWPGITGTNYANWAKGRFTISRDDSKNTLYLQMNSLRAEDTAVYYCAREPVAWGGGLDLWGQGTLVTVSS

[SEQ ID No:85]

Preferably, in this embodiment, the construct comprises the 2136 nucleotide sequence referred to herein as SEQ ID No. 86 (as contained within plasmid IKC 094P) or a fragment or variant thereof, as shown below: ATGCGCCGCCTGGTGCTGCTGCTGTGCATCGGCGCCCTGCTGGGCCACAGCAGCTGCCAGAACCCCGCCAGCCCCCCCGAGGAGGGCAGCCCCGACC

CCGACAGCACCGGCGCCCTGGTGGAGGAGGAGGACCCCTTCTTCAAGG

TGCCCGTGAACAAGCTGGCCGCCGCCGTGAGCAACTTCGGCTACGACCT

GTACCGCGTGCGCAGCAGCACCAGCCCCACCACCAACGTGCTGCTGAGC

CCCCTGAGCGTGGCCACCGCCCTGAGCGCCCTGAGCCTGGGCGCCGAG

CAGCGCACCGAGAGCATCATCCACCGCGCCCTGTACTACGACCTGATCA

GCAGCCCCGACATCCACGGCACCTACAAGGAGCTGCTGGACACCGTGAC

CGCCCCCCAGAAGAACCTGAAGAGCGCCAGCCGCATCGTGTTCGAGAA

GAAGCTGCGCATCAAGAGCAGCTTCGTGGCCCCCCTGGAGAAGAGCTAC

GGCACCCGCCCCCGCGTGCTGACCGGCAACCCCCGCCTGGACCTGCAG

GAGATCAACAACTGGGTGCAGGCCCAGATGAAGGGCAAGCTGGCCCGC

AGCACCAAGGAGATCCCCGACGAGATCAGCATCCTGCTGCTGGGCGTGG

CCCACTTCAAGGGCCAGTGGGTGACCAAGTTCGACAGCCGCAAGACCA

GCCTGGAGGACTTCTACCTGGACGAGGAGCGCACCGTGCGCGTGCCCAT

GATGAGCGACCCCAAGGCCGTGCTGCGCTACGGCCTGGACAGCGACCTG

AGCTGCAAGATCGCCCAGCTGCCCCTGACCGGCAGCATGAGCATCATCT

TCTTCCTGCCCCTGAAGGTGACCCAGAACCTGACCCTGATCGAGGAGAG

CCTGACCAGCGAGTTCATCCACGACATCGACCGCGAGCTGAAGACCGTG

CAGGCCGTGCTGACCGTGCCCAAGCTGAAGCTGAGCTACGAGGGCGAG

GTGACCAAGAGCCTGCAGGAGATGAAGCTGCAGAGCCTGTTCGACAGC

CCCGACTTCAGCAAGATCACCGGCAAGCCCATCAAGCTGACCCAGGTGG

AGCACCGCGCCGGCTTCGAGTGGAACGAGGACGGCGCCGGCACCACCC

CCAGCCCCGGCCTGCAGCCCGCCCACCTGACCTTCCCCCTGGACTACCA

CCTGAACCAGCCCTTCATCTTCGTGCTGCGCGACACCGACACCGGCGCC

CTGCTGTTCATCGGCAAGATCCTGGACCCCCGCGGCCCCCGCGTGCGCC

GCGGCAGCGGCGCCACCAACTTCAGCCTGCTGAAGCAGGCCGGCGACG

TGGAGGAGAACCCCGGCCCCATGCGCCGCCTGCTGATCCTGGCCCTGGT

GGGCGCCGCCGTGGCCGACATCCAGATGACCCAGAGCCCCAGCAGCCT

GAGCGCCAGCGTGGGCGACCGCGTGACCATCACCTGCCAGGCCAGCCA

GAGCATCAACAACCAGCTGAGCTGGTACCAGCAGAAGCCCGGCAAGGC

CCCCAAGCTGCTGATCTACTACGCCAGCACCCTGGCCAGCGGCTACCCC

AGCCGCTTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGACCATCA

GCAGCCTGCAGCCCGAGGACTTCGCCACCTACTACTGCCAGGGCAGCTA

CTACAGCGGCGGCTGGGACTACGGCTTCGGCCAGGGCACCAAGGTGGA

GATCAAGCGCGGCGGCGGCGGCGGCAGCGGCGGCGGCAGCGGCGGCG

GCGGCAGCGGCGGCGGCGGCAGCGAGGTGCAGCTGGTGGAGAGCGGC

GGCGGCCTGGTGCAGCCCGGCGGCAGCCTGCGCCTGAGCTGCGCCGCC

AGCGGCTTCAGCTTCAGCGGCCGCTACTGGATCCAGTGGGTGCGCCAGG

CCCCCGGCAAGGGCCTGGAGTGGGTGGCCAGCGTGTGGCCCGGCATCA

CCGGCACCAACTACGCCAACTGGGCCAAGGGCCGCTTCACCATCAGCCG

CGACGACAGCAAGAACACCCTGTACCTGCAGATGAACAGCCTGCGCGC

CGAGGACACCGCCGTGTACTACTGCGCCCGCGAGCCCGTGGCCTGGGGC

GGCGGCCTGGACCTGTGGGGCCAGGGCACCCTGGTGACCGTGAGCAGC

TGA

[SEQ ID No:86]

In another embodiment, the amino acid sequence of PEDF-furin-P2A-anti-Bb SCVF is referred to herein as SEQ ID No. 65, or a fragment or variant thereof, as shown below: MRRLVLLLCIGALLGHSSCQNPASPPEEGSPDPDSTGALVEEEDPFFKVPVNKLAAAVSNFGYDLYRVRSSTSPTTNVLLSPLSVATALSALSLGAEQRTESIIHRALYYDLISSPDIHGTYKELLDTVTAPQKNLKSASRIVFEKKLRIKSSFVAPLEKSYGTRPRVLTGNPRLDLQEINNWVQAQMKGKLARSTKEIPDEISILLLGVAHFKGQWVTKFDSRKTSLEDFYLDEERTVRVPMMSDPKAVLRYGLDSDLSCKIAQLPLTGSMSIIFFLPLKVTQNLTLIEESLTSEFIHDIDRELKTVQAVLTVPKLKLSYEGEVTKSLQEMKLQSLFDSPDFSKITGKPIKLTQVEHRAGFEWNEDGAGTTPSPGLQPAHLTFPLDYHLNQPFIFVLRDTDTGALLFIGKILDPRGPRVRRGSGATNFSLLKQAGDVEENPGPMRRLLILALVGAAVADVQITQSPSYLAASPGETITINCRASKSISKYLAWYQDKPGKTNKLLIYSGSTLQSGIPSRFSGSGSGTDFTLTISSLEPEDFAMYYCQQHDEYPWTFGGGTKLEIKRGGGGGSGGGGSGGGGSGGGGSQVQLQQSGAELAKPGASVRMSCKASGYTFTNYWIHWVKQRPGQGLEWIGYINPNTGYNDYNQKFKDKATLTADKSSSTVYMQLSSLTSEDSAVYYCARGGQLGLRRAMDYWGQGTSVTVSS

[SEQ ID No:65]

Preferably, in this embodiment, the construct comprises the 2130 nucleotide sequence referred to herein as SEQ ID No. 66 (as contained within plasmid IKC 093P) or a fragment or variant thereof, as shown below: ATGCGCCGCCTGGTGCTGCTGCTGTGCATCGGCGCCCTGCTGGGCCACAGCAGCTGCCAGAACCCCGCCAGCCCCCCCGAGGAGGGCAGCCCCGACCCCGACAGCACCGGCGCCCTGGTGGAGGAGGAGGACCCCTTCTTCAAGGTGCCCGTGAACAAGCTGGCCGCCGCCGTGAGCAACTTCGGCTACGACCTGTACCGCGTGCGCAGCAGCACCAGCCCCACCACCAACGTGCTGCTGAGCCCCCTGAGCGTGGCCACCGCCCTGAGCGCCCTGAGCCTGGGCGCCGAGCAGCGCACCGAGAGCATCATCCACCGCGCCCTGTACTACGACCTGATCAGCAGCCCCGACATCCACGGCACCTACAAGGAGCTGCTGGACACCGTGACCGCCCCCCAGAAGAACCTGAAGAGCGCCAGCCGCATCGTGTTCGAGAAGAAGCTGCGCATCAAGAGCAGCTTCGTGGCCCCCCTGGAGAAGAGCTACGGCACCCGCCCCCGCGTGCTGACCGGCAACCCCCGCCTGGACCTGCAGGAGATCAACAACTGGGTGCAGGCCCAGATGAAGGGCAAGCTGGCCCGCAGCACCAAGGAGATCCCCGACGAGATCAGCATCCTGCTGCTGGGCGTGGCCCACTTCAAGGGCCAGTGGGTGACCAAGTTCGACAGCCGCAAGACCAGCCTGGAGGACTTCTACCTGGACGAGGAGCGCACCGTGCGCGTGCCCATGATGAGCGACCCCAAGGCCGTGCTGCGCTACGGCCTGGACAGCGACCTGAGCTGCAAGATCGCCCAGCTGCCCCTGACCGGCAGCATGAGCATCATCTTCTTCCTGCCCCTGAAGGTGACCCAGAACCTGACCCTGATCGAGGAGAGCCTGACCAGCGAGTTCATCCACGACATCGACCGCGAGCTGAAGACCGTGCAGGCCGTGCTGACCGTGCCCAAGCTGAAGCTGAGCTACGAGGGCGAGGTGACCAAGAGCCTGCAGGAGATGAAGCTGCAGAGCCTGTTCGACAGCCCCGACTTCAGCAAGATCACCGGCAAGCCCATCAAGCTGACCCAGGTGGAGCACCGCGCCGGCTTCGAGTGGAACGAGGACGGCGCCGGCACCACCCCCAGCCCCGGCCTGCAGCCCGCCCACCTGACCTTCCCCCTGGACTACCACCTGAACCAGCCCTTCATCTTCGTGCTGCGCGACACCGACACCGGCGCCCTGCTGTTCATCGGCAAGATCCTGGACCCCCGCGGCCCCCGCGTGCGCCGCGGCAGCGGCGCCACCAACTTCAGCCTGCTGAAGCAGGCCGGCGACGTGGAGGAGAACCCCGGCCCCATGCGCCGCCTGCTGATCCTGGCCCTGGTGGGCGCCGCCGTGGCCGACGTCCAGATCACCCAGAGCCCCAGCTACCTGGCCGCCAGCCCCGGCGAGACCATCACCATCAACTGCCGCGCCAGCAAGAGCATCAGCAAGTACCTGGCCTGGTACCAGGACAAGCCCGGCAAGACCAACAAGCTGCTGATCTACAGCGGCAGCACCCTGCAGAGCGGCATCCCCAGCCGCTTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGACCATCAGCAGCCTGGAGCCCGAGGACTTCGCCATGTACTACTGCCAGCAGCACGACGAGTACCCCTGGACCTTCGGCGGCGGCACCAAGCTGGAGATCAAGCGCGGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCCAGGTGCAGCTGCAGCAGAGCGGCGCCGAGCTGGCCAAGCCCGGCGCCAGCGTGCGCATGAGCTGCAAGGCCAGCGGCTACACCTTCACCAACTACTGGATCCACTGGGTGAAGCAGCGCCCCGGCCAGGGCCTGGAGTGGATCGGCTACATCAACCCCAACACCGGCTACAACGACTACAACCAGAAGTTCAAGGACAAGGCCACCCTGACCGCCGACAAGAGCAGCAGCACCGTGTACATGCAGCTGAGCAGCCTGACCAGCGAGGACAGCGCCGTGTACTACTGCGCCCGCGGCGGCCAGCTGGGCCTGCGCCGCGCCATGGACTACTGGGGCCAGGGCACCAGCGTGACCGTGAGCAGCTGA

[SEQ ID No:66]

In another embodiment, the amino acid sequence of PEDF-furin-P2A-anti-Bb SCVF is referred to herein as SEQ ID No. 67, or a fragment or variant thereof, as shown below: MRRLVLLLCIGALLGHSSCQNPASPPEEGSPDPDSTGALVEEEDPFFKVPVNKLAAAVSNFGYDLYRVRSSTSPTTNVLLSPLSVATALSALSLGAEQRTESIIHRALYYDLISSPDIHGTYKELLDTVTAPQKNLKSASRIVFEKKLRIKSSFVAPLEKSYGTRPRVLTGNPRLDLQEINNWVQAQMKGKLARSTKEIPDEISILLLGVAHFKGQWVTKFDSRKTSLEDFYLDEERTVRVPMMSDPKAVLRYGLDSDLSCKIAQLPLTGSMSIIFFLPLKVTQNLTLIEESLTSEFIHDIDRELKTVQAVLTVPKLKLSYEGEVTKSLQEMKLQSLFDSPDFSKITGKPIKLTQVEHRAGFEWNEDGAGTTPSPGLQPAHLTFPLDYHLNQPFIFVLRDTDTGALLFIGKILDPRGPRRSKRSGSGATNFSLLKQAGDVEENPGPMRRLLILALVGAAVADVQITQSPSYLAASPGETITINCRASKSISKYLAWYQDKPGKTNKLLIYSGSTLQSGIPSRFSGSGSGTDFTLTISSLEPEDFAMYYCQQHDEYPWTFGGGTKLEIKRGGGGGSGGGGSGGGGSGGGGSQVQLQQSGAELAKPGASVRMSCKASGYTFTNYWIHWVKQRPGQGLEWIGYINPNTGYNDYNQKFKDKATLTADKSSSTVYMQLSSLTSEDSAVYYCARGGQLGLRRAMDYWGQGTSVTVSS

[SEQ ID No:67]

