RU2824048C2 - Codon-optimized complement factor i - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: group of inventions relates to agents for use in gene therapy. Disclosed is a polynucleotide coding factor H-like protein 1 (FHL1), a vector containing said polynucleotide, a cell for expressing FHL1 and their use for the treatment or prevention of complement-mediated disorder associated with complement feedback cycle hyperactivity, including eye diseases such as age-related macular degeneration (AMD) or diabetic retinopathy. Nucleotide sequence coding FHL1 is at least 90% identical to the sequence SEQ ID NO: 12.

EFFECT: inventions make it possible to deliver to the patient higher doses of the corresponding protein without increasing the amount of the introduced vector.

37 cl, 6 dwg, 2 tbl, 1 ex

Description

FIELD OF INVENTION

The present invention relates to means for use in gene therapy. In particular, the present invention relates to polynucleotides encoding complement factor I (CFI) and factor H-like protein 1 (FHL1), vectors containing said polynucleotides, and their use in the treatment or prevention of complement-mediated and complement-related disorders, including eye diseases such as age-related macular degeneration (AMD).

LEVEL OF TECHNOLOGY

The macula lutea is a small area of the retina of the eye, about 3 to 5 millimeters in size, adjacent to the optic nerve. It is the most sensitive area of the retina, containing the fovea, a depressed area that provides high visual acuity and contains a dense concentration of cones, the photoreceptors responsible for color vision.

Age-related macular degeneration (AMD) is the most common cause of functional blindness in developed countries in individuals over 50 years of age (Seddon, J.M., Epidemiology of age-related macular degeneration. In: Ogden, T.E., et al., eds. Ryan S.J., ed-in-chief. Retina Vol II. 3rd ed. St. Louis, Mo.: Mosby; 2001: 1039-1050). AMD is associated with neovascularization originating from the choroidal vasculature and extending into the subretinal space. In addition, AMD is characterized by progressive degeneration of the retina, retinal pigment epithelium (RPE), and the underlying choroid (the highly vascular tissue that lies beneath the RPE, between the retina and the sclera).

Multiple factors, including oxidative stress, inflammation with possible autoimmune component, genetic background (eg, mutations), and environmental or behavioral factors such as smoking and diet, may contribute to the pathogenesis of AMD.

The clinical progression of AMD is characterized by stages according to changes in the macula. The hallmark of early AMD is the appearance of drusen, which are collections of extracellular debris beneath the retina and appear as yellow spots on the retina during clinical examination and on fundus images. Drusen are classified as small (<63 μm), medium (63-124 μm), and large (>124 μm). They are also considered hard or soft depending on how their edges appear on ophthalmologic examination. While hard drusen have well-defined edges, soft drusen have less defined fluid edges. The Age-Related Eye Disease Study (AREDS) Photographic Fundus Severity Scale is one of the main classification systems used for this condition.

AMD is divided into "dry" and "wet" (exudative or neovascular) forms. Dry AMD is more common than wet AMD, but the dry form can transition into the wet form, and both occur simultaneously in a significant number of cases. Dry AMD is typically characterized by progressive apoptosis of cells in the RPE layer and overlying photoreceptor cells, and often of underlying cells in the choroidal capillary layer. Confluent areas of RPE cell death accompanied by atrophy of the overlying photoreceptors are called geographic atrophy. Patients with this form of AMD experience slow and progressive deterioration of central vision.

Wet AMD is characterized by bleeding and/or fluid leakage from abnormal vessels that have grown from the choroidal vessels (choriocapillaries) beneath the RPE and macula, which can cause sudden and disabling vision loss. It has been estimated that most of the vision loss experienced by patients is due to this choroidal neovascularization (CNV) and its secondary complications. A subtype of neovascular AMD is called retinal angiomatous proliferation (RAP). Here, the angiomatous proliferation originates from the retina and extends posteriorly into the subretinal space, eventually communicating with choroidal new vessels in some cases.

The complement system (CS) is implicated in the early pathogenesis of AMD based on the identification of CS components in drusen from eyes of patients with AMD. At least 129 types of proteins deposited in drusen have been identified in AMD, including different types of apolipoproteins (E, B, or A-1), several amyloid peptides (P, Aβ, or SA-1), TIMP-3, serum albumin, and several proteins associated with cellular function (e.g., ATP synthase β subunit, scavenger receptor B2, and retinol dehydrogenase). AMD-derived drusen also contain nearly all complement proteins, including regulatory proteins (CFH, complement receptor 1 (CR1), vitronectin, and clusterin), CS activation and degradation products (C1q, C3, C3a, C3b, and C5a), and elements of the CS terminal pathway containing MAC components (i.e., 5, 6, 8 (α, β, and γ), and 9) in distinct and complex form. Drusen accumulation may activate CS, induce local production of inflammatory mediators, and attract leukocytes, which in turn potentiate the local inflammatory state present in AMD.

Current treatment options for AMD include benzoporphyrin photodynamic therapy (Arch Ophthalmol (1999) 117:1329–1345) and a number of therapies targeting the vascular endothelial growth factor (VEGF) pathway. Examples of such VEGF-targeted therapies include the aptamer pegaptanib (N Engl J Med (2004) 351:2805–2816) and antibodies such as ranibizumab (N Engl J Med (2006) 355:1432–1444) and bevacizumab (BMJ (2010) 340:c2459). However, not all patients respond to anti-VEGF antibody treatment and either do not regain vision or progress to documented blindness.

A therapy has been developed to treat geographic atrophy and has been used in a phase III clinical trial. Lampalizumab is a humanized monoclonal antibody that inhibits complement factor D and is administered by intravitreal injection to halt the rate of progression of geographic atrophy. However, in a randomized phase III clinical trial involving 906 participants, lampalizumab failed to reduce the increase in GA compared to placebo over 48 weeks.

Accordingly, there is a significant need in the art for new approaches to treating eye diseases such as AMD.

Due to the ubiquitous nature of the complement system, an overactive or dysfunctional complement system has been implicated in the pathology of many chronic inflammatory conditions for which treatment options either do not exist or require symptom management with regular interventions over several years. Consequently, there is a general need to develop gene therapy approaches that provide new or alternative treatments for complement-mediated and complement-associated disorders, particularly chronic inflammatory conditions and even more specifically those associated with hyperreactivity of the complement C3b feedback loop (Figure 1).

ESSENCE OF THE INVENTION

The applicant has identified codon-optimized sequences of complement factor I (CFI) and factor H-like protein 1 (FHL1) that provide significantly increased expression of the encoded CFI and FHL1 proteins compared to the wild-type sequences.

The improved sequences encoding CFI and FHL1 developed by the Applicant allow higher doses of the respective proteins to be delivered to the patient without increasing the amount of vector administered. Thus, the present invention provides improvements in terms of productivity (i.e., protein delivery can be achieved with smaller amounts of vector produced), drug efficacy, and safety. In particular, since a higher dose of the encoded protein can be achieved with the same amount (e.g., volume) of vector delivered, the risk of damage to the tissue into which the vector is administered is reduced. For example, when the vector is delivered to the eye by subretinal injection, the risk of retinal damage or detachment caused by the injection of large volumes of the drug is reduced. In addition, the risk of off-target effects arising from the spread of large volumes of the drug to adjacent tissues is reduced. In addition, the use of the claimed nucleotide sequences in gene therapy can provide a single dose treatment, ensuring long-term stable protein expression and avoiding the need for monthly or regular injections. The nucleotide sequences and compositions of the present invention have the additional advantage that they can provide a one-time or "one-shot" therapy that avoids repeated or regular surgical interventions.

In one aspect, the invention relates to an isolated polynucleotide comprising a nucleotide sequence encoding complement factor I (CFI), wherein the nucleotide sequence is at least 85% identical to the sequence of SEQ ID NO: 10.

In some embodiments, the nucleotide sequence encoding the CFI is at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 10.

In preferred embodiments, the nucleotide sequence encoding CFI is SEQ ID NO: 10.

In another aspect, the present invention relates to an isolated polynucleotide comprising a nucleotide sequence encoding factor I H-like protein 1 (CFI), wherein the nucleotide sequence is at least 75% identical to the sequence of SEQ ID NO: 12.

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

In preferred embodiments, the nucleotide sequence encoding FHL1 is SEQ ID NO: 12.

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

In some embodiments, the AAV ITRs are AAV2 or AAV8 ITRs. In preferred embodiments, the AAV ITRs are AAV2 ITRs.

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

In some embodiments, the vector is an adeno-associated virus (AAV), retroviral, lentiviral, or adenoviral vector.

In preferred embodiments, the vector is an AAV vector.

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

In some embodiments, the AAV vector particle comprises an AAV2 or AAV8 genome.

In some embodiments, the AAV vector particle comprises AAV2 or AAV8 capsid proteins.

In some embodiments, the AAV vector particle comprises an AAV2 genome and AAV2 capsid proteins (AAV2/2). In other embodiments, the AAV vector particle comprises an AAV2 genome and AAV8 capsid proteins (AAV2/8). In other embodiments, the AAV vector particle comprises an AAV8 genome and AAV8 capsid proteins (AAV8/8).

In some embodiments, the nucleotide sequence encoding CFI is operably linked to a CMV promoter. In some embodiments, the nucleotide sequence encoding CFI is operably linked to a regulatory element, such as a WPRE regulatory element. In preferred embodiments, the WPRE regulatory element is a WPRE3 regulatory element. In some embodiments, the nucleotide sequence encoding CFI is operably linked to a polyadenylation (poly-A) signal, such as a bovine growth hormone poly-A signal.

In preferred embodiments, the nucleotide sequence encoding CFI is operably linked to a CMV promoter; a WPRE regulatory element (preferably a WPRE3 regulatory element); and a bovine growth hormone poly-A signal.

In some embodiments, the nucleotide sequence encoding FHL1 is operably linked to a CMV promoter. In some embodiments, the nucleotide sequence encoding FHL1 is operably linked to a regulatory element, such as a WPRE regulatory element. In preferred embodiments, the WPRE regulatory element is a WPRE3 regulatory element. In some embodiments, the nucleotide sequence encoding FHL1 is operably linked to a poly-A signal, such as a bovine growth hormone poly-A signal.

In preferred embodiments, the nucleotide sequence encoding FHL1 is operably linked to a CMV promoter; a WPRE regulatory element (preferably a WPRE3 regulatory element); and a bovine growth hormone poly-A signal.

In another aspect, the present invention provides a cell comprising a polynucleotide of the present invention.

In another aspect, the present invention provides a cell transduced with a vector of the present invention.

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

In a particular embodiment, the pharmaceutical composition is suitable for systemic administration, such as by infusion through a peripheral vein.

In a particular embodiment, the pharmaceutical composition is suitable for local administration, such as intrathecal administration.

In preferred embodiments, the pharmaceutical composition is intended for intraocular administration, such as by intravitreal injection, suprachoroidal injection, or subretinal injection.

In another aspect, the present invention provides a polynucleotide, vector or cell of the present invention for use in therapy.

In a specific embodiment, the polynucleotide, vector, or cell of the present invention is used to treat complement-mediated disorders, particularly chronic inflammatory conditions.

In a preferred embodiment, the polynucleotide, vector or cell of the present invention is used to treat a disorder associated with hyperactivity of the complement C3b feedback loop.

In another aspect, the present invention provides a polynucleotide, vector or cell of the present invention for use in the treatment or prevention of an eye disease.

In another aspect, the present invention provides a polynucleotide, vector or cell of the present invention for use in the treatment or prevention of a complement-mediated disorder of the eye.

In another aspect, the present invention provides a method of treating or preventing a complement-mediated disorder of the eye, comprising administering a polynucleotide, vector or cell of the present invention to a subject in need thereof.

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

In some embodiments, the disorder is associated with excessive activity of the C3b complement feedback loop and/or insufficient activity of the C3b degradation loop (see Fig. 1).

In some embodiments, the disorder is a chronic complement-mediated inflammatory condition of the eye.

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

In preferred embodiments, the disorder is AMD. In some embodiments, the AMD is dry AMD.

In some embodiments, the subject has been diagnosed with AMD or is at risk of developing AMD.

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

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

(b) who is heterozygous or homozygous for a SNP associated with age-related macular degeneration (AMD), preferably a rare variant of complement factor I.

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

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

(b) who does not carry the rare allele for the complement factor I variant.

In another aspect, the invention provides a polynucleotide, vector, or cell of the invention for use in the treatment or prevention of age-related macular degeneration (AMD). In preferred embodiments, AMD is dry AMD.

In another aspect, the present invention provides a polynucleotide, vector or cell of the present invention for use in the treatment or prevention of diabetic retinopathy.

In some embodiments, the formation of geographic atrophy is prevented or reduced and/or the degree of geographic atrophy is reduced.

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

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

In some embodiments, administration of the polynucleotide, vector, or cell increases the level of C3b inactivation and iC3b degradation activity in the subject or in the eye, such as in the retinal pigment epithelium (RPE) of the subject, optionally to a level that exceeds the normal level for the subject or its eye or RPE.

In another aspect, the invention provides a polynucleotide, vector, or cell of the invention for use in improving or restoring vision or visual acuity, for example, in a subject suffering from an ocular disease, such as an ocular disease described herein. In another aspect, the invention provides a polynucleotide, vector, or cell of the invention for use in reducing vision loss or visual acuity, for example, vision loss or visual acuity associated with a visual disorder, such as a visual disorder described herein.

In another aspect, the invention provides a polynucleotide, vector, or cell of the invention for use in improving or restoring reading speed in a subject, such as a subject suffering from an ocular disease, such as an ocular disease described herein. In another aspect, the invention provides a polynucleotide, vector, or cell of the invention for use in reducing a decrease in reading speed in a subject, such as a decrease in reading speed associated with an ocular disease, such as an ocular disease described herein.

In another aspect, the invention provides a polynucleotide, vector, or cell of the invention for use in reducing or preventing loss of photoreceptors and/or retinal pigment epithelium (RPE), such as loss of photoreceptors and/or RPE associated with an ocular disease, such as an ocular disease described herein.

In some embodiments, the polynucleotide, vector, or cell is administered intraocularly.

In some embodiments, the polynucleotide, vector, or cell is administered to the eye of the subject via subretinal, direct retinal, suprachoroidal, or intravitreal injection.

In some embodiments, the polynucleotide, vector, or cell is administered to the eye of the subject via subretinal injection.

In some embodiments, a polynucleotide or vector of the invention does not comprise an hAAT promoter. In some embodiments, a polynucleotide or vector of the invention does not comprise an ApoR enhancer. In other embodiments, a polynucleotide or vector of the invention does not comprise two ApoR enhancers.

In some embodiments, the vector of the invention does not comprise the AAV2 genome and the AAV8 capsid protein, i.e., the vector of the invention is not an AAV2/8 vector.

In some embodiments, the polynucleotide, vector, or cell of the invention is not administered systemically. In other embodiments, the polynucleotide, vector, or cell of the invention is not administered intravenously.

BRIEF DESCRIPTION OF GRAPHIC MATERIALS

Fig. 1

C3b feedback (amplification) and degradation (suppression) cycles of the vertebrate alternative complement pathway (“I” = complement factor I; “H” = complement factor H; “B” = complement factor B; and “D” = complement factor D).

Fig. 2

Western blot analysis of ARPE19 cell supernatants after transfection with plasmids containing codon-optimized CFI and FHL1.

Fig. 3

ELISA analysis of ARPE19 cell supernatants after transfection with plasmids containing codon-optimized CFI.

Fig. 4

ELISA analysis of ARPE19 cell supernatants after transfection with plasmids containing codon-optimized FHL1.

Fig. 5

ELISA analysis of ARPE19 cell supernatants after transduction with AAV vectors with codon-optimized CFI.

Fig. 6

ELISA analysis of ARPE19 cell supernatants after transduction with AAV vectors with codon-optimized FHL1.

DETAILED DESCRIPTION OF THE ESSENCE OF THE INVENTION

The terms "comprising", "includes" and "consisting of" as used herein are synonymous with "including" or "includes"; or "containing" or "contains" and are inclusive or open-ended and do not exclude additional, unlisted members, elements or steps. The terms "comprising", "includes" and "consisting of" also include the term "consisting of".

COMPLEMENT SYSTEM

The complement system is an integral part of the humoral immune system and is involved in tissue inflammation, cell opsonization, and cytolysis. It provides protection against microorganisms and facilitates the removal of exogenous and endogenous cellular decay products from host tissues.

The complement cascade consists of four activation pathways. All pathways ultimately terminate in the central cleavage of factor C3 and the formation of its active fragments C3a and C3b. C3a is an anaphylatoxin that triggers a number of chemotactic and proinflammatory responses, such as inflammatory cell recruitment and increased microvascular permeability, whereas C3b is responsible for the opsonization of foreign surfaces covalently linked to C3b. Opsonization by activated C3 fragments (C3b and iC3b) has three main functions: (i) clearance of cellular debris by phagocytic cells (e.g., macrophages or microglia) and stimulation of the adaptive immune system (B and T cells); (ii) enhancement of complement activation through the formation of surface-associated C3 convertase; and (iii) assembly of C5 convertase.

The C5 convertase assembly is responsible for the cleavage of C5, resulting in the formation of the cytolytic membrane attack complex (MAC), which is capable of generating perforations in the cell membrane, thereby promoting cell lysis and eliminating unnecessary cells. Through all of these actions, the innate complement cascade supports and stimulates the function of downstream immune mechanisms that protect the integrity of host tissue. Overall, activation of the complement pathway results in a proinflammatory response, including the formation of MAC, which mediates cell lysis, the release of chemokines to attract inflammatory cells to the site of injury, and increased capillary permeability, which facilitates the extravasation of infiltrating leukocytes. Under physiological conditions, complement activation is efficiently controlled by the coordinated action of soluble and membrane-bound complement regulatory molecules (CRMs). Soluble complement regulators such as C1 inhibitor, anaphylatoxin inhibitor, C4b binding protein (C4BP), complement factor H (CFH), complement factor I (CFI), clusterin, and vitronectin limit complement action in human tissues at several sites in the complement cascade. In addition, each individual cell is protected from complement homologous attack by surface proteins such as complement receptor 1 (CR1, CD35), membrane cofactor protein (CD46), and glycosylphosphatidylinositol-anchored proteins such as decay-accelerating factor (CD55) or the CD59 molecule. It should be noted that host cells and tissues that are inadequately protected from complement attack may undergo background cell lysis.

The present invention relates to the treatment or prevention of a complement-mediated disorder of the eye. For example, a complement-mediated disorder may be a disorder associated with a defect in the regulation of the alternative pathway, and in particular with excessive activity of the complement feedback loop C3b and/or insufficient activity of the C3b destruction loop.

In some embodiments, prior to administration of a polynucleotide, vector, cell, or pharmaceutical composition of the invention, the subject has low levels (e.g., lower than normal levels) of complement factor I activity, such as low levels of complement factor I activity in the eye and/or low levels of complement factor I activity in the serum. The subnormal level of complement factor I activity may be due to subnormal expression of normally functioning complement factor I or at least partial (e.g., heterozygous) expression (at normal or subnormal levels) of a non- or subfunctional variant of complement factor I. (Such a subject may carry one or more copies of an AMD-associated SNP, such as a subject may be homo- or heterozygous for one of the rare variants of complement factor I discussed below.) Thus, the subject may have a low concentration (e.g., below normal concentration) of complement factor I in the eye and/or serum. In humans, a normal level of complement factor I activity (C3b inactivating and iC3b degrading activity) may be equivalent to the level provided by 30-40 pg/mL complement factor I in the subject's serum. Thus, in a subject with low complement factor I activity, the serum complement factor I activity may correspond to less than 30 μg/mL and more than 0 μg/mL complement factor I, e.g., 0-20 or 0-10 μg/mL (these are serum complement factor I concentration ranges that a subject with a low complement factor I concentration may encompass).

Thus, a subject that is amenable to treatment according to the present invention may suffer from a complement-mediated eye disorder such as AMD, particularly the dry form of AMD (e.g., characterized by geographic atrophy), or may be at risk of developing such a disorder. For example, the subject may be homozygous or heterozygous, susceptible to one or more SNPs associated with a complement-mediated disorder.

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

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

Alternatively, the subject may have normal levels of endogenous complement factor I activity or concentration, such as in the eye and/or serum, and/or may not have a rare complement factor I allele variant.

In some embodiments, administration of a polynucleotide, vector, cell, or pharmaceutical composition of the invention thereby increases the level of C3b inactivation and iC3b degradation activity in the eye of a subject. In other embodiments, administration of a polynucleotide, vector, cell, or pharmaceutical composition of the invention thereby increases the level of C3b inactivation and iC3b degradation activity in the eye of a subject to a level that exceeds the normal level in the eye. More particularly, the level of C3b inactivation and iC3b degradation activity is increased in the RPE of the eye.

It should be appreciated that the C3b inactivation and iC3b degradation activity in a subject following expression of complement factor I from a polynucleotide or vector of the present invention may include the C3b inactivation and iC3b degradation activity of the subject's endogenous complement factor I (i.e., the subject's complement factor I not produced by expression from a polynucleotide or vector) and the C3b inactivation and iC3b degradation activity produced by expression from a polynucleotide or vector of the present invention, such that the total level of C3b inactivation and iC3b degradation activity in the subject exceeds the normal level.

In some embodiments, the level of C3b inactivation and iC3b degradation activity in a subject, such as in the eye, is increased to a level that is at least 5%, 10%, 15%, 20%, or 25% greater than the normal level.

In other embodiments, the level of C3b inactivation and iC3b degradation activity in a subject, such as in the eye, is increased to a level that is twice the normal level or 80%, 60%, 40%, or 20% higher than the normal level.

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

In some embodiments, administration of a polynucleotide, vector, cell, or pharmaceutical composition of the invention does not result in a detectable increase in the level of C3b inactivation and iC3b degradation activity in the plasma/serum of a subject. In other embodiments, administration of a polynucleotide, vector, cell, or pharmaceutical composition of the invention does not result in a detectable increase in the level of C3b inactivation and iC3b degradation activity in the plasma/serum of a subject to a level above normal.

In the preceding section, except where clearly not applicable, reference to complement factor I and the activity of inactivating C3b and degrading iC3b may be replaced by complement factor H or factor H-like protein 1, and the ability to act as a cofactor for complement factor I-mediated cleavage of C3b and to increase the rate of dissociation of C3 convertase and C5 convertase, respectively. In some embodiments, prior to administration of a polynucleotide, vector, cell, or pharmaceutical composition of the invention, the subject has low levels (e.g., lower than normal levels) of complement factor H, such as low levels of complement factor H in the eye and/or low levels of complement factor H in the serum. For humans, a normal level of complement factor H may be about 200-500 μg/mL in the subject's serum. Thus, a subject with low levels of complement factor H may have serum levels less than 200 μg/mL and greater than 0 μg/mL, such as 0-100 μg/mL. Alternatively, a subject may have normal levels of endogenous complement factor H, such as in the eye and/or serum.

COMPLEMENT FACTOR I (CFI)

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

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

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

iC3b is unable to bind factor B and thus cannot support amplification of the complement cascade or activation via the alternative pathway. Therefore, once C3b has been cleaved to iC3b, neither initiation of the alternative pathway nor activation of the terminal complement cascade occurs.

iC3b is able to exert proinflammatory effects by binding to and activating complement receptor 3 (CR3) (CD11b/CD18) on polymorphonuclear leukocytes (mainly neutrophils), NK cells and mononuclear phagocytes such as macrophages.

Complement factor I is able to convert iC3b to C3d,g via protease activity requiring the cofactor CR1. C3d,g cannot bind to CR3. Since the reaction of iC3b with the complement receptor CR3 is the major mechanism by which complement activation induces inflammation, cleavage of iC3b to C3d,g is essential for reducing complement-induced inflammation (Lachmann (2009) Adv. Immunol. 104: 115–149).

The unique ability of complement factor I to both promote the cleavage of C3b to iC3b and accelerate the cleavage of iC3b - combined with its relatively low concentration in human serum, which impacts the amount required to be delivered for therapeutic efficacy - makes it a particularly advantageous target.