Preferably, in this embodiment, the construct comprises the 2136 nucleotide sequence referred to herein as SEQ ID No. 68 (as contained within plasmid IKC 129P) or a fragment or variant thereof as shown below: ATGCGCCGCCTGGTGCTGCTGCTGTGCATCGGCGCCCTGCTGGGCCACAGCAGCTGCCAGAACCCCGCCAGCCCCCCCGAGGAGGGCAGCCCCGACCCCGACAGCACCGGCGCCCTGGTGGAGGAGGAGGACCCCTTCTTCAAGGTGCCCGTGAACAAGCTGGCCGCCGCCGTGAGCAACTTCGGCTACGACCTGTACCGCGTGCGCAGCAGCACCAGCCCCACCACCAACGTGCTGCTGAGCCCCCTGAGCGTGGCCACCGCCCTGAGCGCCCTGAGCCTGGGCGCCGAGCAGCGCACCGAGAGCATCATCCACCGCGCCCTGTACTACGACCTGATCAGCAGCCCCGACATCCACGGCACCTACAAGGAGCTGCTGGACACCGTGACCGCCCCCCAGAAGAACCTGAAGAGCGCCAGCCGCATCGTGTTCGAGAAGAAGCTGCGCATCAAGAGCAGCTTCGTGGCCCCCCTGGAGAAGAGCTACGGCACCCGCCCCCGCGTGCTGACCGGCAACCCCCGCCTGGACCTGCAGGAGATCAACAACTGGGTGCAGGCCCAGATGAAGGGCAAGCTGGCCCGCAGCACCAAGGAGATCCCCGACGAGATCAGCATCCTGCTGCTGGGCGTGGCCCACTTCAAGGGCCAGTGGGTGACCAAGTTCGACAGCCGCAAGACCAGCCTGGAGGACTTCTACCTGGACGAGGAGCGCACCGTGCGCGTGCCCATGATGAGCGACCCCAAGGCCGTGCTGCGCTACGGCCTGGACAGCGACCTGAGCTGCAAGATCGCCCAGCTGCCCCTGACCGGCAGCATGAGCATCATCTTCTTCCTGCCCCTGAAGGTGACCCAGAACCTGACCCTGATCGAGGAGAGCCTGACCAGCGAGTTCATCCACGACATCGACCGCGAGCTGAAGACCGTGCAGGCCGTGCTGACCGTGCCCAAGCTGAAGCTGAGCTACGAGGGCGAGGTGACCAAGAGCCTGCAGGAGATGAAGCTGCAGAGCCTGTTCGACAGCCCCGACTTCAGCAAGATCACCGGCAAGCCCATCAAGCTGACCCAGGTGGAGCACCGCGCCGGCTTCGAGTGGAACGAGGACGGCGCCGGCACCACCCCCAGCCCCGGCCTGCAGCCCGCCCACCTGACCTTCCCCCTGGACTACCACCTGAACCAGCCCTTCATCTTCGTGCTGCGCGACACCGACACCGGCGCCCTGCTGTTCATCGGCAAGATCCTGGACCCCCGCGGCCCCCGCCGCAGCAAGCGCAGCGGCAGCGGCGCCACCAACTTCAGCCTGCTGAAGCAGGCCGGCGACGTGGAGGAGAACCCCGGCCCCATGCGCCGCCTGCTGATCCTGGCCCTGGTGGGCGCCGCCGTGGCCGACGTCCAGATCACCCAGAGCCCCAGCTACCTGGCCGCCAGCCCCGGCGAGACCATCACCATCAACTGCCGCGCCAGCAAGAGCATCAGCAAGTACCTGGCCTGGTACCAGGACAAGCCCGGCAAGACCAACAAGCTGCTGATCTACAGCGGCAGCACCCTGCAGAGCGGCATCCCCAGCCGCTTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGACCATCAGCAGCCTGGAGCCCGAGGACTTCGCCATGTACTACTGCCAGCAGCACGACGAGTACCCCTGGACCTTCGGCGGCGGCACCAAGCTGGAGATCAAGCGCGGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCCAGGTGCAGCTGCAGCAGAGCGGCGCCGAGCTGGCCAAGCCCGGCGCCAGCGTGCGCATGAGCTGCAAGGCCAGCGGCTACACCTTCACCAACTACTGGATCCACTGGGTGAAGCAGCGCCCCGGCCAGGGCCTGGAGTGGATCGGCTACATCAACCCCAACACCGGCTACAACGACTACAACCAGAAGTTCAAGGACAAGGCCACCCTGACCGCCGACAAGAGCAGCAGCACCGTGTACATGCAGCTGAGCAGCCTGACCAGCGAGGACAGCGCCGTGTACTACTGCGCCCGCGGCGGCCAGCTGGGCCTGCGCCGCGCCATGGACTACTGGGGCCAGGGCACCAGCGTGACCGTGAGCAGCTGA

[SEQ ID No:68]

In another embodiment, the amino acid sequence of PEDF-furin-P2A-sCD 46 is referred to herein as SEQ ID No. 69, or a fragment or variant thereof, as shown below: MRRLVLLLCIGALLGHVFCQNPASPPEEGSPDPDSTGALVEEEDPFFKVPVNKLAAAVSNFGYDLYRVRSSTSPTTNVLLSPLSVATALSALSLGAEQRTESIIHRALYYDLISSPDIHGTYKELLDTVTAPQKNLKSASRIVFEKKLRIKSSFVAPLEKSYGTRPRVLTGNPRLDLQEINNWVQAQMKGKLARSTKEIPDEISILLLGVAHFKGQWVTKFDSRKTSLEDFYLDEERTVRVPMMSDPKAVLRYGLDSDLSCKIAQLPLTGSMSIIFFLPLKVTQNLTLIEESLTSEFIHDIDRELKTVQAVLTVPKLKLSYEGEVTKSLQEMKLQSLFDSPDFSKITGKPIKLTQVEHRAGFEWNEDGAGTTPSPGLQPAHLTFPLDYHLNQPFIFVLRDTDTGALLFIGKILDPRGPRRSKRSGSGATNFSLLKQAGDVEENPGPMRRFLTVISFLLYFGCAFACEEPPTFEAMELIGKPKPYYEIGERVDYKCKKGYFYIPPLATHTICDRNHTWLPVSDDACYRETCPYIRDPLNGQAVPANGTYEFGYQMHFICNEGYYLIGEEILYCELKGSVAIWSGKPPICEKVLCTPPPKIKNGKHTFSEVEVFEYLDAVTYSCDPAPGPDPFSLIGESTIYCGDNSVWSRAAPECKVVKCRFPVVENGKQISGFGKKFYYKATVMFECDKGFYLDGSDTIVCDSNSTWDPPVPKCLKVLPPSSTKPPALSHSVSTSSTTKSPASSASGPRPTYKPPVSNYPGYPKPEEGILDSLDV

[SEQ ID No:69]

Preferably, in this embodiment, the construct comprises the 2328 nucleotide sequence referred to herein as SEQ ID No. 70 (as contained within plasmid IKC 159P) or a fragment or variant thereof, as shown below: ATGCGCCGCCTGGTGCTGCTGCTGTGCATCGGCGCCCTGCTGGGCCACGTGTTCTGCCAGAACCCCGCCAGCCCCCCCGAGGAGGGCAGCCCCGACCCCGACAGCACCGGCGCCCTGGTGGAGGAGGAGGACCCCTTCTTCAAGGTGCCCGTGAACAAGCTGGCCGCCGCCGTGAGCAACTTCGGCTACGACCTGTACCGCGTGCGCAGCAGCACCAGCCCCACCACCAACGTGCTGCTGAGCCCCCTGAGCGTGGCCACCGCCCTGAGCGCCCTGAGCCTGGGCGCCGAGCAGCGCACCGAGAGCATCATCCACCGCGCCCTGTACTACGACCTGATCAGCAGCCCCGACATCCACGGCACCTACAAGGAGCTGCTGGACACCGTGACCGCCCCCCAGAAGAACCTGAAGAGCGCCAGCCGCATCGTGTTCGAGAAGAAGCTGCGCATCAAGAGCAGCTTCGTGGCCCCCCTGGAGAAGAGCTACGGCACCCGCCCCCGCGTGCTGACCGGCAACCCCCGCCTGGACCTGCAGGAGATCAACAACTGGGTGCAGGCCCAGATGAAGGGCAAGCTGGCCCGC

AGCACCAAGGAGATCCCCGACGAGATCAGCATCCTGCTGCTGGGCGTGG

CCCACTTCAAGGGCCAGTGGGTGACCAAGTTCGACAGCCGCAAGACCA

GCCTGGAGGACTTCTACCTGGACGAGGAGCGCACCGTGCGCGTGCCCAT

GATGAGCGACCCCAAGGCCGTGCTGCGCTACGGCCTGGACAGCGACCTG

AGCTGCAAGATCGCCCAGCTGCCCCTGACCGGCAGCATGAGCATCATCT

TCTTCCTGCCCCTGAAGGTGACCCAGAACCTGACCCTGATCGAGGAGAG

CCTGACCAGCGAGTTCATCCACGACATCGACCGCGAGCTGAAGACCGTG

CAGGCCGTGCTGACCGTGCCCAAGCTGAAGCTGAGCTACGAGGGCGAG

GTGACCAAGAGCCTGCAGGAGATGAAGCTGCAGAGCCTGTTCGACAGC

CCCGACTTCAGCAAGATCACCGGCAAGCCCATCAAGCTGACCCAGGTGG

AGCACCGCGCCGGCTTCGAGTGGAACGAGGACGGCGCCGGCACCACCC

CCAGCCCCGGCCTGCAGCCCGCCCACCTGACCTTCCCCCTGGACTACCA

CCTGAACCAGCCCTTCATCTTCGTGCTGCGCGACACCGACACCGGCGCC

CTGCTGTTCATCGGCAAGATCCTGGACCCCCGCGGCCCCCGCCGCAGCA

AGCGCAGCGGCAGCGGCGCCACCAACTTCAGCCTGCTGAAGCAGGCCG

GCGACGTGGAGGAGAACCCCGGCCCCATGCGCCGCTTCCTGACCGTGAT

CAGCTTCCTGCTGTACTTCGGCTGCGCCTTCGCCTGCGAGGAGCCCCCC

ACCTTCGAGGCCATGGAGCTGATCGGCAAGCCCAAGCCCTACTACGAGA

TCGGCGAGCGCGTGGACTACAAGTGCAAGAAGGGCTACTTCTACATCCC

CCCCCTGGCCACCCACACCATCTGCGACCGCAACCACACCTGGCTGCCC

GTGAGCGACGACGCCTGCTACCGCGAGACCTGCCCCTACATCCGCGACC

CCCTGAACGGCCAGGCCGTGCCCGCCAACGGCACCTACGAGTTCGGCTA

CCAGATGCACTTCATCTGCAACGAGGGCTACTACCTGATCGGCGAGGAG

ATCCTGTACTGCGAGCTGAAGGGCAGCGTGGCCATCTGGAGCGGCAAGC

CCCCCATCTGCGAGAAGGTGCTGTGCACCCCCCCCCCCAAGATCAAGAA

CGGCAAGCACACCTTCAGCGAGGTGGAGGTGTTCGAGTACCTGGACGC

CGTGACCTACAGCTGCGACCCCGCCCCCGGCCCCGACCCCTTCAGCCTG

ATCGGCGAGAGCACCATCTACTGCGGCGACAACAGCGTGTGGAGCCGCG

CCGCCCCCGAGTGCAAGGTGGTGAAGTGCCGCTTCCCCGTGGTGGAGA

ACGGCAAGCAGATCAGCGGCTTCGGCAAGAAGTTCTACTACAAGGCCAC

CGTGATGTTCGAGTGCGACAAGGGCTTCTACCTGGACGGCAGCGACACC

ATCGTGTGCGACAGCAACAGCACCTGGGACCCCCCCGTGCCCAAGTGCC

TGAAGGTGCTGCCCCCCAGCAGCACCAAGCCCCCCGCCCTGAGCCACA

GCGTGAGCACCAGCAGCACCACCAAGAGCCCCGCCAGCAGCGCCAGCG

GCCCCCGCCCCACCTACAAGCCCCCCGTGAGCAACTACCCCGGCTACCC

CAAGCCCGAGGAGGGCATCCTGGACAGCCTGGACGTGTAA

[SEQ ID No:70]

In another embodiment, the amino acid sequence of sCD 46-furin-P2A-PEDF is referred to herein as SEQ ID No. 71, or a fragment or variant thereof, as shown below: MRRFLTVISFLLYFGCAFACEEPPTFEAMELIGKPKPYYEIGERVDYKCKKGYFYIPPLATHTICDRNHTWLPVSDDACYRETCPYIRDPLNGQAVPANGTYEFGYQMHFICNEGYYLIGEEILYCELKGSVAIWSGKPPICEKVLCTPPPKIKNGKHTFSEVEVFEYLDAVTYSCDPAPGPDPFSLIGESTIYCGDNSVWSRAAPECKVVKCRFPVVENGKQISGFGKKFYYKATVMFECDKGFYLDGSDTIVCDSNSTWDPPVPKCLKVLPPSSTKPPALSHSVSTSSTTKSPASSASGPRPTYKPPVSNYPGYPKPEEGILDSLDVRRSKRSGSGATNFSLLKQAGDVEENPGPMRRLVLLLCIGALLGHVFCQNPASPPEEGSPDPDSTGALVEEEDPFFKVPVNKLAAAVSNFGYDLYRVRSSTSPTTNVLLSPLSVATALSALSLGAEQRTESIIHRALYYDLISSPDIHGTYKELLDTVTAPQKNLKSASRIVFEKKLRIKSSFVAPLEKSYGTRPRVLTGNPRLDLQEINNWVQAQMKGKLARSTKEIPDEISILLLGVAHFKGQWVTKFDSRKTSLEDFYLDEERTVRVPMMSDPKAVLRYGLDSDLSCKIAQLPLTGSMSIIFFLPLKVTQNLTLIEESLTSEFIHDIDRELKTVQAVLTVPKLKLSYEGEVTKSLQEMKLQSLFDSPDFSKITGKPIKLTQVEHRAGFEWNEDGAGTTPSPGLQPAHLTFPLDYHLNQPFIFVLRDTDTGALLFIGKILDPRGP

[SEQ ID No:71]

Preferably, in this embodiment, the construct comprises the 2328 nucleotide sequence referred to herein as SEQ ID No. 72 (as contained within plasmid IKC 145P) or a fragment or variant thereof, as shown below: ATGCGCCGCTTCCTGACCGTGATCAGCTTCCTGCTGTACTTCGGCTGCGC

CTTCGCCTGCGAGGAGCCCCCCACCTTCGAGGCCATGGAGCTGATCGGC

AAGCCCAAGCCCTACTACGAGATCGGCGAGCGCGTGGACTACAAGTGCA

AGAAGGGCTACTTCTACATCCCCCCCCTGGCCACCCACACCATCTGCGAC

CGCAACCACACCTGGCTGCCCGTGAGCGACGACGCCTGCTACCGCGAG

ACCTGCCCCTACATCCGCGACCCCCTGAACGGCCAGGCCGTGCCCGCCA

ACGGCACCTACGAGTTCGGCTACCAGATGCACTTCATCTGCAACGAGGG

CTACTACCTGATCGGCGAGGAGATCCTGTACTGCGAGCTGAAGGGCAGC

GTGGCCATCTGGAGCGGCAAGCCCCCCATCTGCGAGAAGGTGCTGTGCA

CCCCCCCCCCCAAGATCAAGAACGGCAAGCACACCTTCAGCGAGGTGG

AGGTGTTCGAGTACCTGGACGCCGTGACCTACAGCTGCGACCCCGCCCC

CGGCCCCGACCCCTTCAGCCTGATCGGCGAGAGCACCATCTACTGCGGC

GACAACAGCGTGTGGAGCCGCGCCGCCCCCGAGTGCAAGGTGGTGAAG

TGCCGCTTCCCCGTGGTGGAGAACGGCAAGCAGATCAGCGGCTTCGGCA

AGAAGTTCTACTACAAGGCCACCGTGATGTTCGAGTGCGACAAGGGCTT

CTACCTGGACGGCAGCGACACCATCGTGTGCGACAGCAACAGCACCTGG

GACCCCCCCGTGCCCAAGTGCCTGAAGGTGCTGCCCCCCAGCAGCACCA

AGCCCCCCGCCCTGAGCCACAGCGTGAGCACCAGCAGCACCACCAAGA

GCCCCGCCAGCAGCGCCAGCGGCCCCCGCCCCACCTACAAGCCCCCCGT

GAGCAACTACCCCGGCTACCCCAAGCCCGAGGAGGGCATCCTGGACAG

CCTGGACGTGCGCCGCAGCAAGCGCAGCGGCAGCGGCGCCACCAACTT

CAGCCTGCTGAAGCAGGCCGGCGACGTGGAGGAGAACCCCGGCCCCAT

GCGCCGCCTGGTGCTGCTGCTGTGCATCGGCGCCCTGCTGGGCCACGTG

TTCTGCCAGAACCCCGCCAGCCCCCCCGAGGAGGGCAGCCCCGACCCC

GACAGCACCGGCGCCCTGGTGGAGGAGGAGGACCCCTTCTTCAAGGTG

CCCGTGAACAAGCTGGCCGCCGCCGTGAGCAACTTCGGCTACGACCTGT

ACCGCGTGCGCAGCAGCACCAGCCCCACCACCAACGTGCTGCTGAGCC

CCCTGAGCGTGGCCACCGCCCTGAGCGCCCTGAGCCTGGGCGCCGAGC

AGCGCACCGAGAGCATCATCCACCGCGCCCTGTACTACGACCTGATCAG

CAGCCCCGACATCCACGGCACCTACAAGGAGCTGCTGGACACCGTGACC

GCCCCCCAGAAGAACCTGAAGAGCGCCAGCCGCATCGTGTTCGAGAAG

AAGCTGCGCATCAAGAGCAGCTTCGTGGCCCCCCTGGAGAAGAGCTACG

GCACCCGCCCCCGCGTGCTGACCGGCAACCCCCGCCTGGACCTGCAGG

AGATCAACAACTGGGTGCAGGCCCAGATGAAGGGCAAGCTGGCCCGCA

GCACCAAGGAGATCCCCGACGAGATCAGCATCCTGCTGCTGGGCGTGGC

CCACTTCAAGGGCCAGTGGGTGACCAAGTTCGACAGCCGCAAGACCAG

CCTGGAGGACTTCTACCTGGACGAGGAGCGCACCGTGCGCGTGCCCATG

ATGAGCGACCCCAAGGCCGTGCTGCGCTACGGCCTGGACAGCGACCTGA

GCTGCAAGATCGCCCAGCTGCCCCTGACCGGCAGCATGAGCATCATCTT

CTTCCTGCCCCTGAAGGTGACCCAGAACCTGACCCTGATCGAGGAGAGC

CTGACCAGCGAGTTCATCCACGACATCGACCGCGAGCTGAAGACCGTGC

AGGCCGTGCTGACCGTGCCCAAGCTGAAGCTGAGCTACGAGGGCGAGG

TGACCAAGAGCCTGCAGGAGATGAAGCTGCAGAGCCTGTTCGACAGCC

CCGACTTCAGCAAGATCACCGGCAAGCCCATCAAGCTGACCCAGGTGGA

GCACCGCGCCGGCTTCGAGTGGAACGAGGACGGCGCCGGCACCACCCC

CAGCCCCGGCCTGCAGCCCGCCCACCTGACCTTCCCCCTGGACTACCAC

CTGAACCAGCCCTTCATCTTCGTGCTGCGCGACACCGACACCGGCGCCC

TGCTGTTCATCGGCAAGATCCTGGACCCCCGCGGCCCCTAA

[SEQ ID No:72]

In another embodiment, the amino acid sequence of [ PEDF-furin-P2A-CFHL 1] is referred to herein as SEQ ID No. 87, or a fragment or variant thereof, as set forth below: MRRLVLLLCIGALLGHVFCQNPASPPEEGSPDPDSTGALVEEEDPFFKVPVNKLAAAVSNFGYDLYRVRSSTSPTTNVLLSPLSVATALSALSLGAEQRTESIIHRALYYDLISSPDIHGTYKELLDTVTAPQKNLKSASRIVFEKKLRIKSSFVAPLEKSYGTRPRVLTGNPRLDLQEINNWVQAQMKGKLARSTKEIPDEISILLLGVAHFKGQWVTKFDSRKTSLEDFYLDEERTVRVPMMSDPKAVLRYGLDSDLSCKIAQLPLTGSMSIIFFLPLKVTQNLTLIEESLTSEFIHDIDRELKTVQAVLTVPKLKLSYEGEVTKSLQEMKLQSLFDSPDFSKITGKPIKLTQVEHRAGFEWNEDGAGTTPSPGLQPAHLTFPLDYHLNQPFIFVLRDTDTGALLFIGKILDPRGPRRSKRSGSGATNFSLLKQAGDVEENPGPMRRLLTFISILALVGAFAEDCNELPPRRNTEILTGSWSDQTYPEGTQAIYKCRPGYRSLGNVIMVCRKGEWVALNPLRKCQKRPCGHPGDTPFGTFTLTGGNVFEYGVKAVYTCNEGYQLLGEINYRECDTDGWTNDIPICEVVKCLPVTAPENGKIVSSAMEPDREYHFGQAVRFVCNSGYKIEGDEEMHCSDDGFWSKEKPKCVEISCKSPDVINGSPISQKIIYKENERFQYKCNMGYEYSERGDAVCTESGWRPLPSCEEKSCDNPYIPNGDYSPLRIKHRTGDEITYQCRNGFYPATRGNTAKCTSTGWIPAPRCTLKPCDYPDIKHGGLYHENMRRPYFPVAVGKYYSYYCDEHFETPSGSYWDHIHCTQDGWSPAVPCLRKCYFPYLENGYNQNYGRKFVQGKSIDVACHPGYALPKAQTTVTCMENGWSPTPRCIR

[SEQ ID No:87]

Preferably, in this embodiment, the construct comprises the 2670 nucleotide sequence referred to herein as SEQ ID No. 88 (as contained within plasmid IKC 161P) or a fragment or variant thereof as shown below: ATGCGCCGCCTGGTGCTGCTGCTGTGCATCGGCGCCCTGCTGGGCCACGTGTTCTGCCAGAACCCCGCCAGCCCCCCCGAGGAGGGCAGCCCCGACCCCGACAGCACCGGCGCCCTGGTGGAGGAGGAGGACCCCTTCTTCAAGGTGCCCGTGAACAAGCTGGCCGCCGCCGTGAGCAACTTCGGCTACGACCTGTACCGCGTGCGCAGCAGCACCAGCCCCACCACCAACGTGCTGCTGAGCCCCCTGAGCGTGGCCACCGCCCTGAGCGCCCTGAGCCTGGGCGCCGAGCAGCGCACCGAGAGCATCATCCACCGCGCCCTGTACTACGACCTGATCAGCAGCCCCGACATCCACGGCACCTACAAGGAGCTGCTGGACACCGTGACCGCCCCCCAGAAGAACCTGAAGAGCGCCAGCCGCATCGTGTTCGAGAAGAAGCTGCGCATCAAGAGCAGCTTCGTGGCCCCCCTGGAGAAGAGCTACGGCACCCGCCCCCGCGTGCTGACCGGCAACCCCCGCCTGGACCTGCAGGAGATCAACAACTGGGTGCAGGCCCAGATGAAGGGCAAGCTGGCCCGCAGCACCAAGGAGATCCCCGACGAGATCAGCATCCTGCTGCTGGGCGTGGCCCACTTCAAGGGCCAGTGGGTGACCAAGTTCGACAGCCGCAAGACCAGCCTGGAGGACTTCTACCTGGACGAGGAGCGCACCGTGCGCGTGCCCATGATGAGCGACCCCAAGGCCGTGCTGCGCTACGGCCTGGACAGCGACCTGAGCTGCAAGATCGCCCAGCTGCCCCTGACCGGCAGCATGAGCATCATCT

TCTTCCTGCCCCTGAAGGTGACCCAGAACCTGACCCTGATCGAGGAGAG

CCTGACCAGCGAGTTCATCCACGACATCGACCGCGAGCTGAAGACCGTG

CAGGCCGTGCTGACCGTGCCCAAGCTGAAGCTGAGCTACGAGGGCGAG

GTGACCAAGAGCCTGCAGGAGATGAAGCTGCAGAGCCTGTTCGACAGC

CCCGACTTCAGCAAGATCACCGGCAAGCCCATCAAGCTGACCCAGGTGG

AGCACCGCGCCGGCTTCGAGTGGAACGAGGACGGCGCCGGCACCACCC

CCAGCCCCGGCCTGCAGCCCGCCCACCTGACCTTCCCCCTGGACTACCA

CCTGAACCAGCCCTTCATCTTCGTGCTGCGCGACACCGACACCGGCGCC

CTGCTGTTCATCGGCAAGATCCTGGACCCCCGCGGCCCCCGCCGCAGCA

AGCGCAGCGGCAGCGGCGCCACCAACTTCAGCCTGCTGAAGCAGGCCG

GCGACGTGGAGGAGAACCCCGGCCCCATGCGCCGCCTGCTGACCTTCAT

CAGCATCCTGGCCCTGGTGGGCGCCTTCGCCGAGGACTGCAACGAGCTG

CCCCCCCGCCGCAACACCGAGATCCTGACCGGCAGCTGGAGCGACCAG

ACCTACCCCGAGGGCACCCAGGCCATCTACAAGTGCCGCCCCGGCTACC

GCAGCCTGGGCAACGTGATCATGGTGTGCCGCAAGGGCGAGTGGGTGG

CCCTGAACCCCCTGCGCAAGTGCCAGAAGCGCCCCTGCGGCCACCCCG

GCGACACCCCCTTCGGCACCTTCACCCTGACCGGCGGCAACGTGTTCGA

GTACGGCGTGAAGGCCGTGTACACCTGCAACGAGGGCTACCAGCTGCTG

GGCGAGATCAACTACCGCGAGTGCGACACCGACGGCTGGACCAACGAC

ATCCCCATCTGCGAGGTGGTGAAGTGCCTGCCCGTGACCGCCCCCGAGA

ACGGCAAGATCGTGAGCAGCGCCATGGAGCCCGACCGCGAGTACCACTT

CGGCCAGGCCGTGCGCTTCGTGTGCAACAGCGGCTACAAGATCGAGGGC

GACGAGGAGATGCACTGCAGCGACGACGGCTTCTGGAGCAAGGAGAAG

CCCAAGTGCGTGGAGATCAGCTGCAAGAGCCCCGACGTGATCAACGGC

AGCCCCATCAGCCAGAAGATCATCTACAAGGAGAACGAGCGCTTCCAGT

ACAAGTGCAACATGGGCTACGAGTACAGCGAGCGCGGCGACGCCGTGT

GCACCGAGAGCGGCTGGCGCCCCCTGCCCAGCTGCGAGGAGAAGAGCT

GCGACAACCCCTACATCCCCAACGGCGACTACAGCCCCCTGCGCATCAA

GCACCGCACCGGCGACGAGATCACCTACCAGTGCCGCAACGGCTTCTAC

CCCGCCACCCGGGGCAACACCGCCAAGTGCACCAGCACCGGCTGGATC

CCCGCCCCCCGCTGCACCCTGAAGCCCTGCGACTACCCCGACATCAAGC

ACGGCGGCCTGTACCACGAGAACATGCGCCGCCCCTACTTCCCCGTGGC

CGTGGGCAAGTACTACAGCTACTACTGCGACGAGCACTTCGAGACCCCC

AGCGGCAGCTACTGGGACCACATCCACTGCACCCAGGACGGCTGGAGC

CCCGCCGTGCCCTGCCTGCGCAAGTGCTACTTCCCCTACCTGGAGAACG

GCTACAACCAGAACTACGGCCGCAAGTTCGTGCAGGGCAAGAGCATCG

ACGTGGCCTGCCACCCCGGCTACGCCCTGCCCAAGGCCCAGACCACCGT

GACCTGCATGGAGAACGGCTGGAGCCCCACCCCCCGCTGCATCCGC

[SEQ ID No:88]

In another embodiment, the amino acid sequence of [ CFHL 1-furin-P2A-PEDF ] is referred to herein as SEQ ID No:89, or a fragment or variant thereof, as set forth below: MRRLLTFISILALVGAFAEDCNELPPRRNTEILTGSWSDQTYPEGTQAIYKCRPGYRSLGNVIMVCRKGEWVALNPLRKCQKRPCGHPGDTPFGTFTLTGGNVFEYGVKAVYTCNEGYQLLGEINYRECDTDGWTNDIPICEVVKCLPVTAPENGKIVSSAMEPDREYHFGQAVRFVCNSGYKIEGDEEMHCSDDGFWSKEKPKCVEISCKSPDVINGSPISQKIIYKENERFQYKCNMGYEYSERGDAVCTESGWRPLPSCEEKSCDNPYIPNGDYSPLRIKHRTGDEITYQCRNGFYPATRGNTAKCTSTGWIPAPRCTLKPCDYPDIKHGGLYHENMRRPYFPVAVGKYYSYYCDEHFETPSGSYWDHIHCTQDGWSPAVPCLRKCYFPYLENGYNQNYGRKFVQGKSIDVACHPGYALPKAQTTVTCMENGWSPTPRCIRRRSKRSGSGATNFSLLKQAGDVEENPGPMRRLVLLLCIGALLGHVFCQNPASPPEEGSPDPDSTGALVEEEDPFFKVPVNKLAAAVSNFGYDLYRVRSSTSPTTNVLLSPLSVATALSALSLGAEQRTESIIHRALYYDLISSPDIHGTYKELLDTVTAPQKNLKSASRIVFEKKLRIKSSFVAPLEKSYGTRPRVLTGNPRLDLQEINNWVQAQMKGKLARSTKEIPDEISILLLGVAHFKGQWVTKFDSRKTSLEDFYLDEERTVRVPMMSDPKAVLRYGLDSDLSCKIAQLPLTGSMSIIFFLPLKVTQNLTLIEESLTSEFIHDIDRELKTVQAVLTVPKLKLSYEGEVTKSLQEMKLQSLFDSPDFSKITGKPIKLTQVEHRAGFEWNEDGAGTTPSPGLQPAHLTFPLDYHLNQPFIFVLRDTDTGALLFIGKILDPRGP

Preferably, in this embodiment, the construct comprises the 2670 nucleotide sequence referred to herein as SEQ ID No:90 (as contained within plasmid IKC 174P) or a fragment or variant thereof as shown below: ATGCGCCGCCTGCTGACCTTCATCAGCATCCTGGCCCTGGTGGGCGCCTTCGCCGAGGACTGCAACGAGCTGCCCCCCCGCCGCAACACCGAGATCCTGACCGGCAGCTGGAGCGACCAGACCTACCCCGAGGGCACCCAGGCCATCTACAAGTGCCGCCCCGGCTACCGCAGCCTGGGCAACGTGATCATGGTGTGCCGCAAGGGCGAGTGGGTGGCCCTGAACCCCCTGCGCAAGTGCCAGAAGCGCCCCTGCGGCCACCCCGGCGACACCCCCTTCGGCACCTTCACCCTGACCGGCGGCAACGTGTTCGAGTACGGCGTGAAGGCCGTGTACACCTGCAACGAGGGCTACCAGCTGCTGGGCGAGATCAACTACCGCGAGTGCGACACCGACGGCTGGACCAACGACATCCCCATCTGCGAGGTGGTGAAGTGCCTGCCCGTGACCGCCCCCGAGAACGGCAAGATCGTGAGCAGCGCCATGGAGCCCGACCGCGAGTACCACTTCGGCCAGGCCGTGCGCTTCGTGTGCAACAGCGGCTACAAGATCGAGGGCGACGAGGAGATGCACTGCAGCGACGACGGCTTCTGGAGCAAGGAGAAGCCCAAGTGCGTGGAGATCAGCTGCAAGAGCCCCGACGTGATCAACGGCAGCCCCATCAGCCAGAAGATCATCTACAAGGAGAACGAGCGCTTCCAGTACAAGTGCAACATGGGCTACGAGTACAGCGAGCGCGGCGACGCCGTGTGCACCGAGAGCGGCTGGCGCCCCCTGCCCAGCTGCGAGGAGAAGAGCTGCGACAACCCCTACATCCCCAACGGCGACTACAGCCCCCTGCGCATCAAGCACCGCACCGGCGACGAGATCACCTACCAGTGCCGCAACGGCTTCTACCCCGCCACCCGGGGCAACACCGCCAAGTGCACCAGCACCGGCTGGATCCCCGCCCCCCGCTGCACCCTGAAGCCCTGCGACTACCCCGACATCAAGCACGGCGGCCTGTACCACGAGAACATGCGCCGCCCCTACTTCCCCGTGGCCGTGGGCAAGTACTACAGCTACTACTGCGACGAGCACTTCGAGACCCCCAGCGGCAGCTACTGGGACCACATCCACTGCACCCAGGACGGCTGGAGCCCCGCCGTGCCCTGCCTGCGCAAGTGCTACTTCCCCTACCTGGAGAACGGCTACAACCAGAACTACGGCCGCAAGTTCGTGCAGGGCAAGAGCATCGACGTGGCCTGCCACCCCGGCTACGCCCTGCCCAAGGCCCAGACCACCGTGACCTGCATGGAGAACGGCTGGAGCCCCACCCCCCGCTGCATCCGCCGCCGCAGCAAGCGCAGCGGCAGCGGCGCCACCAACTTCAGCCTGCTGAAGCAGGCCGGCGACGTGGAGGAGAACCCCGGCCCCATGCGCCGCCTGGTGCTGCTGCTGTGCATCGGCGCCCTGCTGGGCCACGTGTTCTGCCAGAACCCCGCCAGCCCCCCCGAGGAGGGCAGCCCCGACCCCGACAGCACCGGCGCCCTGGTGGAGGAGGAGGACCCCTTCTTCAAGGTGCCCGTGAACAAGCTGGCCGCCGCCGTGAGCAACTTCGGCTACGACCTGTACCGCGTGCGCAGCAGCACCAGCCCCACCACCAACGTGCTGCTGAGCCCCCTGAGCGTGGCCACCGCCCTGAGCGCCCTGAGCCTGGGCGCCGAGCAGCGCACCGAGAGCATCATCCACCGCGCCCTGTACTACGACCTGATCAGCAGCCCCGACATCCACGGCACCTACAAGGAGCTGCTGGACACCGTGACCGCCCCCCAGAAGAACCTGAAGAGCGCCAGCCGCATCGTGTTCGAGAAGAAGCTGCGCATCAAGAGCAGCTTCGTGGCCCCCCTGGAGAAGAGCTACGGCACCCGCCCCCGCGTGCTGACCGGCAACCCCCGCCTGGACCTGCAGGAGATCAACAACTGGGTGCAGGCCCAGATGAAGGGCAAGCTGGCCCGCAGCACCAAGGAGATCCCCGACGAGATCAGCATCCTGCTGCTGGGCGTGGCCCACTTCAAGGGCCAGTGGGTGACCAAGTTCGACAGCCGCAAGACCAGCCTGGAGGACTTCTACCTGGACGAGGAGCGCACCGTGCGCGTGCCCATGATGAGCGACCCCAAGGCCGTGCTGCGCTACGGCCTGGACAGCGACCTGAGCTGCAAGATCGCCCAGCTGCCCCTGACCGGCAGCATGAGCATCATCTTCTTCCTGCCCCTGAAGGTGACCCAGAACCTGACCCTGATCGAGGAGAGCCTGACCAGCGAGTTCATCCACGACATCGACCGCGAGCTGAAGACCGTGCAGGCCGTGCTGACCGTGCCCAAGCTGAAGCTGAGCTACGAGGGCGAGGTGACCAAGAGCCTGCAGGAGATGAAGCTGCAGAGCCTGTTCGACAGCCCCGACTTCAGCAAGATCACCGGCAAGCCCATCAAGCTGACCCAGGTGGAGCACCGCGCCGGCTTCGAGTGGAACGAGGACGGCGCCGGCACCACCCCCAGCCCCGGCCTGCAGCCCGCCCACCTGACCTTCCCCCTGGACTACCACCTGAACCAGCCCTTCATCTTCGTGCTGCGCGACACCGACACCGGCGCCCTGCTGTTCATCGGCAAGATCCTGGACCCCCGCGGCCCC