In some embodiments, the complement factor I polypeptide is capable of cleaving C3b to an inactive degradation product. For example, the complement factor I polypeptide may be capable of cleaving C3b to iC3b.

In some embodiments, the complement factor I polypeptide is capable of converting iC3b to an inactive degradation product. For example, the complement factor I polypeptide may be capable of converting iC3b to C3d,g

In preferred embodiments, the complement factor I polypeptide is capable of cleaving C3b to iC3b and processing iC3b to C3d,g

Accordingly, the fragment or derivative of complement factor I may retain at least 50%, 60%, 70%, 80%, 90%, 95% or 100% of the C3b inactivating and iC3b degrading activity of native complement factor I.

The C3b inactivating and iC3b degrading activity of complement factor I or a fragment or derivative thereof may be determined using any suitable method known to one skilled in the art. For example, measurement of complement factor I proteolytic activity is described in Hsiung et al. (Biochem. J. (1982) 203: 293-298). Both the hemolytic and conglutinating assays for CFI activity are described in Lachmann PJ & Hobart MJ (1978) “Complement Technology” in Handbook of Experimental Immunology 3rd edition Ed DM Weir Blackwells Scientific Publications Chapter 5A p17. A more detailed description, including a proteolytic assay, is given by Harrison RA (1996) in “Weir's Handbook of Experimental Immunology” 5th Edition Eds; Herzenberg Leonore A'Weir DM, Herzenberg Leonard A & Blackwell C Blackwells Scientific Publications Chapter 75 36-37. The adhesion assay is highly sensitive and can be used to detect both the first (double) clip converting fixed C3b to iC3b and to determine reactivity with conglutinin; and to detect the last clip on C3dg, starting with fixed iC3b and looking for loss of reactivity with conglutinin. The hemolytic assay is used to convert C3b to iC3b, and the proteolytic assay detects all clips.

In some embodiments, the complement factor I is complement factor I.

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

In some embodiments, the amino acid sequence of complement factor I is SEQ ID NO: 1. In other embodiments, the amino acid sequence of complement factor I is the sequence disclosed at positions 19 to 583 of SEQ ID NO: 1.

MKLLHVFLLFLCFHLRFCKVTYTSQEDLVEKKCLAKKYTHLSCDKVFCQPWQRCIEGTCVCKLPYQCPKNGTAVCATNRRSFPTYCQQKSLECLHPGTKFLNNGTCTAEGKFSVSLKHGNTDSEGIVEVKLVDQDKTMFICKSSWSMREANVACLDLGFQQGADTQRRFKLSDLSINSTECLHVHCRGLETSLA ECTFTKRRTMGYQDFADWCYTQKADSPMDDFFQCVNGKYISQMKACDGINDCGDQSDELCCKACQGKGFHCKSGVCIPSQYQCNGEVDCITGEDEV GCAGFASVTQEETEILTADMDAERRRIKSLLPKLSCGVKNRMHIRRKRIVGGKRAQLGDLPWQVAIKDASGITCGGIYIGGCWILTAAHCLRASKTHRYQIWTTWDWIHPDLKRIVIEYVDRIIFHENYNAGTYQNDIALIEMKKDGNKKDCELPRSIPACVPWSPYLFQPNDTCIVSGWGREKDNERVFSLQWGEVKLI SNCSKFYGNRFYEKEMECAGTYDGSIDACKGDSGGPLVCMDANNVTYVWGWSWGENCGKPEFPGVYTKVANYFDWISYHVGRPFISQYNV

(SEQ ID NO: 1)

In some embodiments, the amino acid sequence of complement factor I is SEQ ID NO: 9, which corresponds to NCBI accession number NP_000195. In other embodiments, the amino acid sequence of complement factor I is the sequence disclosed at positions 19 to 583 of SEQ ID NO: 9.

MKLLHVFLLFLCFHLRFCKVTYTSQEDLVEKKCLAKKYTHLSCDKVFCQPWQRCIEGTCVCKLPYQCPKNGTAVCATNRRSFPTYCQQKSLECLHPGTKFLNNGTCTAEGKFSVSLKHGNTDSEGIVEVKLVDQDKTMFICKSSWSMREANVACLDLGFQQGADTQRRFKLSDLSINSTECLHVHCRGLETSLA ECTFTKRRTMGYQDFADWCYTQKADSPMDDFFQCVNGKYISQMKACDGINDCGDQSDELCCKACQGKGFHCKSGVCIPSQYQCNGEVDCITGEDEV GCAGFASVAQEETEILTADMDAERRRIKSLLPKLSCGVKNRMHIRRKRIVGGKRAQLGDLPWQVAIKDASGITCGGIYIGGCWILTAAHCLRASKTHRYQIWTTWDWIHPDLKRIVIEYVDRIIFHENYNAGTYQNDIALIEMKKDGNKKDCELPRSIPACVPWSPYLFQPNDTCIVSGWGREKDNERVFSLQWGEVKLI SNCSKFYGNRFYEKEMECAGTYDGSIDACKGDSGGPLVCMDANNVTYVWGWSWGENCGKPEFPGVYTKVANYFDWISYHVGRPFISQYNV

(SEQ ID NO: 9)

An example of a wild-type nucleotide sequence encoding complement factor I is the nucleotide sequence having NCBI accession number NM_000204, disclosed herein as SEQ ID NO: 2.

ATGAAGCTTCTTCATGTTTTCCTGTTATTTCTGTGCTTCCACTTAAGGTTTTGCAAGGTCACTTATACATCTCAAGAGGATCTGGTGGAGAAAAAGTGCTTAGCAAAAAAATATACTCACCTCTCCTGCGATAAAGTCTTCTGCCAGCCATGGCAGAGATGCATTGAGGGCACCTGTGTTTGTAAACTACCGTATCAGTGCCCAAAGAATGGCACTGCAGTGTGTGCAACTAACAGGAAGCTTCCC AACATACTGTCAACAAAAGAGTTTGGAATGTCTTCATCCAGGGACAAAGTTTTTAAATAACGGAACATGCACAGCCGAAGGAAAGTTTAGTGTTTCCTTGAAGCATGGAAATACAGATTCAGAGGGAATAGTTGAAGTAAAACTTGTGGACCAAGATAAGACAATGTTCATATGCAAAAGCAGCTGGAGC ATGAGGGAAGCCAACGTGGCCTGCCTTGACCTTGGGTTTCAACAAGGTGCTGATACTCAAAGAAGGTTTAAGTTGTCTGATCTCTCTATAAATTCCACTGAATGTCTACATGTGCATTGCCGAGGATTAGAGACCAGTTTGGCTGAATGTACTTTTACTAAGAGAAGAACTATGGGTTACCAGGATTTCGCTGATGTGGTTTGTTATACACAGAAAGCAGATTCTCCAATGGATGACTTCTT TCAGTGTGTGAATGGGAAATACATTTCTCAGATGAAAGCCTGTGATGGTATCAATGATTGTGGAGACCAAAGTGATGAACTGTGTTGTAAAGCATGCCAAGGCAAAGGCTTCCATTGCAAATCGGGTGTTTGCATTCCAAGCCAGTATCAATGCAATGGTGAGGTGGACTGCATTACAGGGGAAGATGAAGTTGGC TGTGCAGGCTTTGCATCTGTGGCTCAAGAAGAAACAGAAATTTTGACTGCTGACATGGATGCAGAAAGAAGACGGATAAAATCATTATTACCTAAACTATCTTGTGGAGTTAAAAACAGAATGCACATTCGAAGGAAACGAATTGTGGGAGGAAAGCGAGCACAACTGGGAGACCTCCCATGGCAGGTGGCAATTAAGGATGCCAGTGGAATCACCTGTGGGGGAATTTATATTGGTGGCTGTT GGATTCTGACTGCTGCACATTGTCTCAGAGCCAGTAAAACTCATCGTTACCAAATATGGACAACAGTAGTAGACTGGATACACCCCGACCTTAAACGTATAGTAATTGAATACGTGGATAGAATTATTTTCCATGAAAACTACAATGCAGGCACTTACCAAAATGACATCGCTTTGATTGAAATGAAAAAAGAC GGAAACAAAAAAGATTGTGAGCTGCCTCGTTCCATCCCTGCCTGTGTCCCCTGGTCTCCTTACCTATTCCAACCTAATGATACATGCATCGTTTCTGGCTGGGGACGAGAAAAAGATAACGAAAGAGTCTTTTTCACTTCAGTGGGGTGAAGTTAAACTAATAAGCAACTGCTCTAAGTTTTACGGAAATCGTTTCTATGAAAAAGAAATGGAATGTGCAGGTACATATGATGGTTCCATCGAT GCCTGTAAAGGGGACTCTGGAGGCCCCTTAGTCTGTATGGATGCCAACAATGTGACTTATGTCTGGGGTGTTGTGAGTTGGGGGGAAAACTGTGGAAAACCAGAGTTCCCAGGTGTTTACACCAAAGTGGCCAATTATTTTGACTGGATTAGCTACCATGTAGGAAGGCCTTTTATTTCTCAGTACAATGTATAA

(SEQ ID NO: 2)

The complement factor I nucleotide sequences used in the present invention are preferably codon optimized. Different cells vary in their use of particular codons. This codon bias corresponds to a bias in the relative abundance of particular tRNAs in a cell type. By altering the codons in the sequence to match the relative abundance of the corresponding tRNAs, expression can be increased. Similarly, expression can be decreased by deliberately selecting codons for which the corresponding tRNAs are known to be rare in a particular cell type. In this way, an additional degree of translational control is available.

A preferred nucleotide sequence encoding complement factor I is the nucleotide sequence disclosed as SEQ ID NO: 10.

ATGAAACTGCTGCATGTCTTCCTCCTCTTCCTGTGCTTCCACCTCCGTTTCTGTAAAGTCACCTACACTAGCCAGGAGGATCTGGTGGAGAAGAAATGCCTGGCCAAGTATACCCACCTGAGCTGCGACAAAGTGTTCTGCCAGCCCTGGCAACGCTGCATTGAAGGTACTTGTGTGTGCAAGCTGCCCTACCAGTGCCCCAAGAACGGCACGGCCGTGTGTGCCACCAACAGGAGGA GCTTCCCACCTACTGCCAGCAGAAGAGCCTGGAATGCCTCCACCCTGGCACCAAGTTTCTGAACAACGGGACCTGCACAGCCGAGGGGAAATTCAGCGTCTCCCTCAAGCACGGCAATACAGACTCCGAGGGCATTGTGGAAGTGAAGCTGGTGGACCAGGACAAGACCATGTTCATCTGCAAAAGCAGC TGGTCCATGCGGGAGGCCAATGTCGCCTGCCTGGACCTGGGCTTCCAGCAGGGCGCTGATACACAGCGCCGCTTTAAACTCAGTGACCTCAGCATCAACAGCACTGAGTGTCTGCACGTGCACTGCCGGGGCCTGGAGACCAGCCTGGCTGAGTGCACCTTCACCAAGCGCAGGACCATGGGCTACCAGGATTTTGCAGATGTGGTCTGCTACACCCAGAAGGCAGACAGCCCCATGGATG ACTTCTTCCAGTGTGTCAATGGCAAGTACATTTCCCAGATGAAGGCTTGTGACGGGATCAATGATTGCGGGGATCAGAGCGATGAGCTCTGCTGCAAGGCCTGCCAAGGGAAGGGCTTTCACTGTAAGTCTGGGGTGTGCATCCCTTCTCAGTATCAGTGCAACGGAGAGGTGGACTGCATCACTGGGGAG GACGAGGTGGGCTGTGCTGGCTTCGCCTCTGTGGCCCAGGAGGAGACAGAGATCCTCACAGCTGACATGGATGCAGAGCGGCGGCGCATCAAGAGTCTGCTCCCAAAGCTCTCCTGCGGCGTTAAGAATCGCATGCACATCCGGAGGAAGCGGATCGTTGGAGGCAAACGGGCTCAGCTGGGGGACTTGCCGTGGCAGGTGGCCATCAAAGATGCCTCCGGAATCACCTGTGGTGGCATCTA CATCGGCGGCTGCTGGATCCTGACCGCCGCCCACTGCCTTCGGGCCAGCAAGACTCACCGCTACCAGATCTGGACCACGTGGTGGATTGGATTCACCCCGACCTGAAGAGGATTGTCATTGAGTATGTCGACCGCATCATCTTCCATGAAAACTACAATGCCGGGACGTATCAGAACGACATCGCCCTC ATCGAGATGAAGAAGGATGGGAACAAGAAGGACTGTGAGCTGCCTCGCTCCATCCCCGCCTGTGTACCATGGTCTCCGTACCTGTTCCAGCCAAATGACACATGCATCGTGAGCGGCTGGGGCCGCGAGAAAGACAACGAGAGGGTCTTCTCCCTGCAGTGGGGTGAAGTCAAGCTGATCAGCAACTGCTCCAAGTTCTACGGCAACCGCTTCTATGAGAAGGAGATGGAGTGCGCCGGC ACCTATGACGGCAGCATTGACGCGTGCAAGGGAGACAGTGGGGGCCCCCTGGTCTGCATGGACGCCAACAATGTGACCTACGTGTGGGGAGTTGTGTCCTGGGGCGAGAACTGTGGCAAGCCTGAGTTCCCGGGCGTGTACACAAAGGTGGCAAACTATTTTGACTGGATCTCCTATCACGTTGGCAGGCCC TTCATTTCACAGTACAACGTATAA

(SEQ ID NO: 10)

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

In other embodiments, the nucleotide sequence encoding complement factor I is SEQ ID NO: 10.

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

In other embodiments, the nucleotide sequence encoding complement factor I is located at positions 55-1752 of SEQ ID NO: 10.

Another example of a codon-optimized nucleotide sequence encoding complement factor I is SEQ ID NO: 8.

ATGAAGCTGCTGCATGTCTTTCTGCTGTTTCTGTGCTTCCATCTGCGGTTCTGTAAAGTGACCTATACTAGCCAGGAGGATCTGGTGGAGAAGAAGTGTCTGGCCAAGTACACACACCTGAGCTGCGACAAGGTGTTCTGTCAGCCTTGGCAGCGGTGCATCGAGGGCACCTGCGTGTGCAAGCTGCCTTACCAGTGCCCAAAGAACGGCACCGCCGTGTGCGCCACAAATCGGAGAT CTTTTCCAACATATTGCCAGCAGAAGAGCCTGGAGTGTCTGCACCCCGGCACCAAGTTCCTGAACAATGGCACCTGCACAGCCGAGGGCAAGTTTTCTGTGAGCCTGAAGCACGGCAACACAGATAGCGAGGGCATCGTGGAGGTGAAGCTGGTGGACCAGGATAAGACCATGTTCATCTGTAAGAGCTCCTGGTCC ATGAGGGAGGCAAACGTGGCATGCCTGGATCTGGGATTCCAGCAGGGAGCAGACACACAGAGGCGCTTTAAGCTGTCCGACCTGTCTATCAATAGCACCGAGTGCCTGCACGTGCACTGTAGGGGCCTGGAGACATCCCTGGCAGAGTGCACCTTCACAAAGCGGAGAACCATGGGCTACCAGGACTTTGCCGACGTGGTGTGCTATACCCAGAAGGCCGATAGCCCCATGGACGATTTCTT TCAGTGCGTGAACGGCAAGTATATCTCCCAGATGAAGGCCTGCGACGCATCAATGACTGTGGCGATCAGTCTGACGAGCTGTGCTGTAAGGCCTGTCAGGGCAAGGGCTTCCACTGCAAGAGCGGCGTGTGCATCCCTTCCCAGTACCAGTGCAACGGCGAGGTGGATTGTATCACAGGAGAGGACGAAGTGGGA TGCGCAGGATTTGCATCTGTGGCACAGGAGGAGACAGAGATCCTGACAGCCGACATGGATGCCGAGAGGCGCCGGATCAAGTCTCTGCTGCCTAAGCTGAGCTGTGGCGTGAAGAATCGGATGCACATCAGAAGGAAGCGCATCGTGGGAGGCAAGAGGGCACAGCTGGGCGATCTGCCATGGCAGGTGGCCATCAAGGACGCCTCTGGCATCACCTGCGGCGGCATCTACATCGGAGGATGTT GGATCCTGACCGCAGCACACTGCCTGAGAGCAAGCAAGACACACAGGTATCAGATCTGGACCACAGTGGTGGATTGGATCCACCCAGACCTGAAGAGAATCGTGATCGAGTACGTGGATAGGATCATCTTTTCACGAGAACTACAATGCCGGCACATATCAGAACGACATCGCCCTGATCGAGATGAAGAAGGAT GGCAATAAGAAGGACTGTGAGCTGCCCAGATCCATCCCTGCATGCGTGCCATGGAGCCCCTATCTGTTCCAGCCCAACGATACCTGCATCGTGTCCGGATGGGGAAGGGAGAAGGACAATGAGCGGGTGTTTTCTCTGCAGTGGGGCGAGGTGAAGCTGATCTCCAACTGTTCTAAGTTCTACGGCAATAGGTTTTATGAGAAGGAGATGGAGTGCGCCGGCACCTACGATGGCAGCATCGAC GCCTGTAAGGGCGATTCCGGAGGACCACTGGTGTGCATGGACGCAAACAATGTGACATACGTGTGGGGAGTGGTGTCCTGGGGAGAACTGCGGCAAGCCAGAGTTCCCCGGCGTATATACCAAGGTGGCCAATTATTTTGATTGGATTTCCTACCACGTCGGCAGGCCCTTTATTTCCCAGTATAATGTCTAA

(SEQ ID NO: 8)

An advantage of the present invention is that complement factor I is particularly difficult to obtain in the form of a purified protein. Accordingly, the inventors have developed a method for modulating the complement system, for example to provide treatment for age-related macular degeneration (AMD), by administering complement factor I in the form of an AAV vector containing a nucleotide sequence encoding complement factor I. The AAV vector can be administered to a site of interest, such as the eye, to allow in situ translation of the complement factor I polypeptide.

COMPLEMENT FACTOR H (CFH)

Complement factor H (factor H, CFH) is a complement control protein.

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

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

Complement factor H consists of 20 complement control protein (CCP) modules (also called short consensus repeats or sushi domains) connected to each other by short linkers (three to eight amino acid residues) and arranged in an extended head-to-tail arrangement. Each CCP module consists of about 60 amino acids with four disulfide cysteine residues linked in the order 1-3 2-4 and a hydrophobic core built around a nearly invariable tryptophan residue. The CCP modules are numbered 1 through 20 (from the N-terminus of the protein). CCP 1-4 and CCP 19-20 interact with C3b, while CCP 7 and CCP 19-20 bind to GAG and sialic acid (Schmidt et al. (2008) Journal of Immunology 181: 2610-2619).

Gene therapy using complement factor H has been shown to ameliorate induced AMD-like pathology in mice (Cashman et al. (2015) J. Gene Med. 17: 229–243). Mice were co-injected subretinally with (i) an adenoviral vector expressing complement component C3, which has previously been shown to recapitulate many of the pathological features of human AMD; and (ii) an adenoviral vector expressing complement factor H. Compared with controls receiving GFP instead of complement factor H, mice transduced with complement factor H showed a 91% reduction in endothelial cell proliferation and a 69% attenuation of RPE atrophy. Electroretinography showed improved retinal function in complement factor H-treated mice, and rhodopsin and RPE65 immunocytochemistry were consistent with restoration of photoreceptors and RPE in these animals.

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

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

In some embodiments, complement factor H is complement factor H.

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

In some embodiments, the amino acid sequence of complement factor H is SEQ ID NO: 3. In other embodiments, the amino acid sequence of complement factor H is at positions 19 to 1231 of SEQ ID NO: 3.

MRLLAKIICLMLWAICVAEDCNELPPRRNTEILTGSWSDQTYPEGTQAIYKCRPGYRSLGNVIMVCRKGEWVALNPLRKCQKRPCGHPGDTPFGTFTLTGGNVFEYGVKAVYTCNEGYQLLGEINYRECDTDGWTNDIPICEWKCLPVTAPENGKIVSSAMEPDREYHFGQAVRFVCNSGYKIEGDEEMHCSDDGFWSKE KPKCVEISCKSPDVINGSPISQKIIYKENERFQYKCNMGYEYSERGDAVCTESGWRPLPSCEEKSCDNPYIPNGDYSPLRIKHRTGDEITYQCRNGFYPATRGNT AKCTSTGWIPAPRCTLKPCDYPDIKHGGLYHENMRRPYFPVAVGKYYSYYCDEHFETPSGSYWDHIHCTQDGWSPAVPCLRKCYFPYLENGYNQNYGRKFVQGKSIDVACHPGYALPKAQTTVTCMENGWSPTPRCIRVKTCSKSSIDIENGFISESQYTYALKEKAKYQCKLGYVTADGETSGSITCG KDGWSAQPTCIKSCDIPVFMNARTKNDFTWFKLNDTLDYECHDGYESNTGSTTGSIVCGYNGWSDLPICYERECELPKIDVHLVPDRKKDQYKVGEVLKFSCKPGFTIVGPNSVQC YHFGLSPDLPICKEQVQSCGPPPELLNGNVKEKTKEEYGHSEWEYYCNPRFLMKGPNKIQCVDGEWTTLPVCIVEESTCGDIPELEHGWAQLSSPPYYYGDSVEFNCSESFTMIGHRSITCIHGVWTQLPQCVAIDKLKKCKSSNLIILEEHLKNKKEFDHNSNIRYRCRGKEGWIHTVCINGRWDPEVNCSMAQIQLCPP PPQIPNSHNMTTTLNYRDGEKVSVLCQENYLIQEGEEITCKDGRWQSIPLCVEKIPCSQPPQIEHGTINSSRSSQESYAHGTKLSYTCEGGFRISEENETTCYM GKWSSPPQCEGLPCKSPPEISHGWAHMSDSYQYGEEVTYKCFEGFGIDGPAIAKCLGEKWSHPPSCIKTDCLSLPSFENAIPMGEKKDVYKAGEQVTYTCATYYKMDGASNVTCINSRWTGRPTCRDTSCVNPPTVQNAYIVSRQMSKYPSGERVRYQCRSPYEMFGDEEVMCLNGNWTEPPQCKDSTGKCGPPPPIDNGDI TSFPLSVYAPASSVEYQCQNLYQLEGNKRITCRNGQWSEPPKCLHPCVISREIMENYNIALRWTAKQKLYSRTGESVEFVCKRGYRLSSRSHTLRTTCWDGKLE YPTCAKR

(SEQ ID NO: 3)

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

In some embodiments, the nucleotide sequence encoding complement factor H is SEQ ID NO: 4.

ATGAGACTTCTAGCAAAGATTATTTGCCTTATGTTATGGGCTATTTGTGTAGCAGAAGATTGCAATGAACTTCCTCCAAGAAGAAATACAGAAATTCTGACAGGTTCCTGGTCTGACCAAACATATCCAGAAGGCACCCAGGCTATCTATAAATGCCGCCCTGGATATAGATCTCTTGGAAATGTAATAATGGTATGCAGGAAGGGAGAATGGGTTGCTCTTAATCCATTAAGGAAATGTCAGAAAAGGC CCTGTGGACATCCTGGAGATACTCCTTTTGGTACTTTTACCCTTACAGGAGGAAATGTGTTTGAATATGGTGTAAAAGCTGTGTATACATGTAATGAGGGGTATCAATTGCTAGGTGAGATTAATTACCGTGAATGTGACACAGAT GGATGGACCAATGATATTCCTATATGTGAAGTTGTGAAGTGTTTACCAGTGACAGCACCAGAGAATGGAAAAATTGTCAGTAGTGCAATGGAACCAGATCGGGAATACCATTTTGGACAAGCAGTACGGTTTGTATGTAACTCAGGCTACAAGATTGAAGGAGATGAAGAAATGCATTGTTCAGACGATGGTTTTTGGAGTAAAGAGAAACCAAAGTGTGTGAAATTTCATGCAAATCCC CAGATGTTATAAATGGATCTCCTATATCTCAGAAGATTATTTATAAGGAGAATGAACGATTTCAATATAAAATGTAACATGGGTTATGAATACAGTGAAAGAGGAGATGCTGTATGCACTGAATCTGGATGGCGTCCGTTGCCTTCATGTGAAGAA AAATCATGTGATAATCCTTATATTCCAAATGGTGACTACTCACCTTTAAGGATTAAACACAGAACTGGAGATGAAATCACGTACCAGTGTAGAAATGGTTTTTATCCTGCAACCCGGGGAAATACAGCAAAATGCACAAGTACTGGCTGGATACCTGCTCCGAGATGTACCTTGAAACCTTGTGATTATCCAGACATTAAACATGGAGGTCTATATCATGAGAATATGCGTAGACCATACTTTCCAGTAGC TGTAGGAAAATATTACTCCTATTACTGTGATGAACATTTTGAGACTCCGTCAGGAAGTTACTGGGATCACATTCATTGCACACAAGATGGATGGTCGCCAGCAGTACCATGC CTCAGAAAATGTTATTTTCCTTATTTGGAAAATGGATATAATCAAAATCATGGAAGAAAGTTTGTACAGGGTAAATCTATAGACGTTGCCTGCCATCCTGGCTACGCTCTTCCAAAAGCGCAGACCACAGTTACATGTATGGAGAATGGCTGGTCTCCTACTCCCAGATGCATCCGTGTCAAAACATGTTCCAAATCAAGTATAGATATTGAGAATGGGTTTATTTCTGAATCTCAGTATACAT ATGCCTTAAAAGAAAAAGCGAAATATCAATGCAAACTAGGATATGTAACAGCAGATGGTGAAACATCAGGATCAATTACATGTGGGAAAGATGGATGGTCAGCTCAACCCACGTGCATT AAATCTTGTGATATCCCAGTATTTATGAATGCCAGAACTAAAAATGACTTCACATGGTTTAAGCTGAATGACACATTGGACTATGAATGCCATGATGGTTATGAAAGCAATACTGGAAGCACCACTGGTTCCATAGTGTGTGGTTACAATGGTTGGTCTGATTTACCCATATGTTATGAAAGAGAATGCGAACTTCCTAAAATAGATGTACACTTAGTTCCTGATCGCAAGAAAGACCAGTATAAAG TTGGAGAGGTGTTGAAATTCTCCTGCAAACCAGGATTTACAATAGTTGGACCTAATTCCGTTCAGTGCTACCACTTTGGATTGTCTCCTGACCTCCCAATATGTAAAGAGCAAGTA CAATCATGTGGTCCACCTCCTGAACTCCTCAATGGGAATGTTAAGGAAAAAACGAAAGAAGAATATGGACACAGTGAAGTGGTGGAATATTATTGCAATCCTAGATTTCTAATGAAGGGACCTAATAAAATTCAATGTGTTGATGGAGAGTGGACAACTTTACCAGTGTGTATTGTGGAGGAGGTACCTGTGGAGATATACCTGAACTTGAACATGGCTGGGCCCAGCTTTCTTCCCCTCCT TATTACTATGGAGATTCAGTGGAATTCAATTGCTCAGAATCATTTACAATGATTGGACACAGATCAATTACGTGTATTCATGGAGTATGGACCCAACTTCCCCAGTGTGTGGCAATAGAT AAACTTAAGAAGTGCAAATCATCAAATTTAATTATACTTGAGGAACATTTAAAAAACAAGAAGGAATTCGATCATAATTCTAACATAAGGTACAGATGTAGAGGAAAAGAAGGATGGATACACACAGTCTGCATAAATGGAAGATGGGATCCAGAAGTGAACTGCTCAATGGCACAAATACAATTATGCCCACCTCCACCTCAGATTCCCAATTCTCACAATATGACAACCACACTGAATTATCGGGATG GAGAAAAAGTATCTGTTCTTTGCCAAGAAAATTATCTAATTCAGGAAGGAGAAGAAATTACATGCAAAGATGGAAGATGGCAGTCAATACCACTCTGTGTTGAAAAATTCCA TGTTCACAACCACCTCAGATAGAACACGGAACCATTAATTCATCCAGGTCTTCACAAGAAAGTTATGCACATGGGACTAAATTGAGTTATACTTGTGAGGGTGGTTTCAGGATATCTGAAGAAAATGAAACAACATGCTACATGGGAAAATGGAGTTCTCCACCTCAGTGTGAAGGCCTTCCTTGTAAATCTCCACCTGAGATTTCTCATGGTGTTGTAGCTCACATGTCAGACAGTTATCA GTATGGAGAAGAAGTTACGTACAAATGTTTTGAAGGTTTTGGAATTGATGGGCCTGCAATTGCAAAATGCTTAGGAGAAAAATGGTCTCACCCTCCATCATGCATAAAAACAGATTGTCTC AGTTTACCTAGCTTTGAAAATGCCATACCCATGGGAGAAGAAGGATGTGTATAAGGCGGGTGAGCAAGTGACTTACACTTGTGCAACATATTACAAAATGGATGGAGCCAGTAATGTAACATGCATTAATAGCAGATGGACAGGAAGGCCAACATGCAGAGACACCTCCTGTGTGAATCCGCCCACAGTACAAAATGCTTATATAGTGTCGAGACAGATGAGTAAATATCCATCTGGTGAGAGAG TACGTTATCAATGTAGGAGCCCTTATGAAATGTTTGGGGATGAAGAAGTGATGTGTTTAAAATGGAAACTGGACGGAACCACCTCAATGCAAAGATTCTACAGGAAAATGTGGGCCC CCTCCACCTATTGACAATGGGGACATTACTTCATTCCCGTTGTCAGTATATGCTCCAGCTTCATCAGTTGAGTACCAATGCCAGAACTTGTATCAACTTGAGGGTAACAAGCGAATAACATGTAGAAATGGACAATGGTCAGAACCACCAAAATGCTTACATCCGTGTGTAATATCCCGAGAAATTATGGAAAATTATAACATAGCATTAAGGTGGACAGCCAAACAGAAGCTTTATTCGAGA ACAGGTGAATCAGTTGAATTTGTGTGTAAAACGGGGATATCGTCTTTCATCACGTTCTCACACATTGCGAACAACATGTTGGGATGGGAAACTGGAGTATCCAACTTGTGCAAAAAGATAG

(SEQ ID NO: 4)

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

In other embodiments, the nucleotide sequence encoding complement factor H is SEQ ID NO: 4.

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

In other embodiments, the nucleotide sequence encoding complement factor H is located at positions 55-3696 of SEQ ID NO: 4.

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

In other embodiments, the nucleotide sequence encoding complement factor H encodes the amino acid sequence of SEQ ID NO: 3.

In another embodiment, the nucleotide sequence encoding complement factor H encodes an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identical to positions 19-1231 of SEQ ID NO: 3. Preferably, the amino acid sequence substantially retains the functional activity of the protein represented by SEQ ID NO: 3.

In other embodiments, the nucleotide sequence encoding complement factor H encodes the amino acid sequence at positions 19-1231 of SEQ ID NO: 3.

FACTOR H-LIKE PROTEIN 1 (FHL1)

Factor H-like protein 1 (FHL1) is a splice variant of complement factor H that contains the first 7 CCPs of complement factor H followed by a four-amino acid carboxy-terminal tail (Clark, S.J. et al. (2015) J Clin Med 4: 18-31).

In some embodiments, FHL1 is human FHL1.

In some embodiments, the amino acid sequence of FHL1 is SEQ ID NO: 11.

MRLLAKIICLMLWAICVAEDCNELPPRRNTEILTGSWSDQTYPEGTQAIYKCRPGYRSLGNIIMVCRKGEWVALNPLRKCQKRPCGHPGDTPFGTFTLTGGNVFEYGVKAVYTCNEGYQLLGEINYRECDTDGWTNDIPICEWKCLPVTAPENGKIVSSAMEPDREYHFGQAVRFVCNSGYKIEGDEEMHCSDDGFWSKEK PKCVEISCKSPDVI NGSPISQKIIYKENERFQYKCNMGYEYSERGDAVCTESGWRPLPSCEEKSCDNPYIPNGDYSPLRIKHRTGDEITYQCRNGFYPATRGNTAKCTSTGWIPAPRCTLKPCDYPDIKHGGLYHENMRRPYFPVAVGKYYSYYCDEHFETPSGSYWDHIHCTQDGWSPAVPCLRKCYFPYLENGYNQNYGRKFV QGKSIDVACHPGYALPKAQTTVTCM ENGWSPTPRCIRVSFTL

(SEQ ID NO: 11)

The FHL1 nucleotide sequences used in the present invention are preferably codon optimized.

A preferred nucleotide sequence encoding FHL1 is SEQ ID NO: 12.

ATGCGCCTCCTGGCCAAGATCATCTGCCTCATGCTGTGGGCCATCTGCGTGGCTGAGGACTGCAATGAGCTGCCGCCCAGGAGGAACACAGAGATCCTGACAGGGAGCTGGTCTGACCAGACCTACCCTGAGGGCACCCAGGCGATCTACAAGTGCCGGCCGGGCTACAGGAGCCTGGGGAACATCATCATGGTGTGTAGAAAGGGCGAATGGGTGGCCCTCAACCCCCTGAGGAAGTGCCAGAA GCGGCCCTGTGGCCACCCCGGGGACACACCCTTCGGGACCTTCACCCTGACCGGCGGCAATGTGTTTGAGTACGGCGTG AAGGCTGTCTACACATGCAACGAGGGGTACCAGCTGCTGGGCGAGATTAACTACCGGGAGTGTGACACCGATGGGTGGACCAACGACATTCCCATCTGTGAGGTGGTCAAGTGTCTCCCCGTGACAGCCCCAGAAAATGGCAAAATCGTGAGCAGCGCCATGGGAGCCTGACCGCGAATATCACTTTGGGCAGGCCGTGAGGTTTGTGTGCAACTCGGGCTACAAAATTGAAGGTGATGAGGAG ATGCACTGCAGCGATGATGGCTTCTGGTCCAAGGAGAAGCCCAAATGTGTGGAGATCTCTGCAAGTCTCCCGACGTGATC AACGGCAGCCCAATCAGCCAGAAGATTATTTACAAAGAGAACGAGCGCTTCCAGTACAAGTGTAACATGGGCTATGAGTATTCAGAGAGGGGATGCCGTCTGCACTGAGAGCGGCTGGAGACCACTGCCTAGCTGCGAGGAAAAGAGTTGTGACAACCCTTACATCCCAAATGGCGACTACTCCCCTCTGCGGATCAAACACCGGACCGGGGATGAAATCACCTATCAGTGCCGCAATGGATTCT ACCCGGCCACCGCGGCAACACCGCCAAATGCACCAGCACAGGCTGGATCCCCGCCCCCCGCTGTACGCTGAAGCCT TGCGACTATCCAGACATCAAGCACGGAGGCCTGTACCACGAAAACATGCGGCGGCCTTATTTCCCTGTGGCAGTGGGGAAGTACTACAGCTACTACTGCGACGAGCACTTCGAGACCCCCTCTGGCTCCTACTGGGACCACATCCACTGCACACAGGACGGCTGGTCTCCAGCTGTGCCCTGCCTGAGGAAATGCTACTTCCCCTACCTGGAGAACGGATACAACCAGAACTATGGCCGCAAGTTCGTGCA GGGCAAGAGCATCGATGTGGCCTGCCACCCTGGCTACGCCCTGCCCAAGGCCCAGACAACTGTGACCTGCATG GAGAATGGTTGGAGCCCCACCCCGCGCTGCATCCGGGTGTCCTTCACGCTCTGA

(SEQ ID NO: 12)

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

In other embodiments, the nucleotide sequence encoding FHL1 is SEQ ID NO: 12.

POLYNUCLEOTIDE

The polynucleotides of the invention may comprise DNA or RNA, preferably DNA. They may be single-stranded or double-stranded. It will be understood by one skilled in the art that a plurality of different polynucleotides may encode the same polypeptide due to the degeneracy of the genetic code. It will also be understood that those skilled in the art may, using standard techniques, make nucleotide substitutions that do not affect the polypeptide sequence encoded by the polynucleotides of the invention to reflect the codon usage of any particular host organism in which the polypeptides of the invention are to be expressed.

The polynucleotides may be modified by any method available in the art. Such modifications may be made to increase the activity or lifespan of the polynucleotides of the invention in vivo.

Polynucleotides, such as DNA polynucleotides, may be produced recombinantly, synthetically, or by any means available to those skilled in the art. They may also be cloned by standard techniques.

Longer polynucleotides are usually produced using recombinant techniques, such as polymerase chain reaction (PCR) cloning techniques. This would involve creating a pair of primers (e.g., about 15-30 nucleotides) flanking the target sequence to be cloned, contacting the primers with mRNA or cDNA obtained from an animal or human cell, performing the polymerase chain reaction under conditions that cause amplification of the desired region, isolating the amplified fragment (e.g., by purifying the reaction mixture with an agarose gel), and recovering the amplified DNA. The primers may be designed to contain suitable restriction enzyme recognition sites so that the amplified DNA can be cloned into a suitable vector.

STRUCTURE OF THE EYE

The medicinal products described herein can be delivered to the eye of a mammal, preferably a human eye, in connection with the treatment or prevention of an eye disease such as age-related macular degeneration (AMD).

A person skilled in the art of treating eye diseases will have a detailed and complete understanding of the structure of the eye. However, the following structure is described which is of particular relevance to this invention.

Retina

The retina is a multilayered membrane that lines the inner posterior chamber of the eye and receives images of the visual world, which are transmitted to the brain via the optic nerve. In order from the inside of the eye to the outside, the retina consists of layers of the sensorineural retina and the retinal pigment epithelium, with the choroid outside the retinal pigment epithelium.

The neurosensory part of the retina and photoreceptors

The neurosensory retina contains the photoreceptor cells that directly perceive light. It consists of the following layers: internal limiting membrane (ILM); nerve fiber layer; ganglion cell layer; inner plexiform layer; inner nuclear layer; outer plexiform layer; outer nuclear layer (photoreceptor nuclei); external limiting membrane (ELM); and photoreceptors (inner and outer segments of rods and cones).

A person skilled in the art will have a detailed understanding of photoreceptor cells. In short, photoreceptor cells are specialized neurons located in the retina that convert light into biological signals. Photoreceptor cells are made up of rods and cones, which are distributed differently throughout the retina.

Rod cells are distributed mainly in the outer parts of the retina. They are very sensitive and provide vision in low light. The retina of a normal person contains on average about 125 million rods.

Retinal cones are found throughout the retina, but are particularly concentrated in the fovea, the pit in the neurosensory retina that is responsible for high-resolution central vision. Retinal cones are less sensitive than retinal rods. The average human retina contains about 6 to 7 million cones.

Retinal pigment epithelium

The retinal pigment epithelium (RPE) is a pigmented layer of cells located just outside the neurosensory retina. The RPE serves a number of functions, including transporting nutrients and other substances to the photoreceptor cells and absorbing scattered light to improve vision.

Choroid

The choroid is a vascular layer located between the RPE and the outer sclera of the eye. The vascular network of the choroid provides the retina with oxygen and nutrients.

AGE-RELATED MACULAR DEGENERATION (AMD)

The clinical progression of age-related macular degeneration (AMD) is characterized by a stepwise pattern of changes in the macula. The hallmark of early AMD is the appearance of drusen, which are collections of extracellular debris beneath the retina that appear as yellow spots on the retina during clinical examination and on fundus images. Drusen are classified as small (<63 μm), medium (63-124 μm), and large (>124 μm). They are also considered hard or soft based on how their edges appear on ophthalmologic examination. While hard drusen have well-defined edges, soft drusen have less defined fluid edges. The Age-Related Eye Disease Study (AREDS) Photographic Fundus Severity Scale is one of the main classification systems used for this condition.

AMD is divided into "dry" and "wet" (exudative or neovascular) forms. Dry AMD is more common than wet AMD, but the dry form can transition into the wet form, and both occur simultaneously in a significant number of cases. Dry AMD is typically characterized by progressive apoptosis of cells in the RPE layer and overlying photoreceptor cells, and often of underlying cells in the choroidal capillary layer. Confluent areas of RPE cell death accompanied by atrophy of the overlying photoreceptors are called geographic atrophy (GA). Patients with this form of AMD experience a slow and progressive deterioration of central vision.

Wet AMD is characterized by bleeding and/or fluid leakage from abnormal vessels that have grown from the choroidal vessels (choriocapillaries) beneath the RPE and macula, which can cause sudden and disabling vision loss. It has been estimated that most of the vision loss experienced by patients is due to this choroidal neovascularization (CNV) and its secondary complications.

The treatment or prevention of AMD described herein may reduce or prevent the AMD phenotype described above. Preferably, the treatment of AMD may maintain or improve visual function.

In some embodiments, treating or preventing AMD results in preventing or reducing the formation of geographic atrophy. In other embodiments, treating or preventing AMD results in slowing the progression of geographic atrophy. For example, this results in at least a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90% reduction in the increase in GA area over 12 months after administration to the treated eye of the subject compared to the untreated eye over the same period. In other embodiments, treating or preventing AMD results in treating geographic atrophy, such as reducing the degree of geographic atrophy.

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

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

In some embodiments, treating or preventing AMD results in an improvement or restoration of vision or visual acuity. In other embodiments, treating or preventing AMD reduces vision loss or visual acuity.

In some embodiments, treating or preventing AMD results in an improvement or restoration of reading speed in a subject. In other embodiments, treating or preventing AMD reduces a decline in reading speed in a subject.

In some embodiments, treatment or prevention of AMD results in a reduction or prevention of loss of photoreceptors and/or retinal pigment epithelium (RPE).

DIABETIC RETINOPATHY

Diabetic retinopathy is a condition characterized by damage to the blood vessels of the retina, which is caused by high blood sugar levels associated with diabetes. If left untreated, diabetic retinopathy can cause blindness.

Although subjects with mild diabetic retinopathy may have good vision, two types of diabetic retinopathy, namely diabetic macular edema (DMO) and proliferative diabetic retinopathy (PDR), can threaten a subject's vision.

Diabetic macular edema is characterized by fluid leaking from damaged blood vessels at the back of the eye. The leaking fluid accumulates in the macula, causing swelling and blurred vision. This can eventually lead to loss of central vision and the inability to read or drive. Side vision is usually normal.

Proliferative diabetic retinopathy is characterized by the blockage of blood vessels in the retina, which leads to the growth of abnormal, fragile blood vessels on the surface of the retina. This can lead to permanent vision loss due to bleeding in the eye, scarring, and retinal detachment.

VECTORS

A vector is a tool that allows or facilitates the transfer of an object from one environment to another.

Adeno-associated viral (AAV) vectors

In one aspect, the present invention provides an AAV vector comprising a polynucleotide of the present invention.

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

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

An AAV vector may contain an AAV genome or a fragment or derivative thereof.

AAV is known to be capable of packaging genomes up to 5.2 kb in size (Dong, J.-Y. et al. (1996) Human Gene Therapy 7: 2101-2112).

The AAV genome is a polynucleotide sequence that can encode functions necessary for the production of an AAV particle. These functions include those that act in the AAV replication and packaging cycle in a host cell, including encapsidation of the AAV genome into an AAV particle. Naturally occurring AAVs are replication deficient and rely on the provision of auxiliary functions in trans to complete the replication and packaging cycle. Accordingly, the AAV genome of the AAV vector of the present invention is typically replication deficient.

The AAV genome can be in single-stranded form, positive- or negative-sense, or alternatively in double-stranded form. Use of the double-stranded form allows bypassing the step of DNA replication in the target cell and thus can accelerate transgene expression.

The AAV genome may be from any naturally occurring AAV serotype, isolate, or clade. Thus, the AAV genome may be the entire genome of a naturally occurring AAV. As known to those skilled in the art, naturally occurring AAVs may be classified according to different biological systems.

AAVs are usually referred to by their serotype. A serotype corresponds to a variant AAV subtype that, due to its capsid surface antigen expression profile, has distinctive reactivity that can be used to distinguish it from other variant subtypes. Typically, a virus of a particular AAV serotype will not effectively cross-react with neutralizing antibodies specific for any other AAV serotype.

AAV serotypes include AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, and AAV11, as well as recombinant serotypes such as Rec2 and Rec3, recently identified from primate brain. Any of these AAV serotypes may be used in the present invention.

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

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

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

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

The capsid protein may be a mutant capsid protein such as that described in WO 2008/124724, which is incorporated herein by reference.

In some embodiments, the AAV vector comprises an AAV8 capsid with the Y733F mutation.

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

AAV may also refer to a phylogenetic group or clone. This refers to the phylogenetic relationships of naturally occurring AAVs and, generally, to a phylogenetic group of AAVs that can be traced back to a common ancestor and includes all of its descendants. Additionally, AAV may refer to a specific isolate, i.e., a genetic isolate of a specific naturally occurring AAV. The term “genetic isolate” describes a population of AAVs that has undergone limited genetic admixture with other naturally occurring AAVs, thereby defining a recognizable, genetically distinct population.

A person skilled in the art may select a suitable AAV serotype, phylogenetic group, clone or isolate for use in the invention based on his general knowledge. For example, the AAV5 capsid has been shown to efficiently transduce primate cone photoreceptors, as demonstrated by the successful correction of an inherited color vision defect (Mancuso et al. (2009) Nature 461: 784-7).

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

Typically, the AAV genome of a natural AAV serotype, isolate, or phylogenetic group contains at least one inverted terminal repeat (ITR) sequence. The ITR sequence acts in cis to provide a functional origin of replication and to allow integration and excision of the vector from the cellular genome. In preferred embodiments, one or more ITR sequences are flanked by nucleotide sequences encoding complement factor I and/or FHL1. The AAV genome typically also includes packaging genes, such as the rep and/or cap genes, which encode packaging functions for the AAV particle. The rep gene encodes one or more Rep78, Rep68, Rep52, and Rep40 proteins, or variants thereof. The capsid gene encodes one or more capsid proteins, such as VP1, VP2, and VP3, or variants thereof. These proteins comprise the capsid of the AAV particle. Capsid variants are discussed below.

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

As discussed above, the AAV genome used in the AAV vector of the invention may therefore be the complete genome of a naturally occurring AAV. For example, a vector containing the complete AAV genome may be used to produce an AAV vector or vector particle in vitro. However, while in principle such a vector could be administered to patients, in practice this will rarely be the case. Preferably, the AAV genome will be derivatized for administration to patients. Such derivatization is standard in the art, and the invention encompasses the use of any known derivatives of the AAV genome and derivatives that can be prepared using methods known in the art. Derivatization of the AAV genome and AAV capsid are discussed in Coura and Nardi (2007) Virology Journal 4: 99, and in Choi et al. and Wu et al., cited above.

Derivatives of the AAV genome include any truncated or modified forms of the AAV genome that allow expression of a transgene from an AAV vector of the invention in vivo. Typically, the AAV genome may be significantly truncated to include minimal viral sequence while still retaining the above-mentioned function. This is preferred for safety reasons to reduce the risk of recombination of the vector with wild-type virus, as well as to prevent the triggering of a cellular immune response due to the presence of viral gene proteins in the target cell.

Typically, the derivative will include at least one inverted terminal repeat sequence (ITR), preferably more than one ITR, such as two or more ITRs. The one or more ITRs may be from AAV genomes of different serotypes, or may be chimeric or mutant ITRs. A preferred mutant ITR has a deletion of the trs (terminal resolution site). This deletion allows for continued genome replication to create a single-stranded genome that contains both coding and complementary sequences, i.e., a self-complementary AAV genome. This bypasses DNA replication in the target cell and thus allows for accelerated expression of the transgene.