[SEQ ID No:90]

Thus, in a preferred embodiment, the construct encodes an amino acid sequence substantially as shown in SEQ ID No. 55, 57, 59, 61, 63, 65, 67, 69, 71, 85, 87 or 89, or a fragment or variant thereof.

Preferably, the construct comprises a nucleotide sequence substantially as shown in SEQ ID No. 56, 58, 60, 62, 64, 66, 68, 70, 72, 86, 88 or 90 or a fragment or variant thereof.

The inventors have created a series of recombinant expression vectors comprising the constructs of the invention.

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

In one embodiment, the recombinant vector (e.g., termed "IKC 0121V") comprises a nucleotide sequence referred to herein as SEQ ID No:73, or a fragment or variant thereof, as shown below: CGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCAAAGCCCGGGCGTCGGGCGACCTTTGGTCGCCCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCTTGTAGTTAATGATTAACCCGCCATGCTACTTATCTACGTAGCCATGCTCTAGACATGGCTCGACAGATCGAGCTCCACCGGGTACCCTAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGACTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTCGAGGTGAGCCCCACGTTCTGCTTCACTCTCCCCATCTCCCCCCCCTCCCCACCCCCAATTTTGTATTTATTTATTTTTTAATTATTTTGTGCAGCGATGGGGGCGGGGGGGGGGGGGGGGCGCGCGCCAGGCGGGGCGGGGCGGGGCGAGGGGCGGGGCGGGGCGAGGCGGAGAGGTGCGGCGGCAGCCAATCAGAGCGGCGCGCTCCGAAAGTTTCCTTTTATGGCGAGGCGGCGGCGGCGGCGGCCCTATAAAAAGCGAAGCGCGCGGCGGGCGGGAGTCGCTGCGACGCTGCCTTCGCCCCGTGCCCCGCTCCGCCGCCGCCTCGCGCCGCCCGCCCCGGCTCTGACTGACCGCGTTAC

TCCCACAGGTGAGCGGGCGGGACGGCCCTTCTCCTCCGGGCTGTAATTA

GCGCTTGGTTTAATGACGGCTTGTTTCTTTTCTGTGGCTGCGTGAAAGCC

TTGAGGGGCTCCGGGAGGCTAGAGCCTCTGCTAACCATGTTCATGCCTTC

TTCTTTTTCCTACAGCTCCTGGGCAACGTGCTGGTTATTGTGCTGTCTCAT

CATTTTGGCAAAGAATTCGCGACTCGACCCGCTAGCGCCACCATGCGCC

GCCTGGTGCTGCTGCTGTGCATCGGCGCCCTGCTGGGCCACGTGTTCTG

CCAGAACCCCGCCAGCCCCCCCGAGGAGGGCAGCCCCGACCCCGACAG

CACCGGCGCCCTGGTGGAGGAGGAGGACCCCTTCTTCAAGGTGCCCGT

GAACAAGCTGGCCGCCGCCGTGAGCAACTTCGGCTACGACCTGTACCGC

GTGCGCAGCAGCACCAGCCCCACCACCAACGTGCTGCTGAGCCCCCTG

AGCGTGGCCACCGCCCTGAGCGCCCTGAGCCTGGGCGCCGAGCAGCGC

ACCGAGAGCATCATCCACCGCGCCCTGTACTACGACCTGATCAGCAGCC

CCGACATCCACGGCACCTACAAGGAGCTGCTGGACACCGTGACCGCCCC

CCAGAAGAACCTGAAGAGCGCCAGCCGCATCGTGTTCGAGAAGAAGCT

GCGCATCAAGAGCAGCTTCGTGGCCCCCCTGGAGAAGAGCTACGGCACC

CGCCCCCGCGTGCTGACCGGCAACCCCCGCCTGGACCTGCAGGAGATCA

ACAACTGGGTGCAGGCCCAGATGAAGGGCAAGCTGGCCCGCAGCACCA

AGGAGATCCCCGACGAGATCAGCATCCTGCTGCTGGGCGTGGCCCACTT

CAAGGGCCAGTGGGTGACCAAGTTCGACAGCCGCAAGACCAGCCTGGA

GGACTTCTACCTGGACGAGGAGCGCACCGTGCGCGTGCCCATGATGAGC

GACCCCAAGGCCGTGCTGCGCTACGGCCTGGACAGCGACCTGAGCTGC

AAGATCGCCCAGCTGCCCCTGACCGGCAGCATGAGCATCATCTTCTTCCT

GCCCCTGAAGGTGACCCAGAACCTGACCCTGATCGAGGAGAGCCTGAC

CAGCGAGTTCATCCACGACATCGACCGCGAGCTGAAGACCGTGCAGGCC

GTGCTGACCGTGCCCAAGCTGAAGCTGAGCTACGAGGGCGAGGTGACC

AAGAGCCTGCAGGAGATGAAGCTGCAGAGCCTGTTCGACAGCCCCGAC

TTCAGCAAGATCACCGGCAAGCCCATCAAGCTGACCCAGGTGGAGCACC

GCGCCGGCTTCGAGTGGAACGAGGACGGCGCCGGCACCACCCCCAGCC

CCGGCCTGCAGCCCGCCCACCTGACCTTCCCCCTGGACTACCACCTGAA

CCAGCCCTTCATCTTCGTGCTGCGCGACACCGACACCGGCGCCCTGCTG

TTCATCGGCAAGATCCTGGACCCCCGCGGCCCCCGCCGCAGCAAGCGCA

GCGGCAGCGGCGCCACCAACTTCAGCCTGCTGAAGCAGGCCGGCGACG

TGGAGGAGAACCCCGGCCCCATGCGCCGCCTGCTGACCTTCATCAGCAT

CCTGGCCCTGGTGGGCGCCTTCGCCGACATCCAGATGACCCAGAGCCCC

AGCAGCCTGAGCGCCAGCGTGGGCGACCGCGTGACCATCACCTGCCGC

GCCAGCCAGGACGTAAGCACCGCCGTGGCCTGGTACCAGCAGAAGCCC

GGCAAGGCCCCCAAGCTGCTGATCTACAGCGCCAGCTTCCTGTACAGCG

GCGTGCCCAGCCGCTTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCT

GACCATCAGCAGCCTGCAGCCCGAGGACTTCGCCACCTACTACTGCCAG

CAGAGCTACGCCACCCTGCCCACCTTCGAGCAGGGCACCAAGGTGGAG

ATCAAGCGCGGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGC

GGCGGCAGCGGCGGCGGCGGCAGCGAGGTGCAGCTGGTGGAGAGCGG

CGGCGGCCTGGTGCAGCCCGGCGGCAGCCTGCGCCTGAGCTGCGCCGC

CAGCGGCTTCAGCTTCACCAGCAGCAGCGTGAGCCCCGGCAAGGGCCT

GGAGTGGGTGGGCCTGATCTACCCCTACAACGGCTTCAACTACTACGCC

GACAGCGTGAAGGGCCGCTTCACCATCAGCGCCGACACCAGCCTGCAG

ATGAACAGCCTGCGCGCCGAGGACACCGCCGTGTACTACTGCGCCCGCA

ACGCCCTGTACGGCAGCGGCGGCTACTACGCCATGGACTACTGGGGCCA

GGGCACCCTGGTGACCGTGAGCAGCTGATATACTACTAGTACGCGGCCG

CACCGGTGTACAATCAACCTCTGGATTACAAAATTTGTGAAAGATTGACT

GGTATTCTTAACTATGTTGCTCCTTTTACGCTATGTGGATACGCTGCTTTAA

TGCCTTTGTATCATGCTATTGCTTCCCGTATGGCTTTCATTTTCTCCTCCTT

GTATAAATCCTGGTTAGTTCTTGCCACGGCGGAACTCATCGCCGCCTGCC

TTGCCCGCTGCTGGACAGGGGCTCGGCTGTTGGGCACTGACAATTCCGT

GGTGTGAATTCGAGCTAGGTACAGCTTATCGATACCGTCGACAGCAGACA

TGATAAGATACATTGATGAGTTTGGACAAACCACAACTAGAATGCAGTGA

AAAAAATGCTTTATTTGTGAAATTTGTGATGCTATTGCTTTATTTGTAACC

ATTATAAGCTGCAATAAACAAGTTAACAACAACAATTGCATTCATTTTATG

TTTCAGGTTCAGGGGGAGGTGTGGGAGGTTTTTTAAAGCAAGTAAAACC

TCTACAAATGTGGTATGCTCGAGGGCATGCAACAACAACAATTGCATTCA

TTTTATGTTTCAGGTTCAGGGGGAGATGTGGGAGGTTTTTTAAAGCAAGT

AAAACCTCTACAAATGTGGTAAAATCCGATAAGGACTAGAGCATGGCTAC

GTAGATAAGTAGCATGGCGGGTTAATCATTAACTACAAGGAACCCCTAGT

GATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCC

GGGCGACCAAAGGTCGCCCGACGCCCGGGCTTTGCCCGGGCGGCCTCA

GTGAGCGAGCGAGCGCGC

[SEQ ID No:73]

Thus, in one embodiment, the recombinant vector comprises a nucleotide sequence substantially as shown in SEQ ID No. 73 or a fragment or variant thereof.

The recombinant vector may be a recombinant AAV (rAAV) vector. The rAAV may 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.

Advantageously, recombinant AAV2 elicits minimal immune responses in the host organism and mediates long-term transgene expression that can last at least one year in the retina following vector administration.

The term "recombinant AAV (rAAV) vector" may refer to a recombinant AAV-derived nucleic acid. It may comprise at least one terminal repeat.

The capsid coat (capsid coat) of AAV and recombinant vectors is known to contain three capsid proteins, designated VP1, VP2 and VP3, all of which contain a large number of overlapping amino acids, but have unique N-terminal sequences. AAV viruses contain 60 subunits, the ratio of VP1, VP2, and VP3 capsid proteins being 1:1:10[62], which together form an icosahedral structure. Many AAV serotypes have been identified that differ in their amino acid composition and thus confer different binding characteristics to receptors on host cells. Several naturally occurring AAV serotypes have been identified, such as AAV1, AAV2, AAV2.7m8, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11 and AAV12, as well as many artificial variants, wherein further modifications to amino acid sequences have been identified by screening DNA variants from a capsid coding sequence library [63]. Thus, different serotypes may exhibit tropism (tropism), and exchange of various amino acids of one pseudotype may alter tropism or infectivity for different target cells. Thus, a particular AAV pseudotype may be designed to target a particular cell type or to greatly limit infectivity to a particular organ. In some embodiments, the rAAV vector is a vector derived from an AAV serotype (including AAV1, AAV2, AAV2.7m8, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, or AAV 12). The rAAV particle may comprise capsid proteins derived from any AAV serotype, including AAV1, AAV2, aav2.7m8, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAVrh10, AAV11, or AAV12 capsids. The rAAV particles can comprise viral proteins and viral nucleic acids of the same serotype or mixed serotypes.

The capsid proteins or recombinant viral particles of the invention may 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 serotype AAV1, AAV2, AAV2.7m8, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAVrh10, AAV11, or AAV 12), or may be a derivative or chimera of a naturally occurring capsid protein comprising one or more amino acid substitutions, deletions, or additions compared to the amino acid sequence of a naturally occurring capsid protein, e.g., to confer tropism against a tissue type or cell type of interest (e.g., retinal ganglion cells, photoreceptors, or retinal pigment epithelial cells), or to reduce immunogenicity of a recombinant viral particle.

In some embodiments, the capsid protein of a recombinant viral particle of the invention has an amino acid sequence that has at least 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98% or 99% amino acid identity along its entire length to the amino acid sequence of a naturally occurring capsid protein (e.g., an AAV capsid protein of a naturally occurring serotype AAV1, AAV2, AAV2.7m8, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAVrh10, AAV11 or AAV 12).

The constructs and expression vectors described herein are useful for treating retinal disorders, particularly dry age-related macular degeneration and geographic atrophy, and more generally reducing complement activation and retinal cell damage and loss.

Thus, according to 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 for use in therapy.

According to 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 a retinal disorder, or for use in reducing complement activation and retinal cell damage and loss.

According to a fifth aspect, there is provided a method of treating, preventing or ameliorating a retinal disorder in a subject, or for reducing complement activation and retinal cell damage and loss, the method comprising administering to a subject in need of such treatment or having administered to a subject in need of such treatment a therapeutically effective amount of a gene construct according to the first aspect or a recombinant vector according to the second aspect.

Preferably, the genetic construct or recombinant vector according to the invention is used in gene therapy techniques. PEDF receptor agonists encoded by the constructs or vectors activate PEDF receptors, thereby protecting RPE and photoreceptor cells, reducing VEGF release, and preventing the transition from dry AMD to wet AMD. The anti-complement proteins encoded by the constructs or vectors neutralize complement factors, thereby reducing complement activation and reducing GA area and retinal cell loss.

In one embodiment, the retinal disorder treated may be dry age-related macular degeneration or geographic atrophy. Furthermore, the retinal disorder treated may be any pathophysiological condition that causes retinal damage through complement activation.