One or more ITRs will preferably flank the nucleotide sequence encoding complement factor I or FHL1 at either end. The inclusion of one or more ITRs is advantageous to facilitate the formation of a concatemer of the vector of the invention in the host cell nucleus, for example, following conversion of single-stranded vector DNA to double-stranded DNA by host cell DNA polymerases. The formation of such episomal concatemers protects the vector construct during the life of the host cell, thereby allowing for prolonged expression of the transgene in vivo.

In preferred embodiments, the ITR elements will be the only sequences retained from the native AAV genome in the derivative. Thus, the derivative will preferably not include the rep and/or cap genes of the native genome and any other sequences of the native genome. This is preferred for the reasons described above, as well as to reduce the possibility of integration of the vector into the host cell genome. In addition, the reduction in the size of the AAV genome provides increased flexibility in incorporating other sequence elements (such as regulatory elements) into the vector in addition to the transgene.

Therefore, the derivative of the invention may have the following parts removed: one inverted terminal repeat (ITR) sequence, replication (rep) and capsid (cap) genes. However, in some embodiments, the derivatives may further comprise one or more rep and/or cap genes or other viral sequences of the AAV genome. Naturally occurring AAV integrates at a high frequency into a specific region of human chromosome 19 and exhibits a low frequency of random integration, so that the preservation of the integrative capacity of the vector can be tolerated in therapeutic settings.

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

Derivatives with a chimeric, shuffled or modified capsid are typically selected to provide one or more desired functionalities for the AAV vector. Thus, these derivatives may exhibit increased gene delivery efficiency, decreased immunogenicity (humoral or cellular), an altered tropism range and/or improved targeting to a specific cell type compared to an AAV vector containing a natural AAV genome, such as the AAV2 genome. Increased gene delivery efficiency may be achieved through improved binding to a cell surface receptor or coreceptor, improved internalization, improved intracellular and nuclear trafficking, improved unpacking of the viral particle and improved conversion of a single-stranded genome to a double-stranded form. Increased efficiency may also relate to an altered tropism range or targeting to a specific cell population such that the vector dose is not diluted by administration to tissues where it is not needed.

Chimeric capsid proteins include those formed by recombination between two or more capsid encoding sequences of natural AAV serotypes. This can be accomplished, for example, by a marker rescue approach in which non-infectious capsid sequences from one serotype are co-transfected with capsid sequences from another serotype and targeted selection is used to select capsid sequences with desired properties. Capsid sequences from different serotypes can be altered by homologous recombination within the cell to produce new chimeric capsid proteins.

Chimeric capsid proteins also include those generated by engineering capsid protein sequences to transfer domains of specific capsid proteins, surface loops, or specific amino acid residues between two or more capsid proteins, such as between two or more capsid proteins of different serotypes.

Shuffled or chimeric capsid proteins can also be produced by DNA shuffling or error-prone PCR. Hybrid AAV capsid genes can be created by randomly fragmenting sequences of related AAV genes, such as the genes encoding the capsid proteins of several different serotypes, and then reassembling the fragments in a polymerase self-priming reaction that can also induce crossovers in regions of sequence homology. A library of hybrid AAV genes created in this way by shuffling capsid genes from several serotypes can be screened to identify viral clones possessing the desired functionality. Similarly, error-prone PCR can be used to randomly mutate AAV capsid genes to generate a diverse library of variants that can then be selected for a desired property.

Capsid gene sequences can also be genetically modified to introduce specific deletions, substitutions, or insertions relative to the native wild-type sequence. In particular, capsid genes can be modified by inserting an unrelated protein or peptide sequence into the open reading frame of the capsid coding sequence or at the N- and/or C-terminus of the capsid coding sequence.

The unrelated protein or peptide may advantageously be one that acts as a ligand for a particular cell type, thereby providing improved binding to the target cell or increasing the specificity of targeting the vector to a particular cell population. An example would be the use of the RGD peptide to block uptake by the retinal pigment epithelium and thereby enhance transduction of surrounding retinal tissues (Cronin et al. (2008) ARVO Abstract: D1048). The unrelated protein may also be a protein that aids in the purification of the viral particle as part of the manufacturing process, i.e., an epitope or affinity tag. The insertion site is typically chosen so as not to interfere with other functions of the viral particle, e.g., internalization, transport of the viral particle. One skilled in the art can determine suitable insertion sites based on his general knowledge. Specific sites are disclosed in Choi et al., cited above.

The invention further includes providing AAV genome sequences in an order and configuration different from the order and configuration of the native AAV genome. The invention also includes replacing one or more AAV sequences, or genes, with sequences from another virus or with chimeric genes consisting of sequences from more than one virus. Such chimeric genes may consist of sequences of two or more related viral proteins from different virus species.

The AAV vector of the invention may be in the form of a nucleotide sequence comprising an AAV genome or a derivative thereof and a sequence encoding complement factor I or FHL1 or derivatives thereof.

The AAV particles of the invention include transcapsidated forms in which an AAV genome or derivative having an ITR of one serotype is packaged into a capsid of another serotype. The AAV particles of the invention also include mosaic forms in which a mixture of unmodified capsid proteins of two or more different serotypes constitutes the viral capsid. The AAV particle also includes chemically modified forms bearing ligands adsorbed to the surface of the capsid. For example, such ligands may include antibodies for targeting a particular cell surface receptor.

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

An AAV vector may contain multiple copies (e.g., 2, 3, etc.) of the nucleotide sequence referred to herein.

PROMOTERS AND REGULATORY SEQUENCES

The polynucleotide or vector of the invention may also include elements that provide for the expression of the complement factor I or FHL1 transgenes in vitro or in vivo. These may be referred to as expression control sequences. Thus, the polynucleotide or vector typically comprises expression control sequences (e.g., comprising a promoter sequence) operably linked to the nucleotide sequence encoding the transgene.

Any suitable promoter may be used, the selection of which can be readily made by one skilled in the art. The promoter sequence may be constitutively active (i.e., work in any host cell background) or, alternatively, may be active only in a particular host cell environment, allowing targeted expression of the transgene in a particular cell type (e.g., a tissue-specific promoter). The promoter may exhibit inducible expression in response to the presence of another factor, such as a factor present in the host cell. In any case, when the vector is administered for therapy, it is preferred that the promoter be functional in the target cell background.

In some embodiments, it is preferred that the promoter exhibits retinal cell-specific expression so that the transgene can be expressed only in retinal cell populations. Thus, expression of the promoter can be retinal cell-specific, such as limited to only the neurosensory retina and retinal pigment epithelium cells.

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

In preferred embodiments, the polynucleotide or vector comprises a CMV promoter.

An example of a CMV promoter sequence is:

GGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATG CCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTA TTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTCGTTTAGTGAACCG TCAGATCGCCTGGAGACGCCATCCACGCTGTTTTGACCTCCATAGAAGACACCG

(SEQ ID NO: 13)

In some embodiments, the polynucleotide or vector comprises a promoter with a nucleotide sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 13. Preferably, the nucleotide sequence substantially retains the functional activity of the promoter represented by SEQ ID NO: 13.

In other embodiments, the polynucleotide or vector comprises a promoter with the nucleotide sequence of SEQ ID NO: 13.

Another example of a promoter sequence is:

ATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATTGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCA TCGCTATTACCATGGTCGAGGTGAGCCCCACGTTCTGCTTCACTCTCCCCCATCTCCCCCCCCTCCCCACCCCCAATTTTGTATTTATTTATTTTTTAATTATTTTGTGCAGCGATGGGGGCGGGGGGGGGGGGGGCGCGCCAGGCGGGGCGGGGCGGGGCGAGGGCGGGGCGGGGCGAGGCGGAGAGGTGCGGCGGCAGCCAATCAGAGCGGCGCGCTCCGAAAGTTTC CTTTTATGGCGAGGCGGCGGCGGCGGCGGCCCTATAAAAAGCGAAGCGCGCGGCGGGCGGGAGTCGCTGCGCGCTGCCTTCGCCCCGTGCCCCGCTCCGCCGCCGCCTCGCGCCGCCCGCCCCGGCTCTGACTGACCGCGTTACTCCCACAGGTGAGCGGGCGGGACGGCCCTTCTCCTCCGGGCTGTAATTAGCGCTTGGTTTAATGACGGCTTGTTTCTTTTCTGTGGCTG CGTGAAAGCCTTGAGGGGCTCCGGGAGGGCCCTTTGTGCGGGGGGAGCGGCTCGGGGCTGTCCGCGGGGGGACGGCTGCCTTCGGGGGGGACGGGGCAGGGCGGGGTTCGGCTTCTGGCGTGTGACCGGCGGCTCTAGAGCCTCTGCTAACCATGTTCATGCCTTCTTCTTTTTCCTACAGCTCCTGGGCAACGTGCTGGTTATTGTGCTGTCTCATCATTTTGGCAAAGAATT

(SEQ ID NO: 5)

In some embodiments, the polynucleotide or vector comprises a promoter with a nucleotide sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 5. Preferably, the nucleotide sequence substantially retains the functional activity of the promoter represented by SEQ ID NO: 5.

In other embodiments, the polynucleotide or vector comprises a promoter with the nucleotide sequence of SEQ ID NO: 5.

Examples of human sequence-based promoters that can induce the expression of retinal-specific genes include rhodopsin kinase for rods and cones (Allocca et al. (2007) J. Virol. 81: 11372–80), PR2.1 for cones only (Mancuso et al. (2009) Nature 461: 784–7), and/or RPE65 (Bainbridge et al. (2008) N. Engl. J. Med. 358: 2231–9) or VMD2 (Esumi et al. (2004) J. Biol. Chem. 279: 19064–73) for the retinal pigment epithelium.

The polynucleotide or vector of the invention may also comprise one or more additional regulatory sequences that may act pre- or post-transcriptionally. The regulatory sequence may be part of a natural transgene locus or may be a heterologous regulatory sequence. The polynucleotide or vector of the invention may comprise portions of the 5'-UTR or 3'-UTR from a native transgene transcript.

Regulatory sequences are any sequences that facilitate the expression of a transgene, i.e., act to increase transcript expression, improve nuclear export of mRNA, or increase its stability. Such regulatory sequences include, for example, enhancer elements, post-transcriptional regulatory elements, and polyadenylation sites.

The preferred site of polyadenylation is the bovine growth hormone poly-A (bGH poly-A) signal.

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

GTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTTCCTAATAAAATGAGGAAATTGCATCGCATTGTCTGAGTAGGTGTCATTCTATTCTGGGGGGTGGGGTGGGGCAGGACAGCAAGGGGGAGGATTGGGAAGACAATAGCAGGCATGCTGGGGATGCGGTGGGCTCTATGG

(SEQ ID NO: 14)

Another example of the bovine growth hormone poly-A (bGH poly-A) signal is:

TCGCTGATCAGCCTCGACTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTTCCTAATAAAATGAGGAAATTGCATCGCATTGTCTGAGTAGGTGTCATTCTATTCTGGGGGGTGGGGTGGGGCAGGACAGCAAGGGGGAGGATTGGGAAGACAATAGCAGGCATGCTGGGGATGCGGTGGGCTCTATGG CTTCTGAGGCGGAAAGAACCAGCTGGGG

(SEQ ID NO: 6)

In some embodiments, the polynucleotide or vector comprises a polyadenylation signal with a nucleotide sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 14 or 6. Preferably, the nucleotide sequence substantially retains the functional activity of the polyadenylation signal represented by SEQ ID NO: 14 or 6.

In other embodiments, the polynucleotide or vector comprises a polyadenylation signal with a nucleotide sequence of 14 or 6.

In the context of a polynucleotide or vector according to the invention, such regulatory sequences will be cis-acting. However, the invention also includes the use of trans-acting regulatory sequences located on additional genetic constructs.

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

An example of WPRE is:

ATCAACCTCTGGATTACAAAATTTGTGAAAGATTGACTGGTATTCTTAACTATGTTGCTCCTTTTACGCTATGTGGATACGCTGCTTTAATGCCTTTGTATCATGCTATTGCTTCCCGTATGGCTTTCATTTTCTCCTCCTTGTATAAATCCTGGTTGCTGTCTCTTATTGAGGAGTTGTGGCCCGTTGTCAGGCAACGTGGCGTGGTGTGCACTGTGTTTGCTGACGCAACCCCC ACTGGTTGGGGCATTGCCACCACCTGTCAGCTCCTTTCCGGGACTTTCGCTTTCCCCC TCCCTATTGCCACGGCGGAACTCATCGCCGCCTGCCTTGCCCGCTGCTGGACAGGGGCTCGGCTGTTGGGCACTGACAATTCCGTGGTGTTGTCGGGGAAATCATCGTCCTTTCCTTGGCTGCTCGCCTGTGTTGCCACCTGGATTCTGCGCGGGACGTCCTTCTGCTACGTCCCTTCGGCCCTCAATCCAGCGGACCTTCCTTCCCGCGGCCTGCTGCCGGCTCTGCGGCCTCTTCCGCGTC TTCGCCTTCGCCCTCAGACGAGTCGGATCTCCCTTTGGGCCGCCTCCCCGC

(SEQ ID NO: 7)

The WPRE is a tripartite element containing the gamma, alpha, and beta elements in that order. A shortened version of the WPRE that contains only the minimal gamma and alpha elements (referred to as WPRE3; Choi, J.-H. et al. (2014) Molecular Brain 7: 17) can also be used in the invention.

An example of a WPRE3 sequence is:

AATCAACCTCTGGATTACAAAATTTGTGAAAGATTGACTGGTATTCTTAACTATGTTGCTCCTTTTACGCTATGTGGATACGCTGCTTTAATGCCTTTGTATCATGCTATTGCTTCCCGTATGGCTTTCATTTTCTCCTCCTTGTATAAAATCCTGGTTAGTTCTTGCCACGGCGGAACTCATCGCCGCCTGCCTTGCCCGCTGCTGGACAGGGGCTCGGCTGTTGGGCACTGACAATTCCGTGGT

(SEQ ID NO: 15)

In some embodiments, the polynucleotide or vector comprises a post-transcriptional regulatory element with a nucleotide sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 15 or 7. Preferably, the nucleotide sequence substantially retains the functional activity of the post-transcriptional regulatory element represented by SEQ ID NO: 15 or 7.

In other embodiments, the polynucleotide or vector comprises a post-transcriptional regulatory element with the nucleotide sequence of SEQ ID NO: 15 or 7.

Another regulatory sequence that can be used in the polynucleotide or vector of the invention is a DNA sequence having nuclear matrix binding ability (SAR). Additional regulatory sequences can be easily selected by a person skilled in the art.

METHOD OF ADMINISTRATION

The polynucleotide or vector of the invention may be administered systemically (e.g., by infusion into a peripheral vein) and may be administered locally (e.g., into the CNS by intrathecal injection). In preferred embodiments, the polynucleotide or vector is administered intraocularly.

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

In some embodiments, the polynucleotide or vector is administered to the eye of the subject via subretinal, direct retinal, suprachoroidal, or intravitreal injection. In some embodiments, said administration is performed by a robot.

The volume of the administered medicinal composition can, for example, be about 10-500 μl, such as about 50-500, 100-500, 200-500, 300-500, 400-500, 50-250, 100-250, 200-250 or 50-150 μl. The volume can, for example, be about 10, 50, 100, 150, 200, 250, 300, 350, 400, 450 or 500 μl. Preferably, the volume of the administered medicinal composition is 100 μl.

A person skilled in the art will be familiar with and able to perform individual subretinal, direct retinal, suprachoroidal, or intravitreal injections.

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

In some embodiments, the polynucleotide, vector, or pharmaceutical composition comprising the same is administered no more than once or no more than twice during the life of the subject.

Subretinal injection

Subretinal injections are injections into the subretinal space, i.e. under the neurosensory retina. During a subretinal injection, the injected material is directed into the photoreceptor cells and retinal pigment epithelium (RPE) layers and creates space between them.

When the injection is performed through a small retinotomy, retinal detachment may occur. The detached, raised layer of the retina formed by the injected material is called a "bleb."

The hole created by a subretinal injection must be small enough to prevent the injected solution from flowing back into the vitreous cavity after injection. Such reflux would be particularly problematic with drug injection because the drug action would be directed away from the target area. Preferably, the injection creates a self-sealing entry point into the neurosensory retina, i.e., after the injection needle is removed, the hole created by the needle closes so that little or no of the injected material exits through the hole.

To facilitate this process, special subretinal injection needles are commercially available (e.g., DORC 41G Teflon Subretinal Injection Needles, Dutch Ophthalmic Research Center International BV, Zuidland, The Netherlands). These are needles designed for performing subretinal injections.

If no retinal injury occurs during the injection, and as long as a sufficiently small needle is used, virtually all of the injected material remains localized between the detached neurosensory retina and the RPE at the site of the localized retinal detachment (i.e., no reflux into the vitreous cavity). Indeed, the typical short-term persistence of the bleb indicates that there is usually some release of injected material into the vitreous. The bleb may dissipate over a longer period of time as the injected material is absorbed.

Imaging of the eye, particularly the retina, such as with optical coherence tomography, may be performed prior to surgery.

The volume of the administered drug composition can be, for example, about 10-500 μl, such as about 50-500, 100-500, 200-500, 300-500, 400-500, 50-250, 100-250, 200-250 or 50-150 μl. The volume can be, for example, about 10, 50, 100, 150, 200, 250, 300, 350, 400, 450 or 500 μl. Preferably, the volume of the administered drug composition is 100 μl. Larger volumes can increase the risk of retinal stretching, while smaller volumes are difficult to see.

Two-stage subretinal injection

The polynucleotide or vector of the invention can be delivered with increased accuracy and safety using a two-stage method in which a localized retinal detachment is created by a subretinal injection of a first solution. The first solution does not contain a polynucleotide or vector. A second subretinal injection is then used to deliver a drug containing a polynucleotide or vector into the subretinal fluid of the bleb created by the first subretinal injection. Since the injection delivering the drug is not used to detach the retina, a certain volume of solution can be administered in this second stage.

In some embodiments, subretinal injection of a vector comprises the steps of:

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

(b) administering a medicinal composition by subretinal injection into the bleb formed in step (a), wherein the medicinal agent comprises a polynucleotide or a vector.

The volume of the solution introduced in step (a) for at least partially separating the retina can be, for example, about 10-1000 μl, for example, about 50-1000, 100-1000, 250-1000, 500-1000, 10-500, 50-500, 100-500, 250-500 μl. The volume can be, for example, about 10, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900 or 1000 μl.

The volume of the drug composition administered in step (b) can be, for example, about 10-500 μl, such as about 50-500, 100-500, 200-500, 300-500, 400-500, 50-250, 100-250, 200-250 or 50-150 μl. The volume can be, for example, about 10, 50, 100, 150, 200, 250, 300, 350, 400, 450 or 500 μl. Preferably, the volume of the drug composition administered in step (b) is 100 μl. Larger volumes can increase the risk of retinal stretching, while smaller volumes are difficult to see.

The drug-free solution (i.e., the "solution" of step (a)) can be formulated similarly to the drug-free solution, as described below. A preferred drug-free solution is a balanced saline solution (BSS) or similar buffer solution that matches the pH and osmolality of the subretinal space.

Visualization of the retina during surgery

Under certain circumstances, such as during the final stage of retinal degeneration, the retina is difficult to identify because it is thin, transparent, and difficult to see against the background of the destroyed and heavily pigmented epithelium on which it is located. The use of a blue vital dye (e.g., Brilliant Peel®, Geuder; MembraneBlue-Dual®, Dore) can facilitate the identification of the hole in the retina made for the retinal detachment procedure (i.e., step (a) in the two-stage subretinal injection method of the invention), so that the drug can be administered through the same hole without the risk of back reflux into the vitreous cavity.

The use of blue vital dye also allows identification of any areas of the retina where there is a thickened internal limiting membrane or epiretinal membrane, as injection through either of these structures makes it difficult to gain clear access to the subretinal space. Additionally, contraction of either of these structures in the immediate postoperative period may result in stretching of the retinal inlet, which may result in drug leakage into the vitreous cavity.

Suprachoroidal injection

A polynucleotide or vector of the invention can be delivered into the suprachoroidal space using an ab externo approach that utilizes a microcatheter (see, e.g., Peden et al. (2011) PLoS One 6(2):e17140). In this method, a limbal peritomy of the conjunctiva is performed to expose the bare sclera, followed by a sclerotomy to expose the bare choroid. A microcatheter (e.g., the iTrack 250A from iScience Interventional, optionally connected to an illumination system such as the iLumin laser diode microillumination system (iScience Interventional)) is inserted into the suprachoroidal space and advanced in a posterior direction toward the optic disc. Once the tip of the microcatheter has been advanced to the desired position, the injection of the polynucleotide or vector forms a bubble within the retina and choroid.

Thus, in some embodiments, the polynucleotide or vector is delivered suprachoroidally by a method comprising (i) inserting a microcatheter into the suprachoroidal space; (ii) advancing the microcatheter in said space until the tip is near the affected area of the retina; and (iii) injecting the polynucleotide or vector from the tip of the microcatheter to create a bleb.

In some embodiments, the above insertion procedures are performed directly by the robot.

PHARMACEUTICAL COMPOSITIONS AND INJECTION SOLUTIONS

The medicinal products, for example the polynucleotides or vectors of the invention, can be included in pharmaceutical compositions. These compositions can contain, in addition to the medicinal product, a pharmaceutically acceptable carrier, diluent, excipient, buffer, stabilizer or other materials well known in the art. Such materials should be non-toxic and should not interfere with the effectiveness of the active ingredient. The exact nature of the carrier or other material can be determined by a person skilled in the art according to the route of administration, for example by subretinal, direct retinal, suprachoroidal or intravitreal injection.

The pharmaceutical composition is usually in liquid form. Liquid pharmaceutical compositions usually include a liquid carrier such as water, petroleum, animal or vegetable oils, mineral oil or synthetic oil. Physiological saline, magnesium chloride, dextrose or other saccharide solution or glycols such as ethylene glycol, propylene glycol or polyethylene glycol may be included. In some cases, a surfactant such as pluronic acid (PF68) 0.001% may be used.

For intralesional injection, the active ingredient may be in the form of an aqueous solution that is pyrogen-free and has a suitable pH, isotonicity and stability. Suitable solutions can be prepared by a skilled artisan using, for example, isotonic vehicles such as sodium chloride injection, Ringer's injection or Ringer's lactate injection. Preservatives, stabilizers, buffers, antioxidants and/or other additives may be included as needed.

For sustained release, the drug may be included in a pharmaceutical composition that is formulated for slow release, such as in microcapsules formed from biocompatible polymers or in liposomal carrier systems according to methods known in the art.

METHOD OF TREATMENT

It should be appreciated that all references herein to treatment include curative, palliative and prophylactic treatment; although in the context of the invention references to prophylaxis are more often associated with prophylactic treatment. Treatment may also include stopping the progression of a disease.

Preferably, the treatment of mammals, especially humans, is included. However, both human and veterinary treatment are included within the scope of the invention.

VARIANTS, DERIVATIVES, ANALOGUES, HOMOLOGUES AND FRAGMENTS

In addition to the specific proteins and nucleotides mentioned herein, the invention also includes the use of variants, derivatives, analogs, homologues and fragments thereof.

In the context of the invention, a variant of any given sequence is a sequence in which a specific sequence of residues (whether amino acid or nucleic acid residues) has been modified in such a way that the polypeptide or polynucleotide in question substantially retains its function. A sequence variant may be obtained by adding, deleting, substituting, modifying, replacing and/or altering at least one residue present in a natural protein.

The term "derivative" as used herein in relation to proteins or polypeptides of the invention includes any substitution, alteration, modification, replacement, deletion and/or addition of one (or more) amino acid residues from or to a sequence, providing that the resulting protein or polypeptide substantially retains at least one of its endogenous functions.

The term "analog" as used herein with respect to polypeptides or polynucleotides includes any mimetic, that is, a chemical compound that possesses at least one of the endogenous functions of the polypeptides or polynucleotides that it mimics.

Typically, amino acid substitutions may be made, for example, from 1, 2 or 3 to 10 or 20 substitutions, provided that the modified sequence substantially retains the desired activity or capability. Amino acid substitutions may include the use of non-naturally occurring analogs.