In another embodiment, the constructs and vectors may be used to reduce complement activation and retinal cell damage and loss. The constructs and vectors are useful for treating retinal cell damage and loss associated with: retinitis pigmentosa (retinitis pigmentosa), steud's disease (Stargardt disease), diabetic macular degeneration, age-related macular degeneration, and Leber's congenital amaurosis (Leber's congenital amaurosis). In another embodiment, the constructs and vectors may be used to treat retinal cell damage and loss associated with glaucoma.

It will be appreciated that the gene construct according to the first aspect or the recombinant vector according to the second aspect may be used in a medicament which may be used as monotherapy (i.e. the use of the gene construct according to the first aspect of the invention or the vector according to the second aspect of the invention) for the treatment, amelioration or prophylaxis of a retinal disorder, or for the reduction of complement activation and retinal cell damage and loss. Alternatively, the gene construct or recombinant vector according to the invention may be used as an adjunct to or in combination with known therapies for the treatment, amelioration or prevention of retinal disorders, or for the reduction of complement activation and retinal cell damage and loss.

An effective amount of the recombinant viral vector is administered according to the therapeutic objectives. For example, when a low percentage of transduction can achieve a desired therapeutic effect, then the therapeutic goal is typically to meet or exceed the transduction level. In some cases, such a level of transduction may be achieved by transducing only about 1 to 5% of the target cells, in some embodiments by transducing at least about 20% of the target tissue type, in some embodiments by transducing at least about 50%, in some embodiments by transducing at least about 80%, in some embodiments by transducing at least about 95%, in some embodiments by transducing at least about 99% of the cells of the target tissue type. In some embodiments of the invention, the dose of the viral particles administered to the subject is 1x10 ⁸ Up to 1x10 ¹⁴ Between the genome copies.

Recombinant viral particles may be administered by one or more injections, may be administered in the same process, or may be administered at intervals of days, weeks, months or years. In some embodiments, multiple vectors may 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 target tissue cells (e.g., retinal cells of an eye) are transduced. Methods for identifying cells transduced by recombinant viral particles comprising a recombinant viral particle capsid are known in the art. For example, immunohistochemistry or the use of enhanced green fluorescent proteins on the market, etc. can be used to detect transduction of recombinant viral particles.

In some embodiments, the recombinant vector is administered (e.g., by injection or infusion) to one or more locations in the tissue of interest (e.g., the eye). In some embodiments, the recombinant vector is administered (e.g., by injection or infusion) to one, two, three, four, five, six, seven, eight, nine, or ten or more locations in the tissue. In some embodiments, the recombinant vector is administered to more than one location simultaneously or sequentially. In some embodiments, the multiple injections of the recombinant vector are separated by no more than one hour, two hours, three hours, four hours, five hours, six hours, nine hours, twelve hours, or 24 hours.

The genetic constructs or recombinant vectors according to the invention can be combined into compositions having a number of different forms, depending on the manner of use of the composition. Thus, for example, the composition may 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 may be administered to a human or animal in need of treatment. It will be appreciated that the pharmaceutical vehicle (vehicle) according to the invention should be well tolerated by the subject to whom it is administered.

The genetic construct or recombinant vector according to the invention may also be incorporated into a slow-release or delayed-release device. Such devices may be inserted, for example, on or under the skin, and the drug may be released over weeks or even months. The device may be positioned at least adjacent to the treatment site. Such devices may be particularly advantageous when long-term treatment with genetic constructs or recombinant vectors is required, and frequent administration (e.g., at least once daily injection) is often required.

In a preferred embodiment, the medicament according to the invention may be administered to a subject by injection into the blood stream, into a nerve or directly into a site in need of treatment. For example, the drug may be injected at least adjacent to the retina. The injection may be intravitreal, suprachoroidal, subretinal, intraretinal, intravenous (bolus) or infusion) or subcutaneous (bolus or infusion) or intradermal (infusion or infusion).

It will be appreciated that the amount of gene 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 physicochemical properties of the gene construct or recombinant vector and whether it is to be used as a monotherapy or a combination therapy. The frequency of administration will also be affected by the half-life of the transgenic protein in the treated subject. The optimal dose to be administered can be determined by one skilled in the art and will vary with the particular genetic construct or recombinant vector used, the strength of the pharmaceutical composition, the mode of administration, and the progression of retinal edema or retinal damage or retinal neuronal loss resulting therefrom. Depending on other factors of the particular subject being treated (including subject age, weight, sex, diet and time of administration), it may also be desirable to adjust the dosage.

Typically, a daily dose of DNA plasmid of 0.001. Mu.g/kg body weight to 10mg/kg body weight or 1X 10 depending on the gene construct or recombinant vector used ⁸ GC/mL to 1x 10 ¹³ Daily doses of GC/mL of the viral vectors of the invention may be used to treat, ameliorate or prevent a retinal disorder.

The genetic construct or recombinant vector may be administered before, during or after onset of a retinal disorder or retinal capillary dysfunction. Daily doses may be administered in a single administration (e.g., a single daily injection or inhalation nasal spray). Alternatively, the genetic construct or recombinant vector may need to be administered twice or more times a day. For example, the genetic construct or recombinant vector may be administered in two daily doses (or more depending on the severity of the retinal disorder or retinal capillary dysfunction being treated) of between 0.001 μg/kg body weight and 10mg/kg body weight of DNA plasmid, or 1X 10 ⁸ GC/mL to 1x 10 ¹³ Between GC/mL (i.e., assuming a weight of 70 kg). The patient receiving treatment may take a first dose at wake-up and then a second dose at night (if a two dose regimen is employed), or at 3 or 4 hour intervals thereafter. Alternatively, a sustained release device may be used to provide the patient with an optimal dose of a gene construct or recombinant vector according to the invention without the need to administer a repeat agent Amount of the components.

Known procedures, such as those routinely employed by the pharmaceutical industry (e.g., in vivo experiments, clinical trials, etc.), can be used to form specific formulations and precise therapeutic regimens (e.g., daily doses and frequency of administration of the agents) of the genetic constructs or recombinant vectors according to the invention. The inventors believe that they are the first to propose a bicistronic gene construct encoding a promoter operably linked to a coding sequence that will improve retinal survival and reduce GA disease progression by increasing PEDF concentration and attenuating the complement cascade.

According to a sixth aspect, there is provided a pharmaceutical composition comprising a gene construct according to the first aspect or a recombinant vector according to the second aspect, and a pharmaceutically acceptable vehicle.

According to 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 pharmaceutically acceptable vehicle.

The "subject" may be a vertebrate, mammal, or 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 may be used in other veterinary applications. Most preferably, however, the subject is a human.

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

For example, a therapeutically effective amount of the gene construct, recombinant vector or pharmaceutical composition used may be about 1x10 ⁸ From carrier particles to about 1x10 ¹⁵ Individual carrier particles, preferably about 1x10 ¹¹ From carrier particles to about 1x10 ¹² And a plurality of carrier particles.

In some embodiments, the pharmaceutical composition of the invention has a viral titer of 5x10 per milliliter ¹⁰ To 5x10 ¹³ Between the genome copies.

In some embodiments, the pharmaceutical compositions of the inventionThe viral titer of the compound was 5x10 per ml ¹⁰ To 5x10 ¹³ Between the transduction units. The term "transduction 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, e.g., [64]As described in (a).

Reference herein to a "pharmaceutically acceptable vehicle" is to any known compound or combination of known compounds known to those skilled in the art to be useful in formulating pharmaceutical compositions.

In one embodiment, the pharmaceutically acceptable vehicle may be a solid, and the composition may be in the form of a powder or tablet. Solid pharmaceutically acceptable vehicles may include one or more substances which may also act as flavoring agents, lubricants, solubilizers, suspending agents, dyes, fillers, glidants, compression aids, inert binders, sweeteners, preservatives, dyes, coating agents or tablet disintegrating agents. The vehicle may also be an encapsulating material. In powders, the vehicle is a finely divided solid mixed with a finely divided active agent according to the invention. In tablets, the active agent (e.g. a genetic construct or recombinant vector according to the invention) may be mixed in suitable proportions with a vehicle having the necessary compression properties and compacted in the shape and size desired. Powders and tablets preferably contain up to 99% active agent. Suitable solid vehicles include, for example, calcium phosphate, magnesium stearate, talc, sugar, lactose, dextrin, starch, gelatin, cellulose, polyvinylpyrrolidone, low melting waxes and ion exchange resins. In another embodiment, the pharmaceutical vehicle may be a gel and the composition may be in the form of a cream or the like.

However, the pharmaceutical vehicle may be a liquid and the pharmaceutical composition is in the form of a suspension of particles in solution. Liquid vehicles are used for preparing solutions, suspensions, emulsions, syrups, elixirs and pressurized compositions (pressurized composition). The genetic construct or recombinant vector according to the invention may be dissolved or suspended in a pharmaceutically acceptable liquid vehicle, such as water, an ion buffer solution, an organic solvent, a mixture of both or a pharmaceutically acceptable oil or fat. The liquid vehicle may contain other suitable pharmaceutical additives such as solubilizers, emulsifiers, buffers, preservatives, sweeteners, flavoring agents, suspending agents, thickening agents, colorants, viscosity regulators, stabilizers or osmotically adjusted agents. Suitable examples of liquid vehicles for oral and parenteral administration include water (partially containing additives as described above, e.g., cellulose derivatives, preferably sodium carboxymethyl cellulose solution), alcohols (including monohydric alcohols and polyhydric alcohols, e.g., glycols) and their derivatives, and oils (e.g., fractionated coconut oil and arachis oil). For parenteral administration, the vehicle may also be an oily ester, such as ethyl oleate and isopropyl myristate. Sterile liquid vehicles can be used in sterile liquid form compositions for parenteral administration. The liquid vehicle of the pressurized composition may be a halocarbon or other pharmaceutically acceptable propellant (propella).

The liquid pharmaceutical composition is a sterile solution or suspension and can be used, for example, intravitreally, suprachoroidal, subretinal, intraretinal, intracameral, intramuscular, intrathecal, epidural, intraperitoneal, intravenous, and in particular subcutaneous injection. The genetic construct or recombinant vector may be prepared as a sterile solid composition which may be dissolved or suspended at the time of administration using sterile water, saline or other suitable sterile injection medium.

Pharmaceutically acceptable carriers, excipients and diluents are relatively inert or pharmaceutically effective substances that facilitate administration and may be provided in liquid solutions or suspensions, emulsions, or solid forms suitable for dissolution or suspension in a liquid prior to use. For example, the excipient may provide a form of suitable consistency, or act as a diluent. Suitable excipients include, but are not limited to, stabilizers, wetting agents and emulsifiers, salts for altering osmotic pressure, encapsulating agents, pH buffering substances and buffers. Such excipients include any pharmaceutical agent suitable for direct delivery to a subject (e.g., by intravitreal or subretinal) that can be administered without undue toxicity. Pharmaceutically acceptable excipients include, but are not limited to, sorbitol, any of a variety of TWEEN compounds, and liquids such as water, saline, glycerol, and ethanol. Which may include pharmaceutically acceptable salts such as mineral acid salts, e.g., hydrochloride, hydrobromide, phosphate, sulfate, and the like; and salts of organic acids, such as acetates, propionates, malonates or benzoates.

In some embodiments, the pharmaceutically acceptable excipient may include a pharmaceutically acceptable carrier. Such pharmaceutically 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. Saline solutions and aqueous dextrose, polyethylene glycol (PEG) and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. Additional ingredients may also be used, such as preservatives, buffers, tonicity agents, antioxidants and stabilizers, non-ionic wetting or clarifying agents, or viscosity increasing agents. A comprehensive discussion of pharmaceutically acceptable excipients and carriers is provided in Remington's Pharmaceutical Sciences (Ed Remington JP and Gennaro AR; mack Pub. Co. Easton, pa 1990).

It is to be understood that the present invention encompasses any nucleic acid or peptide or variant, derivative or analogue thereof, which comprises essentially amino acids or nucleic acid sequences of any of the sequences mentioned herein, including variants or fragments thereof. The terms "substantially amino acid/nucleotide/peptide sequence", "variant" and "fragment" may be sequences having at least 40% sequence identity to the amino acid/nucleotide-peptide sequence of any of the sequences mentioned herein, e.g. 40% identity to the sequences identified as SEQ ID Nos. 1-90, etc.

Amino acid/polynucleotide/polypeptide sequences having greater than 65%, more preferably greater than 70%, even more preferably greater than 75%, and still more preferably greater than 80% sequence identity to any of the sequences mentioned are also contemplated. Preferably, the amino acid/polynucleotide/polypeptide sequence has at least 85% identity with any of the sequences mentioned, 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 with any of the sequences mentioned herein.

The skilled artisan will understand how to calculate the percent identity between two amino acid/polynucleotide/polypeptide sequences. To calculate the percent identity between two amino acid/polynucleotide/polypeptide sequences, one must first prepare an alignment of the two sequences and then calculate the sequence identity value. The percentage identity of the two sequences may take different values depending on: - (i) methods for aligning sequences, e.g., clustalW,BLAST,FASTA,Smith-Waterman(implemented in a different program), or structural alignment from 3D comparison; and (ii) parameters used in the alignment method, e.g., local alignment versus global alignment, pairing score matrices (e.g., BLOSUM62, PAM250, gonnet, etc.) and gap penalties (gap-penalties), e.g., functional forms and constants, are used.

After alignment, there are many different ways in which the percent identity between two sequences can be calculated. For example, the number of identities may be divided by: (i) the length of the shortest sequence; (ii) alignment length; (iii) the average length of the sequence; (iv) the number of vacancy free positions; or (iv) the number of equivalent positions other than overhang (overlap). Furthermore, it should be appreciated that the percent identity is also strongly dependent on length. Thus, the shorter a pair of sequences, the higher the sequence identity that is expected to occur by chance.

Thus, it should be appreciated that precise alignment of protein or DNA sequences is a complex process. The popular multiplex alignment program ClustalW [65, 66] is a preferred way to generate multiple alignments of proteins or DNA according to the invention. Suitable parameters for ClustalW may be as follows: for DNA alignment: gap open penalty = 15.0, gap extension penalty = 6.66, and Matrix = Identity. For protein alignment: gap open penalty = 10.0, gap extension penalty = 0.2, and Matrix = Gonnet. For DNA and protein alignment: end= -1, gapdst=4. One skilled in the art will appreciate that it may be necessary to alter these and other parameters to achieve optimal sequence alignment.

Preferably, the percent identity between two amino acid/polynucleotide/polypeptide sequences can be calculated from such an alignment based on (N/T) x 100, where N is the number of positions of the sequences sharing the same residue and T is the total number of positions compared, including gaps and including or not including overhangs. Preferably, overhangs are included in the calculation. Thus, the most preferred method for calculating the percent identity between two sequences comprises (i) preparing a sequence alignment using the ClustalW program using a set of appropriate parameters, e.g., as described above; and (ii) inserting the values of N and T into the following formula: sequence identity= (N/T) 100.

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 under stringent conditions to a DNA sequence or its complement. Stringent conditions refer to hybridization of nucleotides to filter-bound DNA or RNA in 3 XSSC/sodium citrate (SSC) at about 45℃followed by at least one wash in 0.2 XSSC/0.1% SDS at about 20-65 ℃. Alternatively, a substantially similar polypeptide may differ from the sequences shown herein by at least 1 but less than 5, 10, 20, 50, or 100 amino acids.