The proteins used in the invention may also have deletions, insertions or substitutions of amino acid residues that cause silent changes and result in a functionally equivalent protein. Intentional amino acid substitutions may be made based on similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity and/or amphipathic nature of the residues, as long as the endogenous function is maintained. For example, negatively charged amino acids include aspartic acid and glutamic acid; positively charged amino acids include lysine and arginine; and amino acids with uncharged polar head groups having similar hydrophilicity values include asparagine, glutamine, serine, threonine and tyrosine.

Conservative substitutions can be made, for example, according to the table below. Amino acids in the same block in the second column and preferably in the same row in the third column can be substituted for each other:

ALIPHATIC Non-polar G A P I L V Polar - uncharged C S T M N Q Polar-charged D E K R H AROMATIC F W Y

As used herein, the term "homolog" means an entity that has a certain homology with a wild-type amino acid sequence and a wild-type nucleotide sequence. The term "homology" may be equated with "identity".

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

A homologous sequence may comprise a nucleotide sequence which may be at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85% or 90% identical, preferably at least 95% or 97% or 99% identical to the sequence in question. Although homology may also be considered in terms of similarity, in the context of the invention it is preferable to express homology in terms of sequence identity.

Preferably, reference to a sequence that has percent identity to any of the SEQ ID NOs detailed herein refers to a sequence that has the claimed percent identity over the entire length of said SEQ ID NO.

Homology comparisons can be made by eye or, more commonly, using readily available sequence comparison programs. These commercially available computer programs can calculate the percent homology or identity between two or more sequences.

Percent homology can be calculated from adjacent sequences, i.e. one sequence is aligned with another sequence, and each amino acid in one sequence is compared directly to the corresponding amino acid in the other sequence, one residue at a time. This is called a "gapless" alignment. Typically, such gapless alignments are performed only for a relatively small number of residues.

Although this is a very simple and consistent method, it does not take into account that, for example, in an otherwise identical pair of sequences, a single insertion or deletion in the nucleotide sequence can cause the alignment of subsequent codons to be disrupted, thus potentially leading to a significant reduction in the percentage of homology when performing a global alignment. Consequently, most sequence comparison methods are designed to produce optimal alignments that account for possible insertions and deletions without unduly reducing the overall homology score. This is achieved by inserting "gaps" into the sequence alignment to attempt to maximize local homology.

However, these more sophisticated methods assign "gap penalties" to each gap that occurs in the alignment, so that for the same number of identical amino acids, the sequence alignment with the fewest possible gaps, reflecting a higher degree of relatedness between the two sequences being compared, will score higher than one with many gaps. Typically, "affine gap costs" are used, which impose a relatively high penalty for the existence of a gap and a smaller penalty for each subsequent residue in the gap. This is the most commonly used gap scoring system. High gap penalties will, of course, result in an optimized alignment with fewer gaps. Most alignment programs allow you to change the gap penalties. However, when using such software to compare sequences, it is preferable to use the default values. For example, when using the GCG Wisconsin Bestfit package, the default gap penalties for amino acid sequences are -12 for a gap and -4 for each extension.

Therefore, in order to calculate the maximum percentage homology, it is first necessary to produce an optimal alignment taking into account gap penalties. A suitable computer program for performing such an alignment is the GCG Wisconsin Bestfit package (University of Wisconsin, USA; Devereux et al. (1984) Nucleic Acids Res. 12: 387). Examples of other software that can perform sequence comparisons include, but are not limited to, the BLAST package (see Ausubel et al. (1999) ibid-Ch. 18), FASTA (Atschul et al. (1990) J. Mol. Biol. 403–410), and the GENEWORKS comparison toolbox. Both BLAST and FASTA are available for offline and online searching (see Ausubel et al. (1999) ibid, pages 7–58 to 7–60). However, for some applications, the GCG Bestfit program may be preferable. Another tool called BLAST 2 Sequences is also available for comparing protein and nucleotide sequences (see FEMS Microbiol. Lett. (1999) 174: 247–50; FEMS Microbiol. Lett. (1999) 177: 187–8).

Although the final percentage of homology can be measured in terms of identity, the alignment process itself is not typically based on an all-or-none comparison of pairs. Instead, a scaled similarity scoring matrix is typically used, which assigns scores to each pairwise comparison based on chemical similarity or evolutionary distance. An example of such a commonly used matrix is the BLOSUM62 matrix, the default matrix for the BLAST suite of programs. The GCG Wisconsin programs typically use either the publicly available defaults or a custom character comparison table if available (see the user manual for more information). For some applications, it is preferable to use the publicly available defaults for the GCG package or, in the case of other software, a default matrix such as BLOSUM62.

Once the software has produced an optimal alignment, the percentage of homology, preferably the percentage of sequence identity, can be calculated. The software usually does this as part of the sequence comparison and generates a numerical result.

"Fragments" of full-length complement factor I or FHL1 are also variants, and the term generally refers to a selected region of a polypeptide or polynucleotide that is of interest either functionally or, for example, in an assay. Thus, "fragment" refers to an amino acid or nucleic acid sequence that is part of a full-length polypeptide or polynucleotide.

Such variants can be produced using standard recombinant DNA techniques such as site-directed mutagenesis. When insertions are to be made, synthetic DNA encoding the insert can be produced, along with 5' and 3' flanking regions corresponding to the natural sequence on either side of the insertion site. The flanking regions will contain convenient restriction sites corresponding to sites in the natural sequence, so that the sequence can be cut with suitable enzyme(s) and the synthetic DNA ligated into the cut. The DNA is then expressed in accordance with the invention to produce the encoded protein. These methods are merely illustrative of numerous standard techniques known in the art for manipulating DNA sequences, and other known techniques may also be used.

A person skilled in the art will understand that they can combine all the features of the invention disclosed in this document without departing from the disclosed scope of the invention.

Preferred features and embodiments of the invention will now be described by means of non-limiting examples.

The practice of the present invention will employ, unless otherwise indicated, conventional techniques of chemistry, biochemistry, molecular biology, microbiology, and immunology, which are within the skill of the art. Such techniques are described in the literature. See, for example, Sambrook, J., Fritsch, E. F. and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press; Ausubel, F. M. et al. (1995 and periodic supplements) Current Protocols in Molecular Biology, Ch. 9, 13 and 16, John Wiley &Sons; Roe, B., Crabtree, J. and Kahn, A. (1996) DNA Isolation and Sequencing: Essential Techniques, John Wiley &Sons; Polak, J. M. and McGee, J. O'D. (1990) In Situ Hybridization: Principles and Practice, Oxford University Press; Gait, M.J. (1984) Oligonucleotide Synthesis: A Practical Approach, IRL Press; and Lilley, D.M. and Dahlberg, J.E. (1992) Methods in Enzymology: DNA Structures Part A: Synthesis and Physical Analysis of DNA, Academic Press. Each of these general texts is incorporated herein by reference.

EXAMPLES

EXAMPLE 1

Codon optimization

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

Table 1

RC number Transgene Codon optimization tool Basic or manually edited AAV size RC128 CFI GeneArt (ThermoFisher) Base 3948 p.o. RC129 CFI GeneArt (ThermoFisher) Hand edited 3948 p.o. RC130 CFI GenScript Base 3948 p.o. RC131 CFI GenScript Hand edited 3948 p.o. RC132 CFI IDT Base 3948 p.o. RC133 CFI IDT Hand edited 3948 p.o. RC134 CFI JCat Base 3948 p.o. RC135 CFI JCat Hand edited 3948 p.o. RC136 CFI COOL Base 3948 p.o. RC137 CFI COOL Hand edited 3948 p.o. RC138 FHL-1 GeneArt (ThermoFisher) Base 3546 p.o. RC139 FHL-1 GeneArt (ThermoFisher) Hand edited 3546 p.o. RC140 FHL-1 GenScript Base 3546 p.o. RC141 FHL-1 GenScript Hand edited 3546 p.o. RC142 FHL-1 IDT Base 3546 p.o. RC143 FHL-1 IDT Hand edited 3546 p.o. RC144 FHL-1 JCat Base 3546 p.o. RC145 FHL-1 JCat Hand edited 3546 p.o. RC146 FHL-1 COOL Base 3546 p.o. RC147 FHL-1 COOL Hand edited 3546 p.o.

For "basic" codon optimization, the CFI or FHL-1 sequence was entered into 5 online codon optimization tools:

1. GeneArt (https://www.thermofisher.com/uk/en/home/life-science/cloning/gene-synthesis/geneart-gene-synthesis/geneoptimizer.html)

2. GenScript (https://www.genscript.com/quick_order/gene_services_gene_synthesis)

3. IDT (https://eu.idtdna.com/CodonOpt)

4. JCat (http://www.jcat.de/)

5. COOL

(http://cool.syncti.org/setup_input_sequence_create_wf1.php?=Start+Using+Codon+ Optimization+On-Line%3E%3E%3E)

All online tools used the standard human genetic code.

For online tools 1–4 described above, one sequence was created for each online tool.

For online tool 5, default settings were used and Homo sapiens was selected as the target expression host. Additionally, 39 genes that are highly expressed in the RPE (Table 2) were entered into the tool (based on Table 4 of Booij, JC et al. (2010) PLoS One 5:e9341).

Table 2

Gene symbol Genbank ID HUGO C6orf105 NM_032744 ADTRP BEST1 NM_004183 BEST1 TMEM27 NM_020665 TMEM27 LRP8 NM_004631 LRP8 LGI1 NM_005097 LGI1 FAM40B AB032996 STRIP2 ERMN AB033015 ERMN LRAT NM_004744 LRAT RLBP1 NM_000326 RLBP1 DUSP6 NM_001946 DUSP6 RBP1 NM_002899 RBP1 SLC16A3 NM_004207 SLC16A3 WFDC1 NM_021197 WFDC1 KIAA0953 AF131834 EFR3B CA14 NM_012113 CA14 RGR NM_002921 RGR STRA6 NM_022369 STRA6 RDH5 NM_002905 RDH5 BMP4 NM_001202 BMP4 CXCL14 NM_004887 CXCL14 LHX2 NM_004789 LHX2 C1QTNF5 NM_015645 C1QTNF5 SLC6A20 NM_020208 SLC6A20 SLC16A8 NM_013356 SLC16A8 CDH3 NM_001793 CDH3 FRZB NM_001463 FRZB SERPINF1 NM_002615 SERPINF1 SPOCK1 NM_004598 SPOCK1 LMO1 NM_002315 LMO1 RDH11 NM_016026 RDH11 SFRP5 NM_003015 SFRP5 SGK1 NM_005627 SGK1 KRT18 NM_000224 KRT18 EZR NM_003379 EZR DHCR7 NM 001360 DHCR7 ITGAV NM_002210 ITGAV GALNT11 NM 022087 GALNT11 PCP4 NM 006198 PCP4 BASP1 NM_006317 BASP1

Online tool 5 generated 70 optimized sequences for CFI and 55 for FHL-1, with the highest ranking sequence used.

For manual codon optimization, the five codon-optimized CFI and FHL-1 core sequences generated above were manually optimized to eliminate cryptic splice sites, miRNA binding sites, remove tandem repeat codons, and check for GC content.

Removing hidden splice site

Cryptococcal splice sites were identified using the www.Fruitfly.org tool. A threshold of 0.4 was used for the analysis, but only sequences with a score > 0.75 were modified.

Splice sites were removed by altering the donor site GT or acceptor site AG when possible. When this was not possible (e.g., for valine-encoding sequences), the 5' adjacent base was altered.

All modified sequences were then analyzed using the www.Fruitfly.org tool to confirm that all splice sites were either removed or reduced to a threshold below 0.75.

Deletion of the microRNA binding site

MicroRNAs were identified using the www.Genecards.org tool.

The following miRNA binding sites were identified for CFI: hsa-mir-335-5p, hsa-mir-181a-5p, hsa-mir-26b-5p.

The following miRNA binding sites have been identified for FHL-1 (based on the complement factor H, CFH, sequence): hsa-mir-146a-5p.

Each codon-optimized sequence (after splice site removal if necessary) was then run through the STarMir tool (http://sfold.wadsworth.org/cgi-bin/starmirtest2.pl) to check if miRNA binding sites were still present. Any miRNA sites that were identified with a logistic probability > 0.75 were changed.

Removal of tandem repeat codons

All sequences were manually checked for tandem repeat codons. Where these were found, the second codon was replaced with the next most frequently used codon in Homo sapiens (using the SnapGene codon usage table).

The wild-type and codon-optimized sequences are detailed below:

GT005: Wild-type CFI sequence:

ATGAAGCTTCTTCATGTTTTCCTGTTATTTCTGTGCTTCCACTTAAGGTTTTGCAAGGTCACTTATACATCTCAAGAGGATCTGGTGGAGAAAAAGTGCTTAGCAAAAAAATATACTCACCTCTCCTGCGATAAAGTCTTCTGCCAGCCATGGCAGAGATGCATTGAGGGCACCTGTGTTTGTAAACTACCGTATCAGTGCCCAAAGAATGGCACTGCAGTGTGTGCAACTAACAGGAAGCTTCCC AACATACTGTCAACAAAAGAGTTTGGAATGTCTTCATCCAGGGACAAAGTTTTTAAATAACGGAACATGCACAGCCGAAGGAAAGTTTAGTGTTTCCTTGAAGCATGGAAATACAGATTCAGAGGGAATAGTTGAAGTAAAACTTGTGGACCAAGATAAGACAATGTTCATATGCAAAAGCAGCTGGAGC ATGAGGGAAGCCAACGTGGCCTGCCTTGACCTTGGGTTTCAACAAGGTGCTGATACTCAAAGAAGGTTTAAGTTGTCTGATCTCTCTATAAATTCCACTGAATGTCTACATGTGCATTGCCGAGGATTAGAGACCAGTTTGGCTGAATGTACTTTTACTAAGAGAAGAACTATGGGTTACCAGGATTTCGCTGATGTGGTTTGTTATACACAGAAAGCAGATTCTCCAATGGATGACTTCTT TCAGTGTGTGAATGGGAAATACATTTCTCAGATGAAAGCCTGTGATGGTATCAATGATTGTGGAGACCAAAGTGATGAACTGTGTTGTAAAGCATGCCAAGGCAAAGGCTTCCATTGCAAATCGGGTGTTTGCATTCCAAGCCAGTATCAATGCAATGGTGAGGTGGACTGCATTACAGGGGAAGATGAAGTTGGC TGTGCAGGCTTTGCATCTGTGGCTCAAGAAGAAACAGAAATTTTGACTGCTGACATGGATGCAGAAAGAAGACGGATAAAATCATTATTACCTAAACTATCTTGTGGAGTTAAAAACAGAATGCACATTCGAAGGAAACGAATTGTGGGAGGAAAGCGAGCACAACTGGGAGACCTCCCATGGCAGGTGGCAATTAAGGATGCCAGTGGAATCACCTGTGGGGGAATTTATATTGGTGGCTGTT GGATTCTGACTGCTGCACATTGTCTCAGAGCCAGTAAAACTCATCGTTACCAAATATGGACAACAGTAGTAGACTGGATACACCCCGACCTTAAACGTATAGTAATTGAATACGTGGATAGAATTATTTTCCATGAAAACTACAATGCAGGCACTTACCAAAATGACATCGCTTTGATTGAAATGAAAAAAGAC GGAAACAAAAAAGATTGTGAGCTGCCTCGTTCCATCCCTGCCTGTGTCCCCTGGTCTCCTTACCTATTCCAACCTAATGATACATGCATCGTTTCTGGCTGGGGACGAGAAAAAGATAACGAAAGAGTCTTTTTCACTTCAGTGGGGTGAAGTTAAACTAATAAGCAACTGCTCTAAGTTTTACGGAAATCGTTTCTATGAAAAAGAAATGGAATGTGCAGGTACATATGATGGTTCCATCGAT GCCTGTAAAGGGGACTCTGGAGGCCCCTTAGTCTGTATGGATGCCAACAATGTGACTTATGTCTGGGGTGTTGTGAGTTGGGGGGAAAACTGTGGAAAACCAGAGTTCCCAGGTGTTTACACCAAAGTGGCCAATTATTTTGACTGGATTAGCTACCATGTAGGAAGGCCTTTTATTTCTCAGTACAATGTATAA

(SEQ ID NO: 16)

RC001: Wild-type FHL-1 sequence:

ATGAGACTTCTAGCAAAGATTATTTGCCTTATGTTATGGGCTATTTGTGTAGCAGAAGATTGCAATGAACTTCCTCCAAGAAGAAATACAGAAATTCTGACAGGTTCCTGGTCTGACCAAACATATCCAGAAGGCACCCAGGCTATCTATAAATGCCGCCCTGGATATAGATCTCTTGGAAATAATAATGGTATGCAGGAAGGGAGAATGGGTTGCTCTTAATCCATTAAGGAAATGTCAGAAAAGGC CCTGTGGACATCCTGGAGATACTCCTTTTGGTACTTTTACCCTTACAGGAGGAAATGTGTTTGAATATGGTGTAAAAGCTGTGTATA CATGTAATGAGGGGTATCAATTGCTAGGTGAGATTAATTACCGTGAATGTGACACAGATGGATGGACCAATGATATTCCTATATGTGAAGTTGTGAAGTGTTTACCAGTGACAGCACCAGAGAATGGAAAAATTGTCAGTAGTGCAATGGAACCAGATCGGGAATACCATTTTGGACAAGCAGTACGGTTTGTATGTAACTCAGGCTACAAGATTGAAGGAGATGAAGAAATGCATTGTT CAGACGATGGTTTTTGGAGTAAAGAGAAACCAAAGTGTGTGGAAATTTCATGCAAATCCCCAGATGTTATAAATGGATCTCCTATATCTCAGAAGATT ATTTATAAGGAGAATGAACGATTTCAATATAAATGTAACATGGGTTATGAATACAGTGAAAGAGGAGATGCTGTATGCACTGAATCTGGATGGCGTCCGTTGCCTTCATGTGAAGAAAAATCATGTGATAATCCTTATATTCCAAATGGTGACTACTCACCTTTAAGGATTAAACACAGAACTGGAGATGAAATCACGTACCAGTGTAGAAATGGTTTTTATCCTGCAACCCGGGGAAATACAGCaAAA TGCACAAGTACTGGCTGGATACCTGCTCCGAGATGTACCTTGAAACCTTGTGATTATCCAGACATTAAACATGGAGGTCTATATCATG AGAATATGCGTAGACCATACTTTCCAGTAGCTGTAGGAAAATATTACTCCTATTACTGTGATGAACATTTTGAGACTCCGTCAGGAAGTTACTGGGATCACATTCATTGCACACAAGATGGATGGTCGCCAGCAGTACCATGCCTCAGAAAATGTTATTTTCCTTATTTGGAAAATGGATATAATCAAAATTATGGAAGAAAGTTTGTACAGGGTAAATCTATAGACGTTGCCTGCCATCCTGGCTACGCT CTTCCAAAAGCGCAGACCACAGTTACATGTATGGAGAATGGCTGGTCTCCTACTCCCAGATGCATCCGTGTCAGCTTTACCCTCTGA

(SEQ ID NO: 17)

RC128: CFI GeneArt- Basic:

ATGAAGCTGCTGCATGTGTTTCTGCTGTTCCTCTGCTTCCACCTGAGGTTCTGCAAAGTGACCTACACCAGCCAAGAGGACCTGGTGGAAAAGAAGTGCCTGGCCAAGTACACCCACCTGAGCTGCGACAAGGTGTTCTGCCAGCCTTGGCAGAGATGCATCGAGGGCACCTGTGTGTGCAAGCTGCCCTATCAGTGCCCCAAGAATGGCACAGCCGTGTGCGCCACCAACAGAAGA AGCTTCCCTACCTACTGCCAGCAGAAAAGCCTGGAATGTCTGCACCCCGGCACCAAGTTTCTGAACAACGGCACCTGTACCGCCGAGGGCAAGTTTAGCGTGTCCCTGAAGCACGGCAACACCGACTCTGAGGGCATCGTGGAAGTGAAGCTGGTGGACCAGGACAAGACCATGTTCATCTGCAAGAGCAGCTGGTCC ATGCGCGAGGCCAATGTGGCTTGTCTGGATCTGGGATTCCAGCAGGGCGCCGACACAGAGAAGATTCAAGCTGAGCGACCTGAGCATCAACAGCACCGAGTGCCTGCATGTGCACTGTAGAGGCCTGGAAACAAGCCTGGCCGAGTGCACCTTCACCAAGAGAAGGACCATGGGCTACCAGGACTTCGCCGACGTCGTGTGCTACACCCAGAAAGCCGACTCTCCCATGGACGATTTCTT CCAGTGCGTGAACGGCAAGTACATCAGCCAGATGAAGGCCTGCGACGGCATCAACGATTGCGGCGATCAGAGCGACGAGCTGTGCTGCAAAGCCTGTCAAGGCAAGGGCTTCCACTGCAAGTCCGGCGTGTGTATCCCTAGCCAGTACCAGTGCAATGGCGAGGTGGACTGTATCACCGGCGAGGATGAAGTGGGC TGTGCCGGATTTGCCACGTGGCCCAAGAGGAAACCGAGATCCTGACCGCCGATATGGACGCCGAGCGGCGGAGAATCAAAAGCCTGCTGCCTAAGCTGTCCTGCGGCGTGAAGAACCGGATGCACATCCGGCGCAAGAGAATCGTCGGAGGCAAAAGAGCACAGCTGGGCGATCTGCCTTGGCAAGTGGCCATCAAGGATGCCAGCGGCATCACATGTGGCGGCATCTACATCGGCGGCTGCTG GATTCTGACAGCCGCTCATTGTCTGCGGGCCAGCAAGACCCACCGGTATCAGATCTGGACCACGTGGTGGACTGGATTCACCCCGACCTGAAGCGGATCGTGATCGAGTACGTGGACCGGATCATCTTCCACGAGAACTACAACGCCGGCACCTACCAGAACGATATCGCCCTGATCGAGATGAAGAAGGAC GGGAACAAGAAGGACTGCGAGCTGCCTAGATCTATCCCCGCCTGTGTTCCTTGGAGCCCCTACCTGTTCCAGCCTAACGATACCTGCATCGTGTCCGGCTGGGGCAGAGAGAAGGATAACGAGAGGGTGTTCAGCCTGCAGTGGGGCGAAGTGAAACTGATCAGCAACTGCAGCAAGTTCTACGGCAACCGGTTCTACGAGAAAGAAATGGAATGCGCCGGCACATACGACGGCTCCATCGATGCCT GTAAAGGCGATTCTGGCGGCCCTCTCGTGTGCATGGATGCCAACAATGTGACCTACGTGTGGGGCGTCGTGTCCTGGGGAGAGAATTGTGGCAAGCCTGAGTTCCCCGGCGTGTACACCAAGGTGGCCAACTACTTCGACTGGATCAGCTACCACGTGGGCAGACCCTTTATCAGCCAGTACAACGTGTGA

(SEQ ID NO: 18)

RC129: CFI GeneArt-Hand Optimized:

ATGAAGCTGCTCCATGTGTTTCTGCTCTTCCTCTGCTTCCACCTGAGGTTCTGCAAAGTGACCTACACCAGCCAAGAGGACCTGGTGGAAAAGAAATGCCTGGCCAAGAAATACACCCACCTGAGCTGCGACAAGGTGTTCTGCCAGCCTTGGCAGAGATGCATCGAGGGCACCTGTGTGTGCAAGCTGCCCTATCAGTGCCCCAAGAATGGCACAGCCGTGTGCGCTACAAACAGAAGGAGCTT CCCTACCTACTGCCAGCAAAAAAGCCTGGAGTGCCTGCACCCCGGCACCAAGTTTCTGAACAATGGCACCTGTACCGCCGAGGGCAAGTTTAGCGTGTCCCTGAAGCACGGCAACACCGACTCTGAGGGCATCGTGGAAGTGAAGCTGGTGGACCAGGACAAGACCATGTTCATCTGCAAGAGCTCC TGGTCCATGCGCGAGGCCAATGTGGCTTGTCTGGATCTGGGATTCCAGCAAGGCGCCGACACACAGAGAAGGTTCAAGCTGAGCGACCTGAGCATCAACAGCACCGAGTGCCTGCATGTGCACTGTAGAGGCCTGGAAACAAGCCTGGCCGAGTGCACCTTCACCAAGAGAAGGACCATGGGCTACCAGGACTTCGCCGACGTCGTGTGCTACACCCAGAAAGCCGACTCTCCCATGGACGA TTTCTTTCAGTGCGTGAACGGCAAGTACATCAGCCAGATGAAGGCCTGCGACGGCATCAACGATTGCGGCGATCAGAGCGACGAGCTGTGCTGTAAAGCCTGTCAAGGCAAGGGCTTCCACTGCAAGTCCGGCGTGTGTATCCCTAGCCAGTACCAGTGCAATGGCGAGGTGGACTGTATCACCGGCGAG GATGAAGTGGGCTGTGCCGGATTTGCCAGCGTGGCCCAAGAGGAAACCGAGATCCTGACCGCCGATATGGACGCCGAGCGGAGGAGAATCAAAAGCCTGCTCCCTAAGCTGTCCTGCGGCGTGAAGAACCGGATGCACATCCGGCGCAAGAGAATCGTCGGAGGCAAAAGAGCACAGCTGGGCGATCTGCCTTGGCAAGTGGCCATCAAGGATGCCAGCGGCATCACATGTGGCGGGATCTACATCG GCGGATGCTGGATTCTGACAGCCGCTCATTGTCTGCGGGCCAGCAAGACCCACCGGTATCAGATCTGGACCACAGTGGTCGACTGGATTCACCCCGACCTGAAGCGGATCGTGATCGAGTACGTGGACCGGATCATTTTCCACGAGAACTACAACGCCGGCACCTACCAGAACGATATCGCCCTG ATCGAGATGAAAAAGGACGGGAACAAGAAAGACTGCGAGCTGCCTAGATCTATCCCCGCCTGTGTTCCTTGGAGCCCCTACCTGTTCCAGCCTAACGATACCTGCATCGTGTCCGGCTGGGGCAGAGAGAAGGATAACGAGAGGGTGTTCAGCCTGCAGTGGGGCGAAGTGAAACTGATCAGCAACTGCAGCAAGTTCTACGGCAACCGGTTCTACGAGAAAGAAATGGAATGCGCCGGCACATACGACG GCTCCATCGATGCCTGTAAAGGCGATTCTGGCGGACCTCTCGTGTGCATGGATGCCAACAATGTGACCTACGTGTGGGGCGTCGTGTCCTGGGGAGAGAATTGTGGCAAGCCTGAGTTCCCCGGCGTGTACACCAAGGTGGCCAACTACTTCGACTGGATCAGCTACCACGTGGGCAGACCC TTTATCAGCCAGTACAACGTGTGA

(SEQ ID NO: 19)

RC130: CFI Genscript-Basic:

ATGAAGCTGCTGCATGTCTTTCTGCTGTTTCTGTGCTTCCATCTGAGGTTCTGCAAGGTCACTTACACTAGCCAGGAGGATCTGGTCGAGAAGAAGTGTCTGGCCAAGTACACACACCTGAGCTGCGACAAGGTGTTCTGTCAGCCTTGGCAGCGGTGCATCGAGGGCACCTGCGTGTGCAAGCTGCCTTACCAGTGCCCAAAGAACGGCACCGCCGTGTGCGCCACAAATCGGAGATCT TTTCCAACATATTGCCAGCAGAAGAGCCTGGAGTGTCTGCACCCCGGCACCAAGTTCCTGAACAATGGCACCTGCACAGCCGAGGGCAAGTTTTCTGTGAGCCTGAAGCACGGCAACACAGATAGCGAGGGCATCGTGGAGGTGAAGCTGGTGGACCAGGATAAGACCATGTTCATCTGTAAGAGCAGCTGGTCC ATGAGGGAGGCAAACGTGGCATGCCTGGATCTGGGATTCCAGCAGGGAGCAGACACACAGAGGCGCTTTAAGCTGTCCGACCTGTCTATCAATAGCACCGAGTGCCTGCACGTGCACTGTAGGGGCCTGGAGACATCCCTGGCAGAGTGCACCTTCACAAAAGCGGAAACAATGGGCTACCAGGACTTTGCCGACGTGGTGTGCTATACCCAGAAGGCCGATAGCCCTATGGACGATTTC TTTCAGTGCGTGAACGGCAAGTATATCTCCCAGATGAAGGCCTGCGACGGCATCAATGACTGTGGCGATCAGTCTGACGAGCTGTGCTGTAAGGCCTGTCAGGGCAAGGGCTTCCACTGCAAGAGCGGCGTGTGCATCCCTTCCCAGTACCAGTGCAACGGCGAGGTGGATTGTATCACAGGAGAGGACGAAGTGGGA TGCGCAGGATTTGCATCTGTGGCACAGGAGGAGACAGAGATCCTGACAGCCGACATGGATGCCGAGAGGCGCCGGATCAAGTCTCTGCTGCCTAAGCTGAGCTGTGGCGTGAAGAATCGGATGCACATCAGAAGGAAGCGCATCGTGGGAGGCAAGCGGGCCCAGCTGGGCGATCTGCCCTGGCAGGTGGCCATCAAGGACGCCTCTGGCATCACCTGCGGCGGCATCTACATCGGCGGCTGTT GGATTCTGACCGCAGCACACTGCCTGAGAGCAAGCAAGACACACAGGTATCAGATCTGGACCACAGTGGTGGATTGGATTCACCCAGACCTGAAGAGAATCGTGATCGAGTACGTGGATAGGATCATCTTCCACGAGAACTACAATGCCGGCACATATCAGAACGACATCGCCCTGATCGAGATGAAGAAGGAT GGCAATAAGAAGGACTGTGAGCTGCCCAGATCCATCCCTGCATGCGTGCCCTGGAGCCCCTATCTGTTCCAGCCCAACGATACCTGCATCGTGTCCGGATGGGGAAGGGAGAAGGACAATGAGCGGGTGTTTTCTCTGCAGTGGGGCGAGGTGAAGCTGATCTCCAACTGTTCTAAGTTCTACGGCAATAGGTTTTATGAGAAGGAGATGGAGTGCGCCGGCACCTACGATGGCAGCATCGAC GCCTGTAAGGGCGATTCCGGAGGACCACTGGTGTGCATGGACGCAAACAATGTGACATACGTGTGGGGAGTGGTGTCCTGGGGAGAGAACTGCGGCAAGCCAGAGTTTCCCGGCGTGTATACCAAGGTGGCCAATTATTTTGATTGGATTTCATACCATGTCGGGAGACCATTCATTAGTCAGTACAACGTGTGA

(SEQ ID NO:20)

RC131: CFI Genscript-Hand Optimized:

ATGAAGCTGCTCCATGTCTTTCTGCTCTTTCTGTGCTTCCATCTGAGGTTCTGCAAGGTCACTTACACTAGCCAGGAGGATCTGGTCGAGAAGAAATGTCTGGCCAAGAAATACACACACCTGAGCTGCGACAAGGTGTTCTGTCAGCCTTGGCAGCGGTGCATCGAGGGCACCTGCGTGTGCAAGCTGCCTTACCAGTGCCCAAAGAACGGCACCGCCGTGTGCGCCACAAATCGGAGATCTTTTC CAACATATTGCCAGCAAAAGAGCCTGGAGTGTCTGCACCCCGGCACCAAGTTCCTGAACAATGGCACCTGCACAGCCGAGGGCAAGTTTTCTGTGAGCCTGAAGCACGGCAACACAGATAGCGAGGGCATCGTGGAGGTGAAGCTGGTGGACCAGGATAAGACCATGTTCATCTGTAAGAGCTCCTGGTCC ATGAGGGAGGCAAACGTGGCATGCCTGGATCTGGGATTCCAGCAAGGAGCAGACACACAGAGGCGCTTTAAGCTGTCCGATCTGAGTATCAATAGCACCGAGTGCCTGCACGTGCACTGTAGGGGCCTGGAGACATCCCTGGCAGAGTGCACCTTTCACAAAGCGGAAACAATGGGCTACCAGGACTTTGCCGACGTGGTCTGCTATACCCAGAAGGCCGATAGCCCTATGGACGATTTC TTTCAGTGCGTGAACGGCAAGTATATCTCCCAGATGAAGGCCTGCGACGGCATCAATGACTGTGGCGATCAGTCTGACGAGCTGTGCTGTAAGGCCTGTCAGGGCAAGGGCTTCCACTGCAAGAGCGGCGTGTGCATCCCTTCCCAGTACCAGTGCAACGGCGAGGTGGATTGTATCACAGGAGAGGACGAAGTGGGA TGCGCAGGATTTGCATCTGTGGCACAGGAGGAAACAGAGATCCTGACAGCCGACATGGATGCCGAGAGGCGCCGGATCAAGTCTCTGCTCCCTAAGCTGAGCTGTGGCGTGAAGAATCGGATGCACATCAGAAGGAAGCGCATCGTGGGAGGCAAGCGGGCCCAGCTGGGCGATCTGCCCTGGCAGGTGGCCATCAAGGACGCCTCTGGCATCACCTGCGGCGGGATCTACATCGGCGGATGTT GGATTCTGACCGCAGCCCACTGCCTGAGAGCAAGCAAGACACACAGATATCAGATCTGGACCACAGTGGTCGATTGGATTCACCCAGACCTGAAGAGAATCGTGATCGAGTACGTGGATAGGATCATTTTCCACGAGAATTACAATGCTGGCACATATCAGAATGATATCGCTCTCATCGAGATGAAGAAAGAT GGCAATAAGAAAGACTGTGAGCTGCCCAGATCCATCCCTGCATGCGTGCCCTGGAGCCCCTATCTGTTCCAGCCCAACGATACCTGCATCGTGTCCGGATGGGGAAGGGAGAAGGACAATGAGCGGGTGTTTTCTCTGCAGTGGGGCGAGGTGAAGCTGATCTCCAACTGTTCTAAGTTCTACGGCAATAGGTTTTATGAGAAGGAGATGGAGTGCGCCGGCACCTACGATGGCAGCATCGAC GCCTGTAAGGGCGATTCCGGAGGCCCACTGGTGTGCATGGACGCAAACAATGTGACATACGTGTGGGGAGTGGTCTCCTGGGGAGAGAACTGCGGCAAGCCAGAGTTTCCCGGCGTGTATACCAAGGTGGCCAATTATTTTGATTGGATTTCATACCATGTCGGGAGACCATTCATTAGTCAATACAACGTTTGA

(SEQ ID NO: 21)

RC132: CFI IDT-Basic:

ATGAAGCTCCTCCACGTCTTCTTGTTGTTTCTCTGTTTCCACCTGAGATTTTGCAAAGTAACTTACACCAGTCAAGAAGACTTGGTCGAGAAGAAGTGTCTCGCCAAAAAGTATACTCACCTGAGCTGTGATAAAGTGTTCTGTCAGCCGTGGCAGCGCTGCATTGAGGGTACATGTGTCTGTAAACTGCCTTATCAGTGTCCGAAGAACGGTACGGCTGTCTGTGCTACTAACAGACGG TCTTTTCCTACTTATTGCCAGCAGAAGAGTTTGGAATGTCTCCACCCTGGTACCAAGTTTCTCAACAATGGCACCTGTACTGCTGAAGGTAAATTCTCCGTCAGTCTCAAGCATGGTAACACTGACAGTGAAGGGATAGTAGAGGTAAAGTTGGTTGACCAGGACAAGACGATGTTCATATGCAAGTCAAGCTGGTCC ATGCGCGAGGCGAATGTCGCTTGTCTTGATTTGGGCTTCCAGCAAGGGGCAGACACACAGAGAAGATTCAAATTGAGCGACCTGAGTATAAATTCAACCGAGTGCCTCCATGTACATTGCAGAGGGCTCGAGACTTCACTTGCCGAATGTACATTTACGAAGAGGCGGACTATGGGATATCAGGACTTTGCCGACGTAGTATGTTATACTCAGAAAGCAGACAGTCCTATGGATGACTTTTTCCAA TGCGTCAACGGCAAATACATCAGTCAAATGAAAGCGTGCGACGGTATCAACGATTGTGGTGACCAGTCTGATGAGCTTTGCTGTAAAGCATGTCAAGGAAAGGGGTTCCATTGCAAGAGTGGTGTATGTATTCCCTCACAATATCAGTGCAATGGGGAAGTCGATTGCATAACAGGTGAGGATGAGGTGGGC TGCGCGGGATTTGCTTCTGTGGCGCAAGAGGAGACTGAGATCCTTACAGCGGATATGGACGCCGAACGAAGACGCATCAAATCTCTCCTTCCCAAACTTTCATGCGGCGTCAAAACCGAATGCATATACGCAGGAAGAGAATTGTTGGGGGAAAGCGGGCACAGCTGGGCGACCTCCCCTGGCAAGTTGCAATAAAGGATGCAAGTGGGATAACGTGCGGGGGCATCTACATCGGGGGGTGCTGGAT CTTGACTGCCGCCCACTGTCTTAGAGCCTCTAAGACCCATAGGTACCAAATCTGGACAACTGTAGTTGACTGGATACATCCGGACCTTAAACGCATAGTTATTGAATACGTTGACCGCATAATATTTCATGAGAATTATAACGCGGGTACCTACAGAATGACATCGCCCTCATCGAGATGAAAAAAGAC GGGAATAAAAAGGACTGCGAGCTGCCGCGCTCTATACCTGCGTGTGTCCCCTGGAGTCCTTATCTTTTCCAACCTAACGATACGTGTATAGTGAGCGGCTGGGGCCGGGAGAAGGACAATGAACGAGTTTTTTCCTTGCAATGGGGAGAAGTGAAGCTTATTTCCAATTGTTCAAAGTTTTATGGAAATAGATTTTATGAAAAAGAAATGGAGTGTGCGGGCACTTATGACGGGTCAATTG ATGCTTGCAAAGGTGATAGCGGGGGCCCACTTGTCTGCATGGACGCTAACAACGTGACTTATGTGTGGGGTGTTGTGTCCTGGGGCGAAAACTGTGGCAAGCCCGAGTTTCCCGGCGTATACACCAAAGTAGCTAATTATTTCGACTGGATTAGTTATCATGTTGGGCGGCCATTTATATCCCAGTATAATGTCTAA

(SEQ ID NO: 22)

RC133: CFI IDT-Hand Optimized:

ATGAAGCTCCTGCACGTCTTCTTGCTGTTTCTCTGTTTCCACCTGAGATTTTGCAAAGTAACTTACACCAGTCAAGAAGACTTGGTCGAGAAGAAATGTCTCGCCAAAAAGTATACTCACCTGAGCTGTGATAAAGTGTTCTGTCAGCCGTGGCAGCGCTGCATTGAGGGTACATGTGTCTGTAAACTGCCTTATCAGTGTCCGAAGAACGGCACGGCTGTCTGTGCTACTAACAGACGGTC TTTTCCTACTTATTGCCAGCAAAAGAGTTTGGAATGTCTCCACCCTGGTACCAAGTTTCTCAACAATGGCACCTGTACTGCTGAAGGCAAATTCTCCGTCAGTCTCAAGCATGGTAACACTGATTCTGAAGGGATAGTAGAAGTAAAGTTGGTTGACCAGGACAAGACGATGTTCATATGCAAGTCAAGC TGGTCCATGCGCGAGGCGAATGTCGCTTGTCTTGATTTGGGCTTCCAGCAAGGGGCAGACACACAGAGAAGATTCAAATTGAGCGACCTGAGTATAAATTCAACCGAGTGCCTCCATGTACATTGCAGAGGGCTCGAGACTTCTCTTGCTGAGTGTACATTTACGAAGAGGCGGACTATGGGATATCAGGACTTTGCTGACGTAGTGTGTGTTATACTCAGAAAGCAGACAGTCCTATGGATGACTTTTT CCAATGCGTCAACGGCAAATACATCAGTCAAATGAAAGCGTGCGACGGTATCAACGATTGTGGTGACCAGTCTGATGAGCTTTGCTGTAAAGCATGTCAAGGAAAGGGGTTCCATTGCAAGAGTGGTGTATGTATTCCCTCACAATATCAGTGCAATGGGGAAGTCGATTGCATAACAGGCGAG GATGAGGTGGGCTGCGCGGGATTTGCTTCTGTGGCGCAAGAGGAAACTGAGATCCTTACAGCGGATATGGACGCCGAACGAAGACGCATCAAATCTCTCCTTCCCAAACTTTCATGCGGCGTCAAAACCGAATGCATATACGCAGGAAGAATTGTTGGGGGAAAGCGGGCACAGCTGGGCGACCTCCCCTGGCAAGTTGCAATAAAGGATGCAAGTGGGATAACGTGCGGGGGCATCTACATCGGG GGCTGCTGGATCTTGACTGCCGCTCACTGTCTTAGAGCCTCTAAGACCCATAGATACCAAATCTGGACAACTGTAGTTGACTGGATACATCCGGACCTTAAACGCATAGTTATTGAATACGTTGACCGCATAATCTTTCATGAGAATTATAACGCGGGCACATACCAAAATGACATCGCCCTG ATCGAGATGAAAAAGGACGGGAATAAAAAGGACTGCGAGCTGCCGCGCTCTATACCTGCGTGTGTCCCCTGGAGTCCTTATCTTTTCCAACCTAACGATACGTGTATAGTGAGCGGCTGGGGCCGGGAGAAGGACAATGAACGAGTTTTTTCCTTGCAATGGGGAGAAGTGAAGCTTATTTCCAATTGTTCAAAGTTTTATGGAAATAGATTTTATGAAAAAGAAATGGAGTGTGCGGGCACT TATGACGGGTCAATTGATGCTTGCAAAGGTGATAGCGGGGGCCCACTTGTCTGCATGGACGCTAACAATGTGACTTATGTGTGGGGTGTTGTGTCCTGGGGCGAAAACTGTGGCAAGCCCGAGTTTCCCGGCGTATACACCAAAGTAGCTAATTATTTCGACTGGATTAGTTATCATGTTGGGCGGCCA TTTATATCCCAGTATAATGTCTAA

(SEQ ID NO: 23)

RC134: CFI JCat-Basic:

ATGAAGCTGCTGCACGTGTTCCTGCTGTTCCTGTGCTTCCACCTGCGCTTCTGCAAGGTGACCTACACCAGCCAGGAGGACCTGGTGGAGAAGAAGTGCCTGGCCAAGTACACCCACCTGAGCTGCGACAAGGTGTTCTGCCAGCCCTGGCAGCGCTGCATCGAGGGCACCTGCGTGTGCAAGCTGCCCTACCAGTGCCCCAAGAACGGCACCGCCGTGTGCGCCACCAACCGCC GCAGCTTCCCCACCTACTGCCAGCAGAAGAGCCTGGAGTGCCTGCACCCCGGCACCAAGTTCCTGAACAACGGCACCTGCACCGCCGAGGGCAAGTTCAGCGTGAGCCTGAAGCACGGCAACACCGACAGCGAGGGCATCGTGGAGGTGAAGCTGGTGGACCAGGACAAGACCATGTTCATCTGCAAGAGCAGCTGGAGC ATGCGCGAGGCCAACGTGGCCTGCCTGGACCTGGGCTTCCAGCAGGGCGCCGACACCCAGCGCCGCTTCAAGCTGAGCGACCTGAGCATCAACAGCACCGAGTGCCTGCACGTGCACTGCCGCGGCCTGGAGACCAGCCTGGCCGAGTGCACCTTCACAAGCGCCGCACCATGGGCTACCAGGACTTCGCCGACGTGGTGTGCTACACCCAGAAGGCCGACAGCCCCATGGACGACTTCTT CCAGTGCGTGAACGGCAAGTACATCAGCCAGATGAAGGCCTGCGACGGCATCAACGACTGCGGCGACCAGAGCGACGAGCTGTGCTGCAAGGCCTGCCAGGGCAAGGGCTTCCACTGCAAGAGCGGCGTGTGCATCCCCAGCCAGTACCAGTGCAACGGCGAGGTGGACTGCATCACCGGCGAGGACGAGGTGGGC TGCGCCGGCTTCGCCAGCGTGGCCCAGGAGGAGACCGAGATCCTGACCGCCGACATGGACGCCGAGCGCCGCCGCATCAAGAGCCTGCTGCCCAAGCTGAGCTGCGGCGTGAAGAACCGCATGCACATCCGCCGCAAGCGCATCGTGGGCGGCAAGCGCCCAGCTGGGCGACCTGCCCTGGCAGGTGGCCATCAAGGACGCCAGCGGCATCACCTGCGGCGGCATCTACATCGGCGGCTGCTGGATC CTGACCGCCGCCCACTGCCTGCGCGCCAGCAAGACCCACCGCTACCAGATCTGGACCACCGTGGTGGACTGGATCCACCCCGACCTGAAGCGCATCGTGATCGAGTACGTGGACCGCATCATCTTCCACGAGAACTACAACGCCGGCACCTACCAGAACGACATCGCCCTGATCGAGATGAAGAAGGAC GGCAACAAGAAGGACTGCGAGCTGCCCCGCAGCATCCCCGCCTGCGTGCCCTGGAGCCCCTACCTGTTCCAGCCCAACGACACCTGCATCGTGAGCGGCTGGGGCCGCGAGAAGGACAACGAGCGCGTGTTCAGCCTGCAGTGGGGCGAGGTGAAGCTGATCAGCAACTGCAGCAAGTTCTACGGCAACCGCTTCTACGAGAAGGAGATGGAGTGCGCCGGCACCTACGACGGCAGCATCGACGC CTGCAAGGGCGACAGCGGCGGCCCCCTGGTGTGCATGGACGCCAACAACGTGACCTACGTGTGGGGCGTGGTGAGCTGGGGCGAGAACTGCGGCAAGCCCGAGTTCCCCGGCGTGTACACCAAGGTGGCCAACTACTTCGACTGGATCAGCTACCACGTGGGCCGCCCCTTCATCAGCCAGTACAACGTGTAA

(SEQ ID NO:24)

RC135: CFI JCat-Hand Optimized:

ATGAAGCTGCTCCACGTGTTCCTGCTCTTCCTGTGCTTCCACCTGCGCTTCTGCAAGGTGACCTACACCAGCCAGGAGGACCTGGTGGAGAAGAAATGCCTGGCCAAGAAATACACCCACCTGAGCTGCGACAAGGTGTTCTGCCAGCCCTGGCAGCGCTGCATCGAGGGCACCTGCGTGTGCAAGCTGCCCTACCAGTGCCCCAAGAACGGCACCGCCGTGTGCGCCACCAACCGCCGGA GCTTCCCCACCTACTGCCAGCAAAAGAGCCTGGAGTGCCTGCACCCCGGCACCAAGTTCCTGAACAATGGCACCTGCACCGCCGAGGGCAAGTTCAGCGTGAGCCTGAAGCACGGCAACACCGACAGCGAGGGCATCGTGGAGGTGAAGCTGGTGGACCAGGACAAGACCATGTTCATCTGCAAGAGCTCCTGGAGC ATGCGCGAGGCCAACGTGGCCTGCCTGGACCTGGGCTTCCAGCAAGGCGCCGACACCCAGCGCCGGTTCAAGCTGAGCGACCTGAGCATCAACAGCACCGAGTGCCTGCACGTGCACTGCCGCGGCCTGGAGACCAGCCTGGCCGAGTGCACCTTCACAAGCGCCGGACCATGGGCTACCAGGACTTCGCCGACGTGGTCTGCTACACCCAGAAGGCTGACTCTCCCATGGACGATTTCTTTC AGTGCGTGAACGGCAAGTACATCAGCCAGATGAAGGCCTGCGACGGCATCAACGACTGCGGCGACCAGAGCGACGAGCTGTGCTGTAAGGCCTGCCAGGGCAAGGGCTTCCACTGCAAGAGCGGCGTGTGCATCCCCAGCCAGTACCAGTGCAACGGCGAGGTGGACTGCATCACCGGCGAGGACGAGGTGGGC TGCGCCGGCTTCGCCAGCGTGGCCCAGGAGGAAACCGAGATCCTGACCGCCGACATGGACGCCGAGCGCAGAAGGATCAAGAGCCTGCTCCCCAAGCTGAGCTGCGGCGTGAAGAACCGCATGCACATCCGCAGAAAGCGCATCGTGGGCGGGAAGCGCCCAGCTGGGCGACCTGCCCTGGCAGGTGGCCATCAAGGACGCCAGCGGCATCACCTGCGGCGGAATCTACATCGGCGGGTGCTGG ATCCTGACCGCCGCTCACTGCCTGCGCGCCAGCAAGACCCACCGCTACCAGATCTGGACCACAGTGGTCGACTGGATCCACCCCGACCTGAAGCGCATCGTGATCGAGTACGTGGACCGCATCATTTTCCACGAGAACTACAACGCCGGCACCTACCAGAACGACATCGCCCTGATCGAGATGAAGAAAGAT GGAAACAAGAAAGACTGCGAGCTGCCCCGCAGCATCCCCGCCTGCGTGCCCTGGAGCCCCTACCTGTTCCAGCCCAACGACACCTGCATCGTGAGCGGCTGGGGCCGCGAGAAGGACAACGAGCGCGTGTTCAGCCTGCAGTGGGGCGAGGTGAAGCTGATCAGCAACTGCAGCAAGTTCTACGGCAACCGCTTCTACGAGAAGGAGATGGAGTGCGCCGGCACCTACGACGGCAGCATCGACGC CTGCAAGGGCGACAGCGGCGGGCCCCTGGTGTGCATGGACGCCAACAATGTGACCTACGTGTGGGGCGTGGTCAGCTGGGGCGAGAACTGCGGCAAGCCCGAGTTCCCCGGCGTGTACACCAAGGTGGCCAACTACTTCGACTGGATCAGCTACCACGTGGGCCGCCCCTTTATCTCTCAATACAACGTCTAA

(SEQ ID NO: 25)

RC136: CFI COOL-Base: See 10 above.