Due to the degeneracy of the genetic code, it is apparent that any of the nucleic acid sequences described herein may be altered or changed to provide a functional variant thereof without substantially affecting the protein sequence it encodes. Suitable nucleotide variants refer to nucleotide variants that change the sequence by replacing different codons within the sequence encoding the same amino acid, thereby producing silent changes. Other suitable variants are variants having homologous nucleotide sequences but including all or part of the sequence, which variants are altered by substitution of different codons, which codons encode amino acids whose side chains have similar biophysical properties as the amino acids replaced thereby producing conservative changes. For example, small nonpolar hydrophobic amino acids include glycine, alanine, leucine, isoleucine, valine, proline, and methionine. Large nonpolar 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. Thus, it will be known which amino acids can be replaced by amino acids having similar biophysical properties, and the skilled person will know the nucleotide sequence encoding these amino acids. All of the features described herein (including any accompanying claims, abstract and drawings), and/or all of the steps of any method or process so disclosed, may be combined with any of the aspects described above, in any combination, except combinations where at least some of such features and/or steps are mutually exclusive.

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

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: -

FIG. 1 is a schematic representation of one embodiment of a viral vector according to the present invention (top of the figure) expressing various transgenic proteins, namely PEDF receptor agonists and anticomplements proteins, and their biological effects in reducing pathophysiology associated with retinal disease, such as geographic atrophy and dry AMD.

FIG. 2 shows a schematic representation of one embodiment of a gene 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 that directs secretion by the cell; the coding sequence of the anticomplementary protein is also preceded by a signal peptide which directs secretion of the cell; both are linked in any orientation by enzymatic cleavage-viral-2A linker sequence/jump site.

Figure 3 shows the intracellular processing of genetic material from gene therapy constructs to produce two mature therapeutic proteins that are capable of protecting retinal cells and neutralizing or attenuating complement activation. Step 1 is transcription of messenger RNA by a single promoter. Step 2 is a ribosome translation jump guided by the viral 2A sequence, producing two separate proteinogens (proteins). Step 3 occurs at the golgi level, where the viral-2A sequence is cleaved from the pro-protein at an upstream cleavage site by furin/enzyme activity. Step 4 is to remove 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.

FIG. 4 shows images of enhanced green fluorescent protein (eGFP) reporter gene expression in HEK293 cells 24 hours post transduction with rAAV2 vectors containing different promoter sequences: chicken beta-actin promoter/cytomegalovirus enhancer promoter (sCAG), introns resulting from fusion of a small stretch of nucleotides derived from 5 'and 3' of rabbit beta-globulin intron (sCAG-intron), cytomegalovirus promoter (CMV), murine phosphoglycerate kinase promoter (mPGK), and human synaptorin-1 promoter (hSYN 1) were added after the sCAG promoter.

FIG. 5 shows cross-section and flat-patch (flat) images of mouse retinas to demonstrate eGFP expression levels three weeks after intravitreal injection of rAAV2 vectors containing different promoter sequences: chicken beta-actin promoter/cytomegalovirus enhancer promoter (sCAG); megavirus enhancer elements plus chicken beta-actin promoter and a small stretch of nucleotides derived from rabbit beta-globulin introns 5 'and 3' (sCAG-introns); a cytomegalovirus promoter (CMV); murine phosphoglycerate kinase-1 promoter (mPGK); and a human synaptophysin-1 (hSYN 1) promoter. The symbol "×" denotes ganglion cell layers.

FIG. 6 shows Western blot showing expression of PEDF protein in supernatants harvested 24 hours after transfection with a series of expression plasmids from HEK293T or ARPE-19 cells, efficient furin cleavage in constructs with optimized sequences compared to the base sequence and release of virus-2A linker from the upstream protein C-terminal (FIG. 6A). IKC036P [ empty (null) control ], IKC030P [ PEDF alone ], IKC093P [ hPEDF-basal furin-virus P2A-anti Bb SCVF ], IKC094P [ hPEDF-basal furin-virus P2A-anti C5 SCVF ], IKC104P [ hPEDF-optimized furin-virus P2A-anti C3b SCVF ], IKC121P [ hPEDF-optimized furin-virus P2A-anti C3b SCVF ], IKC122P [ hPEDF-optimized furin-virus P2A-sccd 55]. FIG. 6B shows the percentage of furin cleavage in HEK293T supernatant and FIG. 6C shows the percentage of furin cleavage in ARPE-19 supernatant.

FIG. 7 is a graphical representation of ELISA assays demonstrating the concentration of PEDF released from HEK293T cells into cell culture medium 24 hours after transfection with a series of bicistronic or control plasmids; IKC036[ empty control ], IKC093P, IKC, 104P, IKC, 121P and IKC122P.

FIG. 8 is a Western blot showing intracellular processing and release of PEDF protein and non-membrane bound anti-complement factors into culture medium after transfection of HEK293T cells with plasmids IKC157P, IKC, P, IKC P and IKC161P compared to the empty control IKC166 plasmid.

Fig. 9 shows that HEK293T cells expressing PEDF and non-membrane bound anti-complement proteins use immunocytochemistry (photoptaining) prior to secretion for release into culture medium after transfection with plasmids IKC093P, IKC094P, IKC157P, IKC158P, IKC P and IKC161P compared to the empty control IKC166 plasmid.

FIGS. 10A and B show the production and release of soluble sCD55 (DAF) from HEK293T cell culture medium or ARPE-19 cell culture medium, respectively, following transfection with the IKC122P plasmid or the control IKC036 empty plasmid.

The data shown in fig. 11 shows neutralization and/or reduction of complement C3b in human serum after incubation of serum with cell growth medium from HEK293T cells transfected with either an empty control plasmid (IKC 036P) or IKC087P, IKC104P, IKC0121P, which secrete single-chain variable fragments capable of binding and neutralizing human C3 b. Note that IKC087P has an unoptimized expression cassette and ELISA antibodies have 80% cross-reactivity with human C3 (about 2/3 of immunoreactivity, thus a 30% decrease in reading would be equivalent to almost 100% neutralization of C3b because SCVF does not bind to C3).

FIG. 12A demonstrates the ability of supernatants harvested from HEK293T cells transfected with the sCD 55-producing IKC122P plasmid construct to reduce production of recombinant C3B (C3 convertase) from parental C3 in the presence of factors B and D compared to an empty control plasmid (IKC 036P) and an IKC121P plasmid construct that expresses a single-stranded variable fragment capable of binding and neutralising human C3B. Figure 12B shows a significant reduction in the percentage of C3 to C3B conversion in the presence of IKC121P compared to the IKC036P control.

FIG. 13A illustrates that supernatants harvested from HEK293T cells transfected with constructs IKC139P and IKC143P producing factor I cofactor sCD46 and CFHL1, respectively, in the presence of low concentrations of recombinant CFI can promote the breakdown of recombinant C3b into two iC3b fragments (68 and 43 kDa). Note that the empty control plasmid IKC036P or PBS control did not produce C3b decomposition. Figure 13B shows the percentage of iC3B fragments in the presence of IKC139P and IKC143P compared to IKC036P and PBS control, which did not show decomposition of C3B to iC 3B.

FIG. 14 shows an embodiment of a plasmid map of the "IKC121P" vector of the present invention.

FIG. 15 shows Western blot data for PEDF and anti-complement transgene expression 48 hours after transduction of HEK293T cells with vector IKC157V, IKC158V, IKC159V, IKC161V, IKC V compared to empty control vector IKC166V, PEDF and anti-complement transgene were expressed and secreted into the culture medium.

FIG. 16 compares the effect of low concentrations of recombinant complement factor I (11 nM) and factor H (0.5 nM) on complement C3b factor (39 nM) decomposition in HEK293T transfected cells and plasmids expressing soluble CD46 (IKC 137P) or complement I (IKC 139P) compared to empty control transfected cells (IKC 036P). Note that supplementation of recombinant complement factor I with cell culture medium harvested from HEK293T cells transfected with IKC137P (complement I) did not increase C3b lysis compared to IKC036P null control. In contrast, supplementation of cell culture medium harvested from HEK293T cells transfected with IKC139P (soluble CD 46) significantly increased enzymatic C3b lysis as seen by the decrease in the C3b alpha chain band and the increase in the iC3b (68 kDa and 43 kDa) bands.

Figure 17 illustrates that intravitreal injection of IKC159V rAAV2 vector increased vitreous PEDF concentration (a) and protected retinal ganglion cells (B and C) when challenged with neurotoxin N-methyl-D-aspartate (NMDA) (3 weeks after delivery of gene therapy, 8 days of challenge). Furthermore, rAAV2 vectors were able to secrete enough soluble CD46 into the vitreous that it was able to significantly break down recombinant complement C3b (ex vivo) (D) compared to the vitreous isolated from animals treated with empty IKC166V vectors.

Figure 18 shows the beneficial effect of the IKC159V rAAV2 vector in preventing the transient decrease in transepithelial resistance of ARPE-19 cells when subjected to mild oxidative stress (hydrogen peroxide) and complement attack (addition of human serum proteins). The data show transient loss of transepithelial resistance and percent change from baseline at 2 hours when IKC159V or IKC166V (empty vector) transduced cells were treated with both hydrogen peroxide and human serum. The comparison is ARPE-19 cells treated with hydrogen peroxide or human serum alone.

Examples

Referring to fig. 1 and 2, the inventors designed and constructed a novel gene construct encoding (i) an agonist of PEDF receptor and (ii) an anticomplementary protein under the control of a single promoter. As shown in FIG. 3, the inventors have also introduced a spacer sequence into the genetic construct (e.g., a viral-2A peptide spacer sequence), which advantageously allows all peptides encoded by the construct to be expressed as a single mRNA transcript under the control of a single promoter. Furthermore, in order to enzymatically remove the viral-2A peptide sequence from the C-terminus of the protein, the inventors introduced a viral-2A removal sequence into the construct, e.g. a furin recognition sequence, see fig. 6.

As shown in FIG. 3, the bicistronic expression cassette produces two mature therapeutic proteins, the PEDF receptor agonist and the anticomplementary protein (FIGS. 7-11 and 15). PEDF receptor agonists are used to protect Retinal Pigment Epithelium (RPE) and other retinal cells (e.g., photoreceptors) from cytotoxic biochemical damage and cell death. In patients with dry AMD and geographic atrophy, endogenous PEDF concentrations in the eye are significantly reduced due to disease pathology, thereby reducing the ability of the retina to function properly. The genetic constructs of the invention will complement retinal PEDF concentrations, restoring retinal defense mechanisms against oxidative damage and other pathophysiological factors that occur in dry AMD. As shown in fig. 17, increasing PEDF concentration will prevent further loss of RPE cells and overlying photoreceptors, thereby slowing or preventing vision loss. Furthermore, as shown in figures 12, 13, 16-18, anticomplementary proteins are able to reduce complement system activation, which have also been shown to play an important role in GA. By increasing retinal PEDF concentration in combination with reduced complement activation to better protect retinal cells from loss, the bicistronic gene construct is able to alleviate or prevent further retinal damage and associated loss of visual acuity.

The inventors then introduced the gene construct into a recombinant expression vector, such as rAAV2 (see, e.g., fig. 14).

Materials and methods

Design and production of DNA plasmids

The tool is usedhttp://www.jcat.de) Or a Genscript online tool to codon optimize the DNA sequence. Synthetic DNA block and cloning was performed using standard molecular biology techniques. After maxi-prep purification, endotoxin content was minimal and all DNA plasmids were amplified overnight in SURE competent cells (Agilent Technologies).

IKC036P is an empty control. IKC030P contains PEDF alone. IKC093P comprises [ hPEDF-basic furin-virus P2A-anti-Bb SCVF ], IKC094P comprises [ hPEDF-basic furin-virus P2A-anti-C5 SCVF ], IKC104P comprises [ hPEDF-optimized furin-virus P2A-anti-C3 b SCVF ], IKC121P comprises [ hPEDF-optimized furin-virus P2A-anti-C3 b SCVF ], and IKC122P comprises [ hPEDF-optimized furin-virus P2A-scVF 55], IKC157P comprises [ hPEDF-optimized furin-virus P2A-anti-C3 bSCVF ], IKC158P [ hPEDF-optimized furin-virus P2A-scCD 46], IKC161P [ hPEDF-optimized furin-virus P2A-sCD46], and IKEDP [ hPEDF-optimized [ hPEDP-1 ] contrast.

Production of recombinant AAV vectors

Recombinant AAV2 vectors were made using DNA plasmids. HEK293 cells (2.5X10) were transduced with a total of 500. Mu.g of three plasmids (Rep-2-Cap 2, pHelper and plasmid containing ORF and ITR) ⁸ ). After freeze thawing HEK293 cells to release viral vector particles, iodixanol gradient ultracentrifugation and desalting were performed subsequently. Suspending the vector in Dulbecco's phosphate from Thermo Fisher/GibcoIn salt buffered saline (DPBS) buffer manufactured according to cGMP standard (catalog number 14190250, from 8g/L NaCl, 1.15g/L Na ₂ HPO ₄ 0.2g/L KCl and 0.2g/LK ₂ HPO ₄ The composition does not contain calcium or magnesium; pH 7.0-7.3, 270-300 mOsm/kg). The following vector titers were obtained by qPCR using primers that recognize the ITR region.

FIG. 4 shows the use of a promoter sequence comprising a different sequence: expression of enhanced green fluorescent protein (eGFP) reporter gene in HEK293 cells 24 hours after transduction of rAAV2 vector of sCAG, sCAG-intron, CMV, hSYN1 and mPGK. As shown in fig. 4, both sCAG and CMV showed high levels of eGFP transgene expression in HEK293 cells.

In addition, figure 5 shows cross-sections and flat-patch images of mouse retinas to demonstrate that intravitreal injections contain different promoter sequences: eGFP expression levels three weeks after rAAV2 vector of sCAG, sCAG-intron, CMV, mPGK and hSYN 1. As can be seen in FIG. 5, the addition of an intron to the sCAG-intron promoter increases retinal expression compared to the same promoter without the intron (i.e., sCAG).

EXAMPLE 1 concentration of PEDF in HEK293T cells after transfection of cells with various plasmid constructs

Briefly, the DNA plasmid was mixed with Opti-MEM (FisherSci; loughborough, leics., U.K.) and lipofectamine 3000 (FisherSci) and added to HEK293T cells cultured to 80% confluence in 24 well plates such that 0.5. Mu.g of plasmid DNA and 0.75. Mu.L lipofectamine were received per well. The cells were incubated at 37℃with 5% CO ₂ Incubate for 24 hours. HEK293T cell incubation medium was collected and centrifuged to remove any cell debris, and subsequently PEDF concentrations produced from the cells were measured using a commercial human PEDF ELISA kit (Abcam; cambridge, u.k.; ab 246535) or by western blotting (Abcam ab180711, dilution 1:1000). The control empty plasmids (IKC 036P or IKC 166P) did not show any further contribution to the small amounts of PEDF produced by HEK293T cells.

Figures 6 and 7 show that 24 hours after transfection with a range of plasmids, HEK293T cells secreted significantly more PEDF than the empty controls (IKC 036P and IKC 166P).

Example 2 detection of furin Activity and Virus-2A peptide cleavage

HEK293T cells were transfected with plasmids as described above. The molecular weight of the test transgenes from the bicistronic constructs (PEDF and anti-complement proteins) was compared to the transgenic constructs that produced only a single transgene. To confirm cleavage of the viral-2A peptide from the C-terminus, the presence of the viral-2A peptide was detected using a 2A antibody (NBP 2-59627) from Bio-Techne (Abingdon, oxon, u.k.).

FIG. 6 shows the expression of PEDF protein (with and without virus-2A) in supernatants harvested from HEK293T and ARPE-19 cells 24 hours after transfection with a series of expression plasmids. Furthermore, FIGS. 6B and 6C illustrate the quantification of efficient furin cleavage and release of the viral-2A linker from the C-terminus of PEDF in HEK293T and ARPE-19 cells transfected with a range of plasmids, indicating that the optimized furin sequences in the IKC104P, IKC P and IKC122P plasmids were significantly more efficient than the base furin sequences in the IKC093P and IKC094P plasmids. PEDF derived from HEK293T cells transfected with IKC030P plasmid was used as a control because it did not have C-terminal furin or viral 2A sequences.