RC137: CFI COOL-Hand Optimized:

ATGAAACTGCTCCATGTCTTCCTCCTGTTCCTGTGCTTCCACCTCCGTTTCTGTAAAGTCACCTACACTAGCCAGGAGGATCTGGTGGAGAAGAAATGCCTGGCCAAGAAAATATACCCACCTGAGCTGCGACAAAGTGTTCTGCCAGCCCTGGCAACGCTGCATTGAAGGCACTTGTGTGTGCAAGCTGCCCTACCAGTGCCCCAAGAACGGCACGGCCGTGTGTGCCACCAACAGGAGAAGCT TCCCCACCTACTGCCAGCAAAAGAGCCTGGAATGCCTCCACCCTGGCACCAAGTTTCTGAACAATGGGACCTGCACAGCCGAGGGGAAATTCAGCGTCTCCCTCAAGCACGGCAATACAGACTCCGAGGGCATTGTGGAAGTGAAGCTGGTGGACCAGGACAAGACCATGTTCATCTGCAAAAGCTCCTGGTCC ATGCGGGAGGCCAATGTCGCCTGCCTGGACCTGGGCTTCCAGCAAGGCGCTGATACACAGCGCAGATTTAAACTCAGTGACCTCAGCATCAACAGCACTGAGTGTCTGCACGTGCACTGCCGGGGCCTGGAGACCAGCCTGGCTGAGTGCACCTTCACCAAGCGCAGGACCATGGGCTACCAGGATTTTTGCAGATGTGGTCTGCTACACCCAGAAGGCAGACAGCCCCATGGATGACTTCTT TCAGTGTGTCAATGGCAAGTACATTTCCCAGATGAAGGCTTGTGACGGGATCAATGATTGCGGGGATCAGAGCGATGAGCTCTGCTGTAAGGCCTGCCAAGGGAAGGGCTTTCACTGCAAGTCTGGGGTGTGCATCCCTTCTCAGTATCAGTGCAACGGAGAGGTGGACTGCATCACTGGGGAGGACGAGGTGGGC TGTGCTGGCTTCGCCTCTGTGGCCCAGGAGGAAACAGAGATCCTCACAGCTGACATGGATGCAGAGCGGAGGCGCATCAAGAGTCTGCTCCCAAAGCTCTCCTGCGGCGTTAAGAATCGCATGCACATCCGGAGGAAGCGGATCGTTGGAGGCAAACGGGCTCAGCTGGGGGACTTGCCGTGGCAGGTGGCCATCAAAGATGCCTCCGGAATCACCTGTGGTGGCATCTACATCGGCGGGGTG CTGGATCCTGACCGCCGCTCACTGCCTTCGGGCCAGCAAGACCCATCGCTACCAGATCTGGACCACAGTGGTCGATTGGATTCACCCCGACCTGAAGAGGATTGTCATTGAGTATGTCGACCGCATCATTTTCCATGAAAACTACAATGCCGGGACGTATCAGAACGACATCGCCCTCATCGAGATGAAGAAAGAT GGGAACAAGAAAGACTGTGAGCTGCCTCGCTCCATCCCCGCCTGTGTACCATGGTCTCCGTACCTGTTCCAGCCAAATGACACATGCATCGTGAGCGGCTGGGGCCGCGAAAGACAACGAGAGGGTCTTCTCCCTGCAGTGGGGTGAAGTCAAGCTGATCAGCAACTGCTCCAAGTTCTACGGCAACCGCTTCTATGAGAAGGAGATGGAGTGCGCCGGCACCTATGACGGCAGCATTGAC GCGTGCAAGGGAGACAGTGGGGGCCCCCTGGTCTGCATGGACGCCAACAATGTGACCTACGTGTGGGGAGTTGTGTCCTGGGGCGAGAACTGTGGCAAGCCTGAGTTCCCGGGCGTGTACACAAAGGTGGCAAACTATTTTGACTGGATCTCCTATCACGTTGGCAGGCCCTTCATTAGCCAGTATAATGTATAA

(SEQ ID NO: 26)

RC138: FHL-1 GeneArt- Basic:

ATGAGACTGCTGGCCAAGATCATCTGCCTGATGCTGTGGGCCATCTGCGTGGCCGAGGATTGCAATGAGCTGCCTCCTCGGAGAAACACCGAGATCCTGACAGGCTCTTGGAGCGACCAGACATACCCTGAGGGAACCCAGGCCATCTACAAGTGCAGACCCGGCTACAGAAGCCTGGGCAACATCATCATGGTCTGCCGGAAAGGCGAGTGGGTCGCCCTGAATCCTCTGCGGAAGTGCCAGAAAAG ACCCTGCGGACACCCTGGCGATACCCCTTTCGGAACCTTTACACTGACCGGCGGCAACGTGTTCGAGTACGGCGTGAAAGCCGTGTACA CCTGTAACGAGGGCTACCAGCTGCTGGGCGAGATCAACTACAGAGAGTGCGATACCGACGCTGGACCAACGACATCCCTATCTGCGAGGTGGTCAAGTGCCTGCCTGTGACAGCCCCTGAGAACGGCAAGATTGTGTCCAGCGCCATGGAACCCGACAGAGAGTACCACTTTGGCCAGGCCGTCAGATTCGTGTGCAACAGCGGCTACAAGATCGAGGGCGACGAGGAAATGCACTGCAGCG ACGATGGCTTCTGGTCCAAAGAAAAGCCTAAGTGCGTGGAAATCAGCTGCAAGAGCCCCGACGTGATCAACGGCAGCCCTATCAGCCAGAAGATT ATCTACAAAGAGAACGAGCGGTTCCAGTACAAGTGTAACATGGGCTACGAGTACAGCGAGAGGGGCGACGCCGTGTGTACAGAATCTGGATGGCGACCTCTGCCTAGCTGCGAGGAAAAGAGCTGCGACAACCCTTACATCCCCCAACGGCGACTACAGCCCACTGCGGATCAAACACAGAACCGGCGACGAGATCACCTACCAGTGCCGGAATGGCTTCTACCCTGCCACCAGAGGCAATACCGCCAAG TGTACAAGCACCGGCTGGATCCCTGCTCCTCGGTGTACACTGAAGCCCTGCGACTACCCCGATATCAAGCACGGCGGACTGTACCACG AGAACATGCGGAGGCCTTACTTCCCTGTGGCCGTGGGCAAGTACTACAGCTACTACTGCGACGAGCACTTCGAGACACCCAGCGGCAGCTACTGGGATCACATCCACTGTACCCAGGACGGCTGGTCACCAGCTGTGCCTTGCCTGAGAAAGTGCTACTTCCCCTACCTGGAAAACGGCTACAACCAGAACTACGGCCGGAAGTTCGTGCAGGGCAAGAGCATCGATGTGGCCTGCCATCCTGGATA CGCCCTGCCTAAGGCTCAGACCACCGTGACCTGCATGGAAAATGGCTGGTCCCCAACACCTCGGTGCATCCGGGTGTCCTTCACACTGTAA

(SEQ ID NO: 27)

RC139: FHL-1 GeneArt-Hand Optimized:

ATGAGACTGCTCGCCAAGATCATTTGCCTGATGCTGTGGGCCATCTGCGTGGCCGAGGATTGCAATGAGCTGCCTCCCCGGAGAAACACCGAGATCCTGACAGGCTCTTGGAGCGACCAGACATACCCTGAGGGAACCCAGGCCATCTACAAGTGCAGACCCGGCTACAGAAGCCTGGGCAACATCATTATGGTCTGCCGGAAAGGCGAGTGGGTCGCCCTGAATCCTCTGCGGAAGTGCCAGAAA AGACCCTGCGGACACCCTGGCGATACCCCTTTCGGAACCTTTACACTGACCGGCGGGAACGTGTTCGAGTACGGCGTGAAAGCCGTGTACA CCTGTAACGAGGGCTACCAGCTGCTCGGCGAGATCAACTACAGAGAGTGCGATACCGACGCTGGACCAACGACATCCCTATCTGCGAGGTGGTCAAGTGCCTGCCTGTGACAGCCCCTGAGAACGGCAAGATTGTGTCCAGCGCCATGGAACCCGACAGAGAGTACCACTTTGGCCAGGCCGTCAGATTCGTGTGCAACAGCGGCTACAAGATCGAGGGCGACGAGGAAATGCACTGCAGCG ACGATGGCTTCTGGTCCAAAGAAAAGCCTAAGTGCGTGGAAATCAGCTGCAAGAGCCCCGACGTGATCAACGGCAGCCCTATCAGCCAGAAGATT ATCTACAAAGAGAACGAGCGGTTCCAGTACAAGTGTAACATGGGCTACGAGTACAGCGAGAGGGGCGACGCCGTGTGTACAGAATCTGGATGGCGACCTCTGCCTAGCTGCGAGGAAAAGAGCTGCGACAACCCTTACATCCCCCAACGGCGACTACAGCCCACTGCGGATCAAACACAGAACCGGCGACGAGATCACCTACCAGTGCCGGAATGGCTTCTACCCTGCCACCAGAGGCAATACCGCCAAG TGTACAAGCACCGGCTGGATCCCTGCTCCTCGGTGTACACTGAAGCCCTGCGACTACCCCGATATCAAGCACGGCGGACTGTACCACG AGAACATGCGGAGGCCTTACTTCCCTGTGGCCGTGGGCAAGTACTATAGCTACTATTGCGACGAGCACTTCGAGACACCCAGCGGCAGCTACTGGGATCACATCCACTGTACCCAGGACGGCTGGTCACCAGCTGTGCCTTGCCTGAGAAAGTGCTACTTCCCCTACCTGGAAAACGGCTACAACCAGAACTACGGCCGGAAGTTCGTGCAGGGCAAGAGCATCGATGTGGCCTGCCATCCTGGATA CGCCCTGCCTAAGGCTCAGACCACAGTGACCTGCATGGAAAATGGCTGGTCCCCAACACCTCGGTGCATCCGGGTGTCCTTCACACTGTAA

(SEQ ID NO:28)

RC140: FHL-1 Genscript-Basic:

ATGCGGCTGCTGGCCAAGATCATCTGCCTGATGCTGTGGGCCATCTGCGTGGCCGAGGACTGTAACGAGCTGCCCCCTCGGAGAAATACAGAGATCCTGACCGGCTCTTGGAGCGATCAGACATATCCTGAGGGCACCCAGGCCATCTACAAGTGCAGGCCAGGCTATCGCTCCCTGGGCAACATCATCATGGTGTGCAGGAAGGGAGAGTGGGTGGCCCTGAATCCTCTGAGGAAGTGCCA GAAGAGGCCATGTGGACACCCAGGCGACACCCTTTTCGGCACCTTTACACTGACCGGCGGCAACGTGTTCGAGTACGGCGTGAAGGCCGTGTATA CATGCAACGAGGGCTACCAGCTGCTGGGCGAGATCAATTACAGAGAGTGTGACACAGATGGCTGGACCAACGATATCCCAATCTGCGAGGTGGTGAAGTGTCTGCCAGTGACCGCCCCCGAGAATGGCAAGATCGTGAGCTCCGCCATGGAGCCCGACAGGGAGTATCACTTCGGCCAGGCCGTGCGCTTCGTGTGCAACTCTGGCTACAAGATCGAGGGCGATGAGGAGATGCACTG TAGCGACGATGGCTTCTGGTCCAAGGAGAAGCCCAAGTGCGTGGAGATCAGCTGTAAGTCCCCAGACGTGATCAATGGCTCTCCCATCAGCCAGAAGATC ATCTATAAGGAGAACGAGAGGTTTCAGTACAAGTGCAATATGGGCTACGAGTATTCCGAGAGGGGCGATGCCGTGTGCACCGAGTCTGGCTGGAGACCACTGCCCTCCTGCGAGGAGAAGTCTTGTGACAACCCATATATCCCCAATGGCGATTACTCTCCCCCTGCGGATCAAGCACAGAACAGGCGACGAGATCACCTATCAGTGCCGGAACGGCTTCTACCCTGCCACAAGAGGCAATACCGCCA AGTGTACAAGCACCGGATGGATCCCTGCACCAAGGTGCACCCTGAAGCCTTGTGACTATCCAGATATCAAGCACGGCGGCCTGTATCACG AGAATATGAGGCGCCCTTACTTCCCAGTGGCCGTGGGCAAGTACTATAGCTACTATTGCGACGAGCACTTTGAGACCCCTTCCGGCTCTTACTGGGACCACATCCACTGTACACAGGATGGATGGTCCCCAGCAGTGCCTTGCCTGAGGAAGTGTTACTTCCCATATCTGGAGAACGGCTACAACCAGAATTATGGCCGCAAGTTTGTGCAGGGCAAGAGCATCGATGTGGCATGCCACCCAGGATA CGCACTGCCTAAGGCACAGACCACAGTGACATGCATGGAGAATGGCTGGTCTCCCACCCCTCGGTGTATCAGAGTGAGCTTTACACTGTGA

(SEQ ID NO: 29)

RC141: FHL-1 Genscript-Hand Optimized:

ATGCGGCTGCTCGCCAAGATCATTTGCCTGATGCTGTGGGCCATCTGCGTGGCCGAGGACTGTAACGAGCTGCCCCCTCGGAGAAATACAGAGATCCTGACCGGCTCTTGGAGCGATCAGACATATCCTGAGGGCACCCAGGCCATCTACAAGTGCAGGCCAGGCTATCGCTCCCTGGGCAACATCATTATGGTGTGCAGGAAGGGAGAGTGGGTGGCCCTGAATCCTCTGAGGAAGTGC CAGAAGAGGCCATGTGGACACCCAGGCGACACCCTTTTCGGCACCTTTACACTGACCGGCGGGAACGTGTTCGAGTACGGCGTGAAGGCCGTGTATA CATGCAACGAGGGCTACCAGCTGCTCGGCGAGATCAATTACAGAGAGTGTGACACAGATGGCTGGACCAACGATATCCCAATCTGCGAGGTGGTCAAGTGTCTGCCAGTGACCGCCCCCGAGAATGGCAAGATCGTGAGCTCCGCCATGGAGCCCGACAGGGAGTATCACTTCGGCCAGGCCGTGCGCTTCGTGTGCAACTCTGGCTACAAGATCGAGGGCGATGAGGAAATGCACTG TAGCGACGATGGCTTCTGGTCCAAGGAGAAGCCCAAGTGCGTGGAGATCAGCTGCAAGTCCCCAGACGTGATCAATGGCTCTCCCATCAGCCAGAAGATC ATTTATAAGGAGAACGAGAGGTTTCAGTACAAGTGCAATATGGGCTACGAGTATTCCGAGAGGGGCGATGCCGTGTGCACCGAGTCTGGCTGGAGACCACTGCCCTCCTGCGAGGAAAAGTCTTGTGACAACCCATATATCCCCAATGGCGATTACTCTCCCCCTGCGGATCAAGCACAGAACAGGCGACGAGATCACCTATCAGTGCCGGAACGGCTTCTACCCTGCCACAAGAGGCAATACCGCCAAG TGTACAAGCACCGGATGGATCCCTGCACCAAGGTGCACCCTGAAGCCTTGTGACTATCCAGATATCAAGCACGGCGGGCTGTATCACG AGAATATGAGGCGCCCTTACTTCCCAGTGGCCGTGGGCAAGTACTATAGCTACTATTGCGACGAGCACTTTGAGACCCCTTCCGGCTCTTACTGGGACCACATCCACTGTACACAGGATGGATGGTCCCCAGCAGTGCCTTGCCTGAGGAAGTGTTACTTCCCATATCTGGAGAACGGCTACAACCAGAATTATGGCCGCAAGTTTGTGCAGGGCAAGAGCATCGATGTGGCATGCCACCCAGGATA CGCACTGCCTAAGGCACAGACCACAGTGACATGCATGGAGAATGGCTGGTCTCCCACCCCTCGGTGTATCAGAGTGAGCTTTACACTGTGA

(SEQ ID NO: 30)

RC142: FHL-1 IDT-Basic:

ATGAGACTGCTTGCGAAAATTATATGCCTGATGCTTTGGGCTATTTGCGTTGCGGAAGACTGTAACGAACTCCCGCCCCGCCGAAATACAGAGATCCTCACAGGCAGTTGGAGCGACCAAACGTACCCTGAAGGTACGCAGGCCATATATAAGTGTAGGCCAGGCTACAGATCACTTGGTAACATAATAATGGTATGTCGGAAAGGAGAGTGGGTCGCTCTCAACCCTCTTAGGAAATGTCAAAAAAGACC CTGTGGGCATCCGGGAGATACGCCTTTCGGGACATTCACTCTCACGGGCGGAAACGTATTCGAATATGGCGTGAAGGCAGTGTATA CCTGCAATGAAGGGTATCAGCTGCTTGGGGAAATTAATTATAGGGAATGTGACACGGATGGGTGGACAAACGATATTCCAATATGCGAAGTAGTTAAATGCCTGCCCGTTACTGCACCGGAGAATGGCAAAATAGTCAGTAGTGCAATGGAGCCGGATCGCGAGTATCATTTTGGTCAGGCCGTGCGGTTCGTATGTAATTCTGGGTACAAGATCGAAGGTGACGAAGAGATGCATTGCTCA GATGACGGCTTTTGGAGTAAAGAAAAGCCTAAGTGTGTTGAAATCAGCTGTAAGAGTCCAGACGTGATTAACGGTTCCCCGATCCTCTCAGAAGATA ATTTACAAGGAAAACGAACGATTCCAATATAAGTGTAACATGGGCTACGAGTATTCCGAGCGAGGTGACGCAGTATGTACGGAAAGCGGGTGGCGACCTCTGCCCTCCTGCGAGGAAAAGAGCTGTGATAATCCGTATATCCCCAACGGTGACTATAGCCCACTGCGCATAAAACATCGGACGGGAGATGAGATTACATACCAATGCCGCAATGGTTTTTACCCCGCCACCCGAGGGAACACGGCAAAGT GCACTTCTACGGGGTGGATTCCAGCTCCTAGGTGCACTCTTAAACCCTGCGACTACCCAGATATCAAGCATGGTGGACTGTATCATG AGAATATGAGGAGACCATACTTTCCAGTTGCAGTGGGCAAGTACTATAGCTATTACTGTGATGAGCACTTTGAAACTCCGTCTGGGAGCTACTGGGATCATATCCATTGTACGCAAGACGGCTGGAGTCCAGCAGTTCCATGCTTGCGGAAATGTTATTTTCCCTACCTCGAAAACGGATATAATCAGAACTATGGGAGGAAGTTTGTTCAAGGCAAAAGCATTGATGTGGCATGTCACCCCGGTTATGC CCTGCCCAAGGCGCAAACCACAGTAACTTGCATGGAGAATGGATGGAGCCCCACACCCAGATGTATACGAGTATCCTTCACGCTTTGA

(SEQ ID NO: 31)

RC143: FHL-1 IDT-Hand Optimized:

ATGAGACTGCTTGCGAAAATTATATGCCTGATGCTTTGGGCTATTTGCGTTGCGGAAGACTGTAACGAACTCCCGCCCCGCCGAAATACAGAGATCCTCACAGGCAGTTGGAGCGACCAAACGTACCCTGAAGGCACGCAGGCCATATAAGTGTAGGCCAGGCTACAGATCACTTGGTAACATAATCATGGTGTGTCGGAAAGGAGAGTGGGTCGCTCTCAACCCTCTTCGCAAATGTCAAAAAAGACCCT GTGGGCATCCGGGAGATACGCCTTTCGGGACATTCACTCTCACGGGCGGAAACGTATTCGAATATGGCGTGAAGGCAGTGTATA CCTGCAATGAAGGGTATCAGCTGCTTGGGGAAATTAATTATAGGGAATGTGACACGGATGGGTGGACAAACGATATTCCAATATGCGAAGTAGTTAAATGCCTGCCCGTTACTGCACCGGAGAATGGCAAAATAGTCAGTAGCGCAATGGAGCCGGATCGCGAGTATCATTTTGGTCAGGCCGTGCGGTTCGTATGTAATTCTGGGTACAAGATCGAAGGTGACGAAGAGATGCATTGCTCA GATGACGGCTTTTGGAGCAAGGAAAAGCCTAAGTGTGTTGAAATCAGCTGTAAGAGTCCAGACGTGATTAACGGTTCCCCGATCCTCTCAGAAGATA ATTTACAAGGAAAACGAACGATTCCAATATAAGTGTAACATGGGCTACGAGTATTCCGAGCGAGGTGACGCAGTATGTACGGAAAGCGGGTGGCGACCTCTGCCCTCCTGCGAGGAAAAGAGCTGTGATAATCCGTATATCCCCAACGGCGACTATAGCCCACTGCGCATAAAACATCGGACGGGAGATGAGATTACATACCAATGCCGCAATGGTTTTTACCCCGCCACCCGAGGGAACACGGCAAAGT GCACTTCTACGGGGTGGATTCCAGCTCCTAGGTGCACTCTTAAACCCTGCGACTACCCAGATATCAAGCATGGTGGACTGTATCATG AGAATATGAGGAGACCATACTTTCCAGTTGCAGTGGGCAAGTACTATAGCTATTACTGTGATGAGCACTTTGAAACTCCGTCTGGGAGCTACTGGGATCATATCCATTGTACGCAAGACGGCTGGAGTCCAGCAGTTCCATGCTTGCGGAAATGTTATTTTCCCTACCTCGAAAACGGATATAATCAGAATTACGGCAGGAAATTTGTGCAAGGCAAAAGCATTGATGTGGCATGTCACCCCGGTTATGC CCTGCCCAAGGCGCAAACCACAGTAACTTGCATGGAGAATGGATGGAGCCCCACACCCAGATGTATACGAGTATCCTTCACGCTTTGA