Example 3 detection of expressed anticomplement proteins

FIGS. 8 and 9 show the production, proper processing and release of PEDF and downstream anti-complement proteins in HEK293T cells after transfection with plasmids. In FIG. 8, PEDF and anticomplementary proteins released into the medium from HEK293T cells transfected with control IKC166P (empty) or IKC157P, IKC158P, IKC P and IKC161P plasmids were assayed by Western blotting using the same antibodies as used in immunocytochemistry (see below) and all diluted 1:1000. The secondary antibody was a goat-anti-rabbit antibody (Abcam, ab 6721) diluted 1:10,000.

FIG. 9 shows pre-release PEDF and anti-complement proteins in transfected HEK293T cells grown on coverslips and stained by immunocytochemistry (anti-Bb, anti-C5 and anti-C3 b SCVF stained with Genscript generated custom rabbit polyclonal antibody (peptide 1), CD55 Abcam ab133684, CD46 Invitrogen PA535311 and CFH Abcam ab 133536). In fig. 15, PEDF and anti-complement proteins released into the culture medium from HEK293T cells transduced with rAAV vectors (including control IKC166V (empty vector) or IKC157V, IKC158V, IKC159V, IKC161V and IKC167V rAAV2 vector) were assessed by western blotting using the same antibodies as described in fig. 8 and 9 above.

FIG. 10 shows the production and secretion of soluble/non-cell membrane bound forms of (DAF) sCD55 in HEK293T and ARPE-19 cells 24 hours after transfection with plasmid IKC122P by Western blot assessment using CD55 antibody (ab-133684 from 1:1000 dilution of Abcam (Cambridge, U.K.) and peroxidase-labeled goat anti-rabbit secondary antibody (1:10,000 dilution, abcam, ab 6721).

EXAMPLE 4 demonstration of anticomplement protein Activity

The activity of anti-complement proteins is shown in figures 11, 12, 13 and 16, wherein neutralization of C3b is shown in figure 11, prevention of C3 convertase (C3 bBb) production is shown in figure 12, complement Factor I (CFI) mediated cleavage of C3b assay is shown in figures 13 and 16.

For the C3b neutralization assay shown in fig. 11, HEK293T cells were transfected with plasmids as described above. After 24 hours, the supernatant was collected and clarified by brief centrifugation. The supernatant (190. Mu.L) was incubated with 10. Mu.L of normal human serum (1:20000 dilution) at room temperature with gentle agitation for 30 minutes. After incubation, the samples were quantified using the human complement C3b ELISA kit (abcam, ab 195461) which had 80% cross-reactivity with human C3 (about 2/3 of immunoreactivity, thus a 30% decrease in reading corresponds to almost 100% neutralization of C3b because SCVF did not bind to C3).

For the C3 convertase assay shown in fig. 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 with HEK293T medium, previously transfected with various plasmids, in veronal buffer for 30 minutes at 37 ℃ in a total volume of 50 μl. After incubation, the production of C3 convertase was measured by SDS-Page electrophoresis and staining of the gel with simplybue Safe stain (thermosusher).

For the C3b cleavage assay shown in fig. 13, HEK293T medium previously transfected with various plasmids was incubated with C3b substrate (42 nM) and recombinant CFI (1.2 nM) (Complement Technologies inc.) for 60 minutes at 37 ℃ in a total volume of 60 μl. The C3b breakdown products (iC 3b 64kDa and 43 kDa) were detected by Western blotting using goat anti-human C3 antibodies (AHP 1752,1:2,000 dilution; bioRad) and peroxidase-labeled donkey anti-goat secondary antibodies (705-035-147,1: 10,000;Jackson ImmunoResearch Europe,Ely,U.K.).

For the C3b cleavage assay shown in fig. 16, medium from transfected HEK293T cells was combined with recombinant complement factors. The concentrations of recombinant complement factor I and factor H used previously were reduced to 11nM and 0.5nM, respectively, while substrate C3b was maintained at about 39nM. Note that the addition of more complement factor I (IKC 137P) from HEK293T medium did not significantly increase C3b decomposition, whereas addition of HEK293T medium containing soluble CD46 cofactor significantly promoted C3b decomposition (IKC 139P). These data indicate that C3b decomposition is more sensitive to cofactor addition than to factor I supplementation.

Example 5-demonstration of anticomplementary protein and PEDF activity of bicistronic rAAV vector in vivo.

The activity of both sCD46 and PEDF proteins after intravitreal delivery of IKC159V (soluble CD46 vector) is shown in figure 17.

Mice were intravitreally injected (2 μl) with either IKC166V (empty control) or IKC159V. After 21 days, their eyes were dissected, vitreous samples (4 to 5 μl) were extracted and C3b decomposition was assessed ex vivo using the C3b cleavage assay method described above. The results show significant breakdown of C3b in the vitreous of eyes treated with IKC159V compared to IKC166V (empty) group.

Another group of mice receiving intravitreal injections (2 μl) of IKC166V (empty control) or IKC159V was used in the NMDA study. 21 days after vehicle injection, mice received further intravitreal injection of NMDA (30 nmol/eye) or vehicle and animals were sacrificed 8 days later. Vitrectomy samples were obtained from vehicle-injected eyes and PEDF concentration was measured using a commercial PEDF ELISA kit (Abcam). Retinal plates were prepared from all eyes and retinal ganglion cell counts were measured using RBPMS immunomarkers. Notably, the retinal ganglion cells of the IKC166V (null) plus NMDA treated group were about 50% lost compared to the almost complete protection of the IKC159V plus NMDA treated group.

Example 6-demonstration of the decrease in trans-epithelial resistance of the bicistronic rAAV2 vector (IKC 159V) against ARPE-19 cells Is of beneficial effect of (1)

For the ARPE-19 cell transepithelial resistance (TER) assay shown in FIG. 18, ARPE-19 cells were grown on transwells (24 wells, greiner) for 2 weeks, and medium was periodically changed until stable monolayers and TER were reached. The medium was then changed to serum-free medium for another 2 weeks before transduction of the monolayers with rAAV vectors for 48 hours. Upon exposure to 1mM H ₂ O ₂ And human serum were taken 1, 2 and 4 hours before and after the TER assay readings. As shown in FIG. 18, the IKC159V rAAV2 vector prevented a transient decrease in transepithelial resistance of ARPE-19 cells when challenged with mild oxidative stress.

Discussion and conclusion

As shown in the examples, the inventors have surprisingly demonstrated that it is possible to combine Open Reading Frames (ORFs) encoding PEDF receptor agonists and anti-complement proteins in a single gene construct.

PEDF receptor agonists restore PEDF concentration, thereby reducing inflammation and protecting RPE and photoreceptor cells. In addition, anti-complement proteins are capable of neutralizing or attenuating the alternative complement pathway, thereby preventing further loss of RPE cells. Advantageously, the genetic constructs of the invention target the AP pathway, which means that the classical and lectin pathways of the complement system are maintained, thereby maintaining an antimicrobial defense system that helps to destroy invading pathogens.

Reference to the literature

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.Risk factors for age-related macular degeneration:Pooled findings from three continents.Ophthalmology 2001,vol.108,PP:697-704.

6.Fraser-Bell S,Donofrio J,Wu J,Klein R,Azen SP,Varma R,Los Angeles Latino Eye Study Group(2005).Sociodemographic factors and age-related macular degeneration in Latinos:the Los Angeles Latino Eye Study.American Journal of Ophthalmology,vol.139,PP:30-38.

7.Young RW(1987).Pathophysiology of age-related macular degeneration.Surv.Ophthalmol.,vol.31,PP:291-306.

8.Pilgrim MG,Lengyel I,Lanzirotti A,Newville M,Fearn S,Emri E,Knowles JC,Messinger JD,Read RW,Guidry C,Curcio CA(2017).Subretinal pigment epithelial deposition of Drusen components including hydroxyapatite in a primary cell culture model.Invest.Ophthamol.Vis.Sci.,vol.58,PP:708-719.

9.Schmitz-Valckenberg S,Fleckenstein M,Gobel AP,Hohman TC,Holz FG(2011).Optical coherence tomography and autofluorescense findings in areas with geographic atrophy due to age-related macular degeneration.Invest.Ophthalmol.Cis.Sci.2011,vol.52(1),PP:1-6.

10.Marsiglia M,Boddu S,Bearelly S,Xu L,Breaux BE Jr,Freund KB,Yannuzzi LA,Smith RT(2013).Association between geographic atrophy progression and reticular pseudodrusenin eyes with dry age-related macular degeneration.Invest Ophthalmol Vis Sci.,vol.54,PP:7362-7369.

11.Kennedy CJ,Raboczy PE,Constable IJ(1995).Lipofuscin of the retinal pigment epithelium:a review.Eye,vol.9,PP:763-771.

12.Feeney-Burns L,Hilderbrand ES,Eldridge S(1984).Aging human RPE:morphometric analysis of macular,equatorial and peripheral cells.Invest.Ophthamol.Vis.Sci.,vol.25,PP:195-200.

13.Dorey CK,Wu G,Ebenstein D,Garsd A,Weiter JJ(1989).Cell loss in the aging retina.Relationship to lipofuscin accumulation and macular degeneration.Invest.Ophthamol.Vis,Sci.,30(8),1691-1699.

14.Delori FC,Dorey DDK,Staurenghi G,Arend O,Goger DG,Weiter JJ(1995).In vivo fluorescence of the ocular fundus exhibits retinal pigment epithelium lipofuscin characteristics.Invest.Ophthamol.Vis.Sci.,vol.36,PP:718-729.

15.Delori FC,(1995).RPE lipofuscin in aging and age-related macular degeneration.In:Coscas G,Piccolino FC,eds.Retinal Pigment Epithelium and Macular Disease:Documenta Ophthalmologica.Dordrecht,The Netherlands,Kluwer；vol.62,PP:37-45.

16.Holz FG,Pauleikhoff D,Klein R,Bird AC(2004).Pathogenesis of lesions in late age-related macular disease.Am J Ophthalmol,vol.137(3),PP:504-510.

17.Eldred G,Lasky MR(1993).Retinal age pigments generated by self-assembling lysosomotrophic detergents.Nature,vol.361,PP:724-726.

18.Sakai N,Decatur J,Nakanishi K,Eldered GE(1996).Ocular age pigment “A2E”:an unprecedented pyridinium bisretinoid.J.Am.Chem.Soc.,vol.118,PP:1559-1560.

19.Ren RX-F,Sakai N,Nakanishi K,Eldred GE(1996).Total synthesis of the ocular age pigment A2E:a convergent pathway.J.Am.Chem.Soc.,vol.119,PP:3619-3620.

20.Parish CA,Hashimoto M,Nakanishi K,Dillon J,Sparrow JR(1998).Isolation and one-step preparation of A2E and iso-A2E,fluorophores from human retinal pigment epithelium.Proc.Natl.Acad.Sci.USA,vol.95,PP:14609-14613.

21.Boyer NP,Higbee D,Currin MB,Blakeley LR,Chen C,Ablonczy Z,Crouch RK,Koutalos Y(2012).Lipofuscin and N-retinylidene-N-retinylethaolamine(A2E)accumulate in retinal pigment epithelium in absence of light exposure.J.Biol.Chem.,vol.287(26),PP:22276-22286.

22.Weiter JJ,Delori FC,Wing GL,Fitch KA(1986).Retinal pigment epithelial lipofuscin and melanin and choroidal melanin in human eyes.Invest.Ophthamol.Vis.Sci.,vol.27,PP:145-152.

23.Lakkaraju A,Finnemann SC,Rodriguez-Boulan E(2007).The lipofuscin fluorphore A2E perturbs cholesterol metabolism in retinal pigment epithelial cells.Proc.Natl.Acad.Sci.USA,vol.104(26),PP:11026-11031.

24.Maeda A,Maeda T,Golczak M,Palczewska G,Dong Z,Golczak M,et al(2008).Retinopathy in mice induced by disrupted all-trans-retinal clearance.J.Biol.Chem.,vol.283(39),PP:26684-26693.

25.Zhang J,Kiser PD,Badiee M,Palczewska G,Dong Z,Golczak M(2015).Molecular pharmacodynamics of emixustat in protection against retinal degeneration.J.Clin.Invest.,vol.125(7),PP:2781-2794.

26.Rosenfeld PJ,Dugel PU,Holz FG,Heier JS,Pearlman JA,Novack RL,Csaky KG,Koester JM,Gregory JK,Kubota R(2018).Emixustat hydrochloride for geographic atrophy secondary to age-related macular degeneration.Ophthalmol.,vol.125(10),PP:1556-1567.

27.Trou LA,Pickering MC,Blom AM(2017).The complement system as a potential therapeutic target in rheumatic disease.Nature Rev.,vol.13,PP:538-547.

28.Anderson DH,Radeke MJ,Gallo NB,Chapin EA,Johnson PT,Curletti CR,Hancox LS,Hu J,Ebright JN,Malek G,Hauser MA,Rickman CB,Bok D,Hageman GS,Johnson LV(2010).The pivotal role of the complement system in aging and age-related macular degeneration:hypothesis re-visited.Prog.Retin.Eye Res.,vol.29,PP:95-112.

29.Scholl HPN,Issa PC,Walier M,Janzer S,Pollok-Kopp B, F,Fritsche LG,Chong NV,Fimmers R,Wienker T,Holz FG,Weber BH,Oppermann M(2008).Systemic complement activation in age related macular degeneration.PLoS ONE,vol.3(7)P:e2593.

30.Sepp T,Khan JC,Thurlby DA,Shahid H,Clayton DG,Moore AT,Bird AC,Yates JR(2006).Complement factor H variant Y402H is a major risk determinant for geographic atrophy and choroidal neovascularization in smokers and nonsmokers.Invest.Ophthalmol.Vis.Sci.,vol.47(2),PP:536-540.

31.Cameron DJ,Yang Z,Gibbs D,Chen H,Kaminoh Y,Jorgensen A,Zeng J,Luo L,Brinton E,Brinton G,Brand JM,Bernstein PS,Zabriskie NA,Tang S,Constantine R,Tong Z,Zhang K.(2007).HTRA1 variant confers similar risks to geographic atrophy and neovascular age-related macular degeneration.Cell Cycle 2007,vol.6(9),PP:1122-1125.

32.Hageman GS,Luthert PJ,Victor Chong NH,Johnson LV,Anderson DH,Mullins RF(2001).An integrated hypothesis that considers drusen as biomarkers of immune-mediated processes at the RPE-Bruch’s membrane interface in aging and age-related macular degeneration.Prog Retin.Eye Res.,vol.20,PP:705-732.

33.Xu H,Chen M(2016).Targeting the complement system for the management of retinal inflammatory and degenerative diseases.Eur.J.Pharmacol.,vol.787,PP:94-104.

34.Nilsson SC,Sim RB,Lea SM,Fremeaux-Bacchi V,Blom Am(2011)Complement factor I in health and disease.Mol.Immunol.Vol.,48(14),PP:1611-1620.

35.Lachmann PJ(2019)The story of complement factor I.Immunology,vol.224(4),PP:511-517.

36.Lukacik P,Roversi P,White J,Esser D,Smith GP,Billington J,Williams PA,Rudd PM,Wormald MR,Harvey DJ,Crispin MDM,Radcliffe CM,Dwek RA,Evans DJ,Morgan BP,Smith RAG,Lea SM(2004).Complement regulation at the molecular level:The structure of decay-accelerating factor.Proc.Natl.Acad.Sci.USA,vol.101(5),PP:1279-1284.

37.Lubin DM,Atkinson JP(1998).Decay-accelerating factor:Biochemistry,molecular-biology,and function.Ann.Rev.Immunol.,vol.7,PP:35-58.

38.Williams P,Chaudry Y,Goodfellow IG,Billington J,Powell R,Spiller OB,Evans DJ,Lea S(2003).Mapping CD55 function.J.Biol.Chem.,vol.12,PP:10691-10696.