(SEQ ID NO: 32)

RC144: FHL-1 JCat- Basic

ATGCGCCTGCTGGCCAAGATCATCTGCCTGATGCTGTGGGCCATCTGCGTGGCCGAGGACTGCAACGAGCTGCCCCCCCGCCGCAACACCGAGATCCTGACCGGCAGCTGGAGCGACCAGACCTACCCCGAGGGCACCCAGGCCATCTACAAGTGCCGCCCCGGCTACCGCAGCCTGGGCAACATCATCATGGTGTGCCGCAAGGGCGAGTGGGTGGCCCTGAACCCCCTGCGCAAGTGCCAG AAGCGCCCCTGCGGCCACCCCGGCGACACCCCCTTCGGCACCTTCACCCTGACCGGCGGCAACGTGTTCGAGTACGGCGTGAAGGCCGTGTACA CCTGCAACGAGGGCTACCAGCTGCTGGGCGAGATCAACTACCGCGAGTGCGACACCGACGGCTGGACCAACGACATCCCCATCTGCGAGGTGGTGAAGTGCCTGCCCGTGACCGCCCCCGAGAACGGCAAGATCGTGAGCAGCGCCATGGAGCCCGACCGCGAGTACCACTTCGGCCAGGCCGTGCGCTTCGTGTGCAACAGCGGCTACAAGATCGAGGGCGACGAGGAGATGCACTGCAGCG ACGACGGCTTCTGGAGCAAGGAGAAGCCCAAGTGCGTGGAGATCAGCTGCAAGAGCCCCGACGTGATCAACGGCAGCCCCATCAGCCAGAAGATC ATCTACAAGGAGAACGAGCGCTTCCAGTACAAGTGCAACATGGGCTACGAGTACAGCGAGCGCGGCGACGCCGTGTGCACCGAGAGCGGCTGGCGCCCCCTGCCCAGCTGCGAGGAGAAGAGCTGCGACAACCCCTACATCCCCCAACGGCGACTACAGCCCCCTGCGCATCAAGCACCGCACCGGCGACGAGATCACCTACCAGTGCCGCAACGGCTTCTACCCCGCCACCCGGCGCAACACCGC CAAGTGCACCAGCACCGGCTGGATCCCCGCCCCCCGCTGCACCCTGAAGCCCTGCGACTACCCCGACATCAAGCACGGCGGCCTGTACCACG AGAACATGCGCCGCCCCTACTTCCCCGTGGCCGTGGGCAAGTACTACAGCTACTACTGCGACGAGCACTTCGAGACCCCCAGCGGCAGCTACTGGGACCACATCCACTGCACCCAGGACGGCTGGAGCCCCGCCGTGCCCTGCCTGCGCAAGTGCTACTTCCCCTACCTGGAGAACGGCTACAACCAGAACTACGGCCGCAAGTTCGTGCAGGGCAAGAGCATCGACGTGGCCTGCCACCCCGG CTACGCCCTGCCCAAGGCCCAGACCACGTGACCTGCATGGAGAACGGCTGGAGCCCCACCCCCCGCTGCATCCGCGTGAGCTTCACCCTGTAA

(SEQ ID NO: 33)

RC145: FHL-1 JCat-Hand Optimized:

ATGCGCCTGCTCGCCAAGATCATTTGCCTGATGCTGTGGGCCATCTGCGTGGCCGAGGACTGCAACGAGCTGCCCCCTCGCCGGAACACCGAGATCCTGACCGGCAGCTGGAGCGACCAGACCTACCCCGAGGGCACCCAGGCCATCTACAAGTGCCGCCCCGGCTACCGCAGCCTGGGCAACATCATTATGGTGTGCCGCAAGGGCGAGTGGGTGGCCCTGAACCCCCTGCGCAAGTGCC AGAAGCGCCCCTGCGGCCACCCCGGGCGACACCCCCTTCGGCACCTTCACCCTGACCGGCGGCAACGTGTTCGAGTACGGCGTGAAGGCCGTGTACA CCTGCAACGAGGGCTACCAGCTGCTCGGCGAGATCAACTACCGCGAGTGCGACACCGACGGCTGGACCAACGACATCCCCATCTGCGAGGTGGTCAAGTGCCTGCCCGTGACCGCCCCCGAGAACGGCAAGATCGTGAGCTCCGCCATGGAGCCCGACCGCGAGTACCACTTCGGCCAGGCCGTGCGCTTCGTGTGCAACAGCGGCTACAAGATCGAGGGCGACGAGGAGATGCACTGCAGCG ACGATGGCTTCTGGAGCAAGGAGAAGCCCAAGTGCGTGGAGATCAGCTGCAAGAGCCCCGACGTGATCAACGGCAGCCCCATCAGCCAGAAGATC ATTTACAAGGAGAACGAGCGCTTCCAGTACAAGTGCAACATGGGCTACGAGTACAGCGAGCGCGGCGACGCCGTGTGCACCGAGAGCGGCTGGCGCCCCCTGCCCAGCTGCGAGGAAAAGAGCTGCGACAACCCCTACATCCCCCAACGGCGACTACAGCCCCCTGCGCATCAAGCACCGCACCGGCGACGAGATCACCTACCAGTGCCGCAACGGCTTCTACCCCGCCACCCGGCGCAACACCGC CAAGTGCACCAGCACCGGCTGGATCCCCGCCCCCCGCTGCACCCTGAAGCCCTGCGACTACCCCGACATCAAGCACGGCGGGCTGTACCACG AGAACATGCGCCGGCCCTACTTCCCCGTGGCCGTGGGCAAGTACTATAGCTACTATTGCGACGAGCACTTCGAGACCCCCAGCGGCAGCTACTGGGACCACATCCACTGCACCCAGGACGGCTGGAGCCCCGCCGTGCCCTGCCTGCGCAAGTGCTACTTCCCCTACCTGGAGAACGGCTACAACCAGAACTACGGCCGCAAGTTCGTGCAGGGCAAGAGCATCGACGTGGCCTGCCACCCCGG CTACGCCCTGCCCAAGGCCCAGACCACAGTGACCTGCATGGAGAACGGCTGGAGCCCCACCCCCCGCTGCATCCGCGTGAGCTTCACCCTGTAA

(SEQ ID NO: 34)

RC146: FHL-1 COOL-Base: see SEQ ID NO:12 above.

RC147: FHL-1 COOL-Hand Optimized:

ATGCGCCTCCTGGCCAAGATCATTTGCCTCATGCTGTGGGCCATCTGCGTGGCTGAGGACTGCAATGAGCTGCCGCCCAGGAGAAACACAGAGATCCTGACAGGGAGCTGGTCTGACCAGACCTACCCTGAGGGCACCCAGGCGATCTACAAGTGCCGGCCGGGCTACAGGAGCCTGGGGAACATCATTATGGTGTGTAGAAAGGGCGAATGGGTGGCCCTCAACCCCCTGAGGAAGTGCCAG AAGCGGCCCTGTGGCCACCCCGGGGACACACCCTTCGGGACCTTCACCCTGACCGGCGGGAATGTGTTTGAGTACGGCGTGAAGGCTGTCTACA CATGCAACGAGGGGTACCAGCTGCTCGGCGAGATTAACTACCGGGAGTGTGACACCGATGGGTGGACCAACGACATTCCCATCTGTGAGGTGGTCAAGTGTCTCCCCGTGACAGCCCCAGAAAATGGCAAAATTGTGAGCTCCGCCATGGAGCCTGACCGCGAATATCACTTTGGGCAGGCCGTGAGGTTTGTGTGCAACTCGGGCTACAAAATTGAAGGTGATGAGGAAATGCACTGCAGCG ATGACGGCTTCTGGTCCAAGGAGAAGCCCAAATGTGTGGAGATCTCCTGCAAGTCTCCCGACGTGATCAACGGCAGCCCAATCAGCCAGAAGATT ATCTACAAAGAGAACGAGCGCTTCCAGTACAAGTGTAACATGGGCTATGAGTATTCAGAGAGGGGAGATGCCGTCTGCACTGAGAGCGGCTGGAGACCACTGCCTAGCTGCGAGGAAAAGAGTTGTGACAACCCTTACATCCCAAATGGCGACTACTCCCCTCTGCGGATCAAACACCGGACCGGGGATGAAATCACCTATCAGTGCCGCAATGGATTCTACCCGGCCACCCGCGGCAACACCGCCAAA TGCACCAGCACAGGCTGGATCCCCGCCCCCCGCTGTACGCTGAAGCCTTGCGACTATCCAGACATCAAGCACGGAGGCCTGTACCACG AAAACATGCGGAGGCCTTATTTCCCTGTGGCAGTGGGGAAGTACTATAGCTACTATTGCGACGAGCACTTCGAGACCCCCTCTGGCTCCTACTGGGACCACATCCACTGCACACAGGACGGCTGGTCTCCAGCTGTGCCCTGCCTGAGGAAATGCTACTTCCCCTACCTGGAGAACGGATACAACCAGAACTATGGCCGCAAGTTCGTGCAGGGCAAGAGCATCGATGTGGCCTGCCACCCTGGCTACGC CCTGCCCAAGGCCCAGACAACTGTGACCTGCATGGAGAATGGTTGGAGCCCCACCCCGCGCTGCATCCGGGTGTCCTTCACGCTCTGA

(SEQ ID NO: 35)

Plasmid generation

All 10 codon-optimized sequences were synthesized and cloned into an AAV vector backbone. The vector also contained the left and right inverted terminal repeat (ITR) sequences of AAV-2 flanking a modified CBA/CAG (chicken beta-actin with CMV enhancer; "CBA") promoter. The promoter drives expression of codon-optimized FHL1 or CFI. In addition, the transgene is followed by a modified woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) sequence and a bovine growth hormone polyA (bGH polyA) sequence located 3' relative to the cDNA.

Transfection

All 20 plasmids were transfected into ARPE19 cells using the following procedure:

Day 1: ARPE19 cells were dissociated and counted using ViCell. Cells were seeded in a 48-well plate at ^6x104 cells/well in 500 µl DMEM, 10% FBS/well.

Day 2: Confluency was checked and was 70-80%. Cells were then transfected with 0.25 µg plasmid DNA using PEI at a DNA:PEI ratio of 1:3 in duplicate:

1. 2 × 0.25 μg DNA was diluted in 2 × 5 μl PBS.

2. 2 x 0.75 µl PEI was diluted in 2 x 5 µl PBS.

3. The PEI mixture was added dropwise to the DNA mixture, mixed and then incubated at room temperature for 20 minutes.

4. 2 x 250 µl DMEM/Glutamax/10% FBS were added to the mixture.

5. The medium was removed from the cells and replaced with 250 µl DNA/PEI complexes per well.

Day 3: The medium was removed and replaced with 125 µl serum-free DMEM/Glutamax medium.

Day 5: The medium was collected, centrifuged at 14,000 rpm for 10 minutes at 4 °C, and the supernatant was then transferred to a fresh tube.

Western blotting

Transfection supernatants were analyzed by Western blotting (primary antibodies to CFI and FHL-1 were: goat antiserum to CFI 1:3000; Quidel A312 1:3000; and secondary antibodies rabbit anti-goat with HRP 1:5000).

The Western blot results are shown in Fig. 2.

CFI IFA

Post-transfection supernatants were analyzed by CFI ELISA using the following procedure:

Day 1: The ELISA plate was coated with 50 µl/well of sheep polyclonal antibody to CFI diluted 1:4000 in 1x coating buffer. The plates were stored at 4°C overnight.

Day 2: The plate was washed 3 times with 200 µl per well of PBS-Tween (0.05%) then applied to the tissue. 200 µl of 1% BSA fraction V in PBS-Tween (0.05%) was added to each well and allowed to block for 2 hours at room temperature.

Samples and a standard curve were prepared during blocking incubation. A standard curve was generated using purified CFI protein (Sigma C5938-1MG) diluted in DMEM 2% FBS. Samples were diluted 1:10, 1:20, and 1:40 in DMEM 2% FBS.

After 2 hours of blocking, the plate was washed 3 times as described above, then 50 µl of sample or standard was loaded into each well and incubated at room temperature for 1 hour.

After 1 hour, the plate was washed as described above, then anti-CFI (Ox21) antibody was diluted 1:2000 in DMEM 5% FBS and 50 µl of this was added to each well and incubated at room temperature for 1 hour.

After 1 hour, the plate was washed as described above, then donkey anti-mouse antibodies with HRP were diluted 1:5000 in DMEM 5% FBS, and 50 µl was applied to each well and incubated at room temperature for 1 hour.

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

The A450 was then recorded, the data was processed and transferred to Microsoft Excel for analysis.

The results of the CFI ELISA are shown in Fig. 3.

FHL1 ELISA

Post-transfection supernatants were analyzed by FHL1 ELISA using the following procedure:

Day 1: The ELISA plate was coated with 50 µl/well of FHL-1 antibody (Biorad, AbD33594.1) diluted to 5 µg/ml in 100 mM carbonate/bicarbonate buffer, pH 9.6. The plates were stored at 4°C overnight.

Day 2: The plate was washed 3 times with 200 µl per well of PBS-Tween (0.05%) then applied to the tissue. 200 µl of 1% BSA fraction V in PBS-Tween (0.05%) was added to each well and allowed to block for 2 hours at room temperature.

Samples and standard curve were prepared during blocking incubation. Standard curve was constructed for FHL1-His protein diluted in DMEM 2% FBS. Samples were diluted with blocking solution 1:5, 1:10 and 1:30.

After 2 hours of blocking, the plate was washed 3 times as described above, then 50 µl of sample or standard was loaded into each well and incubated at room temperature for 1 hour.

After 1 hour, the plate was washed as described above, then anti-CFH antibody (Ox24, Santa Cruz Biotechnologies, sc-53067) was diluted 1:3000 in DMEM 5% FBS, and 50 µl was applied to each well and incubated at room temperature for 1 hour.

After 1 hour, the plate was washed as described above, then donkey anti-mouse antibodies with HRP were diluted 1:5000 in DMEM 5% FBS, and 50 µl was applied to each well and incubated at room temperature for 1 hour.

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

A450 was then recorded, the data was processed and transferred to Microsoft Excel for analysis.

The results of FHL1 ELISA are shown in Fig. 4.

Production of AAV2 vectors

The best four (CFI) and five (FHL1) sequences were used in subsequent studies using AAV.

HEK293 cells were transfected with the selected codon-optimized plasmids together with pRepCap and pHelper according to a typical triple transfection protocol, namely:

Day 1: HEK293 cells were dissociated and counted using ViCell. Cells were seeded in a 10 cm dish at ^6x105 cells/ ^cm2 in 10 ml DMEM 10% FBS per dish.

Day 2: Confluence was checked and was 70-80%.

The medium was replaced with 10 ml DMEM/Glutamax with 5% FBS.

After 4 h, cells were transfected with 5 μg of plasmid using PEI at a DNA:PEI ratio of 1:3.

Day 3: 15 mM butyrate was added to 11 ml of medium in each plate.

Day 5: The supernatant was collected and centrifuged at 1000 rpm for 10 minutes to remove cell debris.

The supernatant was transferred to a fresh tube and 1/5 vol. was added with AAVanced reagent (AAV110A-1, Cambridge Bioscience) (2.75 ml to 11 ml).

The mixture was then stored at 4°C.

Day 8: The supernatant/AAV mixture was centrifuged at 1000 rpm for 30 min at 4 °C.

The supernatant was discarded and the pellet was resuspended in 500 μl PBS. It was then transferred to a 1.5 ml tube and centrifuged for 3 min at 1500 g.

The supernatant was discarded and the remaining pellet was resuspended in 1/100 of the original volume (i.e. 100 µl per 11 ml of supernatant).

The vector was stored at -80 °C.

Transduction of ARPE19 cells

ARPE19 cells were transduced with codon-optimized vectors.

Day 1: ARPE19 cells were dissociated and counted on ViCell, then seeded at 1 x ¹⁰⁵ cells/well in 200 µl DMEM/Glutamax 10% FBS.

Day 2: Vector was added to the cells.

Day 3: The medium was replaced with serum-free medium.

Day 4: The supernatant was collected, centrifuged at 14,000 rpm for 10 minutes at 4 °C, then transferred to a fresh tube.

Total protein concentration was estimated using the Bradford assay.

CFI IFA

Transduction supernatants were analyzed by CFI ELISA according to the protocol described above.

The results of the CFI ELISA are shown in Fig. 5.

FHL-1 ELISA

Transduction supernatants were analyzed by FHL1 ELISA according to the protocol described above.

The results of FHL1 ELISA are shown in Fig. 6.

CONCLUSIONS

Each of RC136 (CFI; SEQ ID NO: 10) and RC146 (FHL-1; SEQ ID NO: 12) yielded higher transgene expression than the wild-type sequences and other codon-optimized sequences tested.

All publications mentioned in the above description are incorporated herein by reference. Various modifications and variations of the disclosed agents, compositions, uses and methods of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been disclosed in connection with specific preferred embodiments, it is to be understood that the claimed invention should not be unduly limited to such specific embodiments. Indeed, various modifications of the disclosed modes of carrying out the invention that are obvious to those skilled in the art are intended to fall within the scope of the following claims.

Claims (37)

1. An isolated polynucleotide for expressing factor H-like protein 1 (FHL1) in vitro or in vivo, comprising a nucleotide sequence encoding factor H-like protein 1 (FHL1), wherein the nucleotide sequence encoding FHL1 is at least 90% identical to the sequence of SEQ ID NO: 12. 2. The isolated polynucleotide according to claim 1, characterized in that the nucleotide sequence encoding FHL1 is SEQ ID NO: 12. 3. An isolated polynucleotide according to claim 1 or 2, characterized in that the polynucleotide additionally contains an inverted terminal repeat (ITR) of an adeno-associated virus (AAV) at its 5' end and an AAV ITR at its 3' end. 4. The isolated polynucleotide according to claim 3, characterized in that the AAV ITRs are AAV2 or AAV8 ITRs. 5. The isolated polynucleotide according to claim 3, characterized in that the AAV ITRs are AAV2 ITRs. 6. An isolated polynucleotide according to any one of claims 1-5, characterized in that the nucleotide sequence encoding FHL1 is operably linked to the CMV promoter, the WPRE regulatory element and the poly-A signal. 7. The isolated polynucleotide according to claim 6, characterized in that the promoter has the nucleotide sequence of SEQ ID NO: 5 or SEQ ID NO: 13 or a nucleotide sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 5 or SEQ ID NO: 13. 8. The isolated polynucleotide according to claim 6 or 7, characterized in that the poly-A signal is the poly-A signal of bovine growth hormone. 9. The isolated polynucleotide according to claim 8, characterized in that the poly-A signal has the nucleotide sequence of SEQ ID NO: 14 or SEQ ID NO: 6 or a nucleotide sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 14 or SEQ ID NO: 6. 10. An isolated polynucleotide according to any one of claims 6 to 9, characterized in that the WPRE regulatory element has a nucleotide sequence of SEQ ID NO: 7 or SEQ ID NO: 15 or a nucleotide sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 15 or SEQ ID NO: 7. 11. A vector for the expression of factor H-like protein 1 (FHL1) in vitro or in vivo, comprising the polynucleotide of any preceding paragraph. 12. The vector according to claim 11, characterized in that the vector is an adeno-associated virus (AAV) vector. 13. The vector according to item 11 or 12, characterized in that the vector is in the form of a viral vector particle. 14. The vector according to claim 13, characterized in that the AAV vector particle contains the capsid proteins of AAV2 or AAV8. 15. The vector according to claim 14, characterized in that the AAV vector particle contains the AAV2 or AAV8 genome and the AAV2 or AAV8 capsid proteins. 16. A cell for expressing factor H-like protein 1 (FHL1) in vitro or in vivo, comprising a polynucleotide according to any one of claims 1-10. 17. A cell for expressing factor H-like protein 1 (FHL1) in vitro or in vivo, transduced with the vector according to any one of claims 11-15 and containing it. 18. A pharmaceutical composition for the treatment or prevention of a complement-mediated disorder associated with hyperactivity of the complement feedback loop C3b, comprising a polynucleotide according to any one of claims 1-10, a vector according to any one of claims 11-15, or a cell according to claim 16 or 17 in combination with a pharmaceutically acceptable carrier, diluent or excipient. 19. Use of a polynucleotide according to any one of claims 1-10, a vector according to any one of claims 11-15, or a cell according to claim 16 or 17 in the treatment or prevention of a complement-mediated eye disorder associated with hyperactivity of the complement C3b feedback loop. 20. The use according to claim 19, wherein the disorder is age-related macular degeneration (AMD) or diabetic retinopathy. 21. The use according to claim 19, wherein the disorder is age-related macular degeneration (AMD). 22. The use according to paragraph 20 or 21, characterized in that the AMD is dry AMD. 23. The use according to any one of paragraphs 19-22, characterized in that the formation of geographic atrophy is prevented or reduced and/or the degree of geographic atrophy is reduced. 24. The use according to any of paragraphs 19-23, characterized in that the progression of geographic atrophy is slowed. 25. The use according to any one of claims 19-24, characterized in that the administration of the polynucleotide, vector or cell increases the level of activity in relation to C3b inactivation and iC3b degradation in the subject. 26. The use according to any one of claims 19-24, characterized in that the administration of the polynucleotide, vector or cell increases the level of activity in relation to C3b inactivation and iC3b degradation in the eye of the subject. 27. The use according to any one of claims 19-24, characterized in that the administration of the polynucleotide, vector or cell increases the level of activity in relation to C3b inactivation and iC3b degradation in the retinal pigment epithelium (RPE) of the subject. 28. The use according to any one of claims 25-27, characterized in that the level of activity with respect to C3b inactivation and iC3b degradation is increased to a level exceeding the normal level. 29. Use of a polynucleotide according to any one of claims 1-10, a vector according to any one of claims 11-15, or a cell according to claim 16 or 17 for improving or restoring reading speed in a subject suffering from a complement-mediated eye disorder associated with hyperactivity of the complement C3b feedback loop, or reducing a decrease in reading speed associated with a complement-mediated eye disorder associated with hyperactivity of the complement C3b feedback loop. 30. The use of a polynucleotide according to any one of claims 1-10, a vector according to any one of claims 11-15, or a cell according to claim 16 or 17 for reducing or preventing photoreceptor loss associated with a complement-mediated eye disorder associated with hyperactivity of the complement C3b feedback loop. 31. The use of a polynucleotide according to any one of claims 1-10, a vector according to any one of claims 11-15, or a cell according to claim 16 or 17 for reducing or preventing the loss of retinal pigment epithelium (RPE) associated with a complement-mediated eye disorder associated with hyperactivity of the complement feedback loop C3b. 32. The use of a polynucleotide according to any one of claims 1-10, a vector according to any one of claims 11-15, or a cell according to claim 16 or 17 for reducing or preventing the loss of photoreceptors and retinal pigment epithelium (RPE) associated with a complement-mediated eye disorder associated with hyperactivity of the complement feedback loop C3b. 33. Use according to any of paragraphs 19-32, characterized in that the polynucleotide, vector or cell is administered intraocularly. 34. The use according to any one of paragraphs 19-33, characterized in that the polynucleotide, vector or cell is introduced into the eye of the subject by subretinal, direct retinal, suprachoroidal or intravitreal injection. 35. The use according to any one of paragraphs 19-34, characterized in that the polynucleotide, vector or cell is introduced into the eye of the subject by subretinal injection. 36. A method for treating or preventing a complement-mediated eye disorder associated with hyperactivity of the complement C3b feedback loop, comprising administering a polynucleotide according to any one of claims 1-10, a vector according to any one of claims 11-15, or a cell according to claim 16 or 17 to a subject in need thereof. 37. A method for providing factor H-like protein 1 (FHL1) to a subject, comprising delivering the polynucleotide of any one of claims 1-10, the vector of any one of claims 11-15, or the cell of claim 16 or 17 to the eye of the subject.