39.Dho SH,Lim JC,Kim LK(2018).Beyond the role of CD55 as a complement component.Innune Netw.,Vol.18(1),e11.

40.Skerka C,Chen Q,Fremeaux-Bacchi V,Roumenina LT(2013).Complement factor H related proteins(CFHRs).Mol.Immunol.,vol.56(3),PP:170-180.

41.Cipriani V,Lores-Motta L,He F,Fathalla D,Tilakaratna V,McHarg S,Bayatti N,Acar IE,Hoyng CB,Fauser S,Moore AT,Yates JRW,de Jong EK,Morgan BP,den Hollander AI,Bishop PN,Clark SJ(2020)Increased circulating levels of Factor H-related Protein 4 are strongly associated with age-related macular degeneration.Nat.Commun.Vol.11(1),PP778.

42.Lako M,Kavangh D,(2021).Revisiting the role of factor H in age-related macular degeneration:Insights from complement-mediated renal disease and rare genetic variants.Surv.Ophthamol.,vol.66(2),PP:378-401.

43.Heinen S,Hartmann A,Lauer N,Wiel U,Dahse H-M,Schirmer S,Gropp K,Enghardt T,Wallich R, S,Mihlan M,/>-Schrehardt U,Zipfel PF,Skerka C(2009).Factor H-related protein 1(CFHR-1)inhibits complement C5 convertase activity and terminal complex formation.Blood,vol.114(12),PP:2439-2447.

44.Geller A,Yan J(2019).The role of membrane bound complement regulatory proteins in tumor development and cancer immunotherapy.Front.Immunol.,vol.10；article 1074.

45.Christiansen D,Milland J,Thorley BR,McKenzie IFC,Mottram PL,Purcell LJ,Loveland BE(1996).Engineering of recombinant soluble CD46:an inhibitor of complement activation.Immunology,vol.87,PP:348-354.

46.Holz FG,Sadda SR,Busbee B et al(2018).Efficacy and safety of lampalizumab for geographic atrophy due to age-related macular degeneration.JAMA Ophthalmol.,vol.136(6),PP:666-677.

47.Dreismann AK,MvClements ME,Barnard AR,Orhan E,Hughes JP,Lachmann PJ,MacLaren RE(2021)Functional expression of complement factor I following AAV-mediated gene delivery in the retina of mice and human cells.Gene Ther.,https://www.nature.com/articles/s41434-021-00239-9

48.Tombran-Tink J,Chader GG,Johnson LV(1991).PEDF:a pigment epithelium-derived factor with potent neuronal differentiative activity.Exp.Eye Res.,vol.53(3),PP:411-414.

49.Tombran-Tink J,Johnson LV(1989).Neuronal differentiation of retinoblastoma cells induced by medium conditioned by human RPE cells.Invest.Ophthalmol.Vis.Sci.,vol.30(8),PP:1700–1707.

50.Subramanian P,Locatelli-Hoops S,Kenealey J,DesJardin J,Notari L,Becerra SP(2013).Pigment epithelium-derived factor(PEDF)prevents retinal cell death via PEDF receptor(PEDF-R).J.Biol.Chem.,vol.288(33),PP:23928-23942.

51.Kenealey J,Subramanian P,Comitato A,Bullock J,Keehan L,Polato F,Hoover D,Marigo V,Becerra SP(2015).Small retinoprotective peptides reveal a receptor-binding region on pigment epithelium-derived factor.J.Biol.Chem.,vol.290(42),PP:25241-25253.

52.Filleur S,Volz K,Nelius T,Mirochnik Y,Huang H,Zaichuk TA,Aymerich MS,Becerra SP,Yap R,Veliceasa D,Shroff EH,Volpert OV(2005).Two functional epitopes of pigment epithelial-derived factor block angiogenesis and induce differentiation in prostate cancer.Cancer Res.,vol.65(12),PP:5144–5152.

53.Tombran-Tink J,Barnstable CJ(2003).PEDF:a multifaceted neurotrophic factor".Nature Reviews.Neuroscience,vol.4(8),PP:628–636.

54.Dawson DW,Volpert OV,Gillis P,Crawford SE,Xu H,Benedict W,Bouck NP(1999).Pigment epithelium-derived factor:a potent inhibitor of angiogenesis.Science,vol.285(5425),PP:245–248.

55.Cao W,Tombran-Tink J,Chen W,Mrazek D,Elias R,McGinnis JF(1999).Pigment epithelial-derived factor protects cultured retinal neurons against hydrogen peroxide-induced cell death.J.Neurosci.Res.,vol.57,PP:789-800.

56.Cao W,Tombran-Tink J,Elias R,Sezate S,Mrazek D,McGinnis JF(2001).In vivo protection of photoreceptors from light damage by pigment epithelium-derived factor.Invest.Ophthamol.Vis.Sci.,vol.42,PP:1646-1652.

57.Aymerich MS,Alberdi EM,Martinez A,Becerra SP(2001).Evidence for the pigment epithelial-derived factor receptors in the neural retina.Invest.Ophthamol.Vis.Sci.,vol.42(13),PP:3287-3293.

58.Wang X,Liu X,Ren Y,Liu Y,Han S,Zhao J,Gou X,He Y(2019).PEDF protects human retinal pigment epithelial cells against oxidative stress via upregulation of UCP2 expression.Mol.Med.Report,vol.19,PP:59-74.

59.Callis,J.,Fromm,M.,and Walbot,V.(1987).Introns increase gene expression in cultured maize cells.Genes Dev.1,1183–1200.

60.Rose AB(2019).Introns as gene regulators:a brick on the accelerator.Front.Genet.,vol.9；672.

61.Haut DD,Pintel DJ(1998).Intron definition is required for excision of the minute virus of mice small intron and definition of the upstream exon.J.Virol.,vol.72(3),PP:1834-1843.

62.Samulski RJ,Chang L-S,Shenk T(1987).A recombinant plasmid from which an infectious adeno-associated virus genome can be excised in vitro and its use to study viral replication.J.Virol.,vol.,61(10),PP:3096-3101.

63.Li C,Samulski RJ(2020).Engineering adeno-associated virus vectors for gene therapy.Nat.Rev.Genetics,vol.21,PP:255-272.

64.Fisher KJ,Gao GP,Weiitzman MD,DeMatteo R,Burda.JF,Wilson JM(1996).Transduction with recombinant adeno-associated virus for gene therapy is limited by leading-strand synthesis.J.Virol.,vol.70(1),PP:520-532.

65.Thompson JD,Higgins DG,Gibson TJ(1994).CLUSTAL W:improving the sensitivity of progressive multiple sequence alignment through sequence weighting,position-specific gap penalties and weight matric choice.Nucleic Acids Res.,22(22),4673-4680.

66.Thompson JD,Gibson TJ,Plewniak F,Jeanmougin F,Higgins DG(1997).The CLUSTAL_X windows interface:flexible strategies for multiple sequence alignment aided by quality analysis tools.Nucleic Acids Res.,25(24),4876-4882

Claims

1. A genetic construct comprising a promoter operably linked to a first coding sequence encoding an agonist of PEDF receptor and a second coding sequence encoding an anticomplementary protein.

2. The genetic construct of claim 1, wherein the promoter is a Cytomegalovirus (CMV) promoter, a fusion of a CMV early enhancer element with a first intron of a chicken β -actin gene (CAG), a vitelliform macular dystrophin-2 (VMD 2) promoter, a human phosphoglycerate kinase-1 (PGK-1) promoter, or an EF1 a promoter, optionally wherein the promoter comprises a 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 according to claim 1 or 2, wherein the first coding sequence comprises a nucleotide sequence encoding PEDF protein.

4. The genetic construct according to any preceding claim, wherein the first coding sequence comprises a nucleotide sequence substantially as shown in any one of SEQ ID nos. 11, 12 or 13 or a fragment or variant thereof, and/or wherein the first coding sequence encodes an amino acid sequence substantially as shown in SEQ ID No. 10 or a fragment or variant thereof.

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

6. The genetic construct according to any preceding claim, wherein the anti-complement protein is capable of targeting the Alternative Pathway (AP) of the complement system, preferably wherein the anti-complement protein minimally affects the Classical Pathway (CP) and/or Lectin Pathway (LP) of the complement system.

7. The genetic construct according to any preceding claim, wherein the anti-complement protein is an anti-C3 b, anti-Bb or anti-C5 antibody or antigen-binding fragment thereof, optionally wherein the anti-complement protein is Single Chain Variable Fragment (SCVF).

8. The genetic construct according to any one of claims 1-6, wherein the anti-complement protein is CD55, preferably soluble CD55 (sCD 55).

9. The genetic construct of any one of claims 1-6, wherein the anti-complement protein is complement factor H-related protein-1 (CFHR 1).

10. The genetic construct according to any one of claims 1-6, wherein the anti-complement protein is CD46, preferably soluble CD46 (sCD 46).

11. The genetic construct of any one of claims 1-6, wherein the anti-complement protein is complement factor H-like protein 1 (CFHL 1).

12. The genetic construct according to claim 7, wherein the second coding sequence comprises a nucleotide sequence substantially as shown in SEQ ID No. 15, 17 or 83, or a fragment or variant thereof, and/or wherein the second coding sequence encodes an amino acid sequence substantially as shown in SEQ ID No. 14, 16 or 82, or a fragment or variant thereof.

13. The genetic construct according to claim 8, wherein the second coding sequence comprises a nucleotide sequence substantially as shown in SEQ ID No. 19 or a fragment or variant thereof, and/or wherein the second coding sequence encodes an amino acid sequence substantially as shown in SEQ ID No. 18 or a fragment or variant thereof.

14. The genetic construct according to claim 9, wherein the second coding sequence comprises a nucleotide sequence substantially as shown in SEQ ID No. 21 or 22 or a fragment or variant thereof, and/or wherein the second coding sequence encodes an amino acid sequence substantially as shown in SEQ ID No. 20 or a fragment or variant thereof.

15. The genetic construct according to claim 10, wherein the second coding sequence comprises a nucleotide sequence substantially as shown in SEQ ID No. 24 or a fragment or variant thereof, and/or wherein the second coding sequence encodes an amino acid sequence substantially as shown in SEQ ID No. 23 or a fragment or variant thereof.

16. The genetic construct according to claim 11, wherein the second coding sequence comprises a nucleotide sequence substantially as shown in SEQ ID No. 81 or a fragment or variant thereof, and/or wherein the second coding sequence encodes an amino acid sequence substantially as shown in SEQ ID No. 80 or a fragment or variant thereof.

17. The genetic construct according to any preceding claim, wherein the genetic construct comprises a spacer sequence disposed between the first coding sequence and the second coding sequence, the spacer sequence encoding a peptide spacer configured to produce PEDF receptor agonist and anticomplementary protein as separate molecules.

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

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

20. The genetic construct according to any one of claims 17-19, wherein the spacer sequence comprises a nucleotide sequence substantially as shown in SEQ ID No. 26, 28, 30 or 32 or a fragment or variant thereof, and/or wherein the spacer sequence encodes an amino acid sequence substantially as shown in SEQ ID No. 25, 27, 29 or 31 or a fragment or variant thereof.

21. The genetic construct according to any one of claims 17-20, wherein the genetic construct comprises a viral-2A removal sequence, optionally wherein the viral-2A removal sequence is placed 5' to the viral-2A sequence.

22. The genetic construct of claim 21, wherein the viral-2A removal sequence is separated from the viral-2A peptide spacer sequence by a linker sequence comprising a tripeptide glycine-serine-glycine sequence (G-S-G).

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

24. The genetic construct according to claim 23, wherein the viral-2A removal sequence comprises a nucleotide sequence substantially as shown 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 shown in SEQ ID No. 34 or 36 or a fragment or variant thereof.

25. The genetic construct according to claim 21 or 22, wherein the viral-2A removal sequence is a gelatinase MMP-2 recognition sequence, optionally wherein the viral-2A removal sequence comprises a nucleotide sequence substantially as shown in SEQ ID No:39 or a fragment or variant thereof, and/or wherein the viral-2A removal sequence encodes an amino acid sequence substantially as shown in SEQ ID No:38 or a fragment or variant thereof.

26. The genetic construct according to claim 21 or 22, wherein the virus-2A removal sequence is a renin recognition sequence, optionally wherein the virus-2A removal sequence comprises a nucleotide sequence substantially as shown in SEQ ID No. 41 or a fragment or variant thereof, and/or wherein the virus-2A removal sequence encodes an amino acid sequence substantially as shown in SEQ ID No. 40 or a fragment or variant thereof.

27. The genetic construct according to any preceding claim, wherein the genetic construct comprises a nucleotide sequence encoding a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE), optionally wherein the WPRE comprises a nucleic acid sequence substantially as set out in SEQ ID nos. 42 or 43 or a fragment or variant thereof.

28. The genetic construct according to any preceding claim, wherein the genetic construct comprises a nucleotide sequence encoding a polyA tail, optionally wherein the polyA tail comprises a nucleic acid sequence substantially as set out in SEQ ID nos 44, 45 or 84 or a fragment or variant thereof.

29. The genetic construct according to any preceding claim, wherein the genetic construct comprises a nucleotide sequence encoding a left and/or right Inverted Terminal Repeat (ITR), optionally wherein the left and/or right inverted terminal repeat comprises a nucleic acid sequence substantially as shown in SEQ ID No. 46 or 47 or a fragment or variant thereof.

30. The genetic construct according to any preceding claim, wherein the genetic construct comprises a non-coding intron, optionally wherein the non-coding intron is located between the promoter and the first coding sequence.

31. The genetic construct of claim 30, wherein the non-coding intron comprises a nucleic acid sequence substantially as shown in SEQ ID nos. 48, 49 or 50 or a fragment or variant thereof.

32. The genetic construct according to any preceding claim, wherein the genetic construct comprises a signal peptide coding sequence, optionally wherein the signal peptide coding sequence comprises a nucleotide sequence substantially as shown in any one of SEQ ID nos. 52 or 54 or a fragment or variant thereof, and/or wherein the signal peptide coding sequence encodes an amino acid sequence substantially as shown in SEQ ID nos. 51 or 53 or a fragment or variant thereof.

33. The genetic construct according to any preceding claim, wherein the genetic construct encodes an amino acid sequence substantially as shown 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 wherein the construct comprises a nucleotide sequence substantially as shown 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 gene construct according to any one of claims 1-33.

35. The recombinant vector of claim 34, wherein the vector 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. The recombinant vector according to any one of claims 34-37, wherein the recombinant vector comprises a nucleotide sequence substantially as set forth in SEQ ID No. 67, or a fragment or variant thereof.

39. The genetic construct according to any one of claims 1 to 33 or the recombinant vector according to any one of claims 34 to 38 for use as a medicament or for use in therapy.

40. The genetic construct according to any one of claims 1-33 or the recombinant vector according to any one of claims 34-38 for use in the treatment, prevention or amelioration of a retinal disorder, or for use in reducing complement activation and retinal cell damage and loss.

41. The genetic construct or vector for use according to claim 40, wherein the retinal disorder treated is: dry age-related macular degeneration, geographic atrophy and/or any pathophysiological condition involving retinal damage through complement activation.

42. The genetic construct or vector for use according to claim 41, wherein the retinal disorder is dry age-related macular degeneration.

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

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

45. A pharmaceutical composition comprising the genetic construct according to any one of claims 1-33 or the recombinant vector according to any one of claims 34-38 and a pharmaceutically acceptable vehicle.

46. A method of preparing the pharmaceutical composition of claim 45, the method comprising contacting the genetic construct of any one of claims 1-33 or the recombinant vector of any one of claims 34-38 with a pharmaceutically acceptable vehicle.

47. The genetic construct or vector for use according to claim 40, wherein the construct or vector is for use in reducing complement activation and retinal cell damage and loss associated with glaucoma.