WO2019193375A1 - Use of fzd7 inhibitors for the treatment of retinal neovascularization - Google Patents

Abstract

Retinal ischemia and abnormal blood vessels growth are major determinants in the pathogenesisof retinopathy of prematurity (ROP) and proliferative diabetic retinopathy (DR). The present study used mice model of oxygen-induced retinopathy (OIR) to investigate the role of Fzd7 during initial vaso-obliteration (VO) and subsequent hypoxia-induced neovascularization (NV) phases. In particular, the inventors performed two blocking strategies: a monoclonal antibody (mAbFzd7) directed against Fzd7 and a soluble Fzd7 receptor (CRD domain). In vivo intravitreal microinjection of mAbFzd7 or CRD receptor in mice after 5 days of exposure to 75% oxygen (P12) resulted in a significant decrease of pathological neovascularization in the treated eye compared to the control eye. Collectively, the results established that Fzd7 acts as an important regulator of retinal neovascularization and offers a promising anti-angiogenic strategy for the treatment of ischemic retinopathies. Accordingly, the present invention relates to use of Fzd7 inhibitors for the treatment of retinal neovascularization.

Description

USE OF FZD7 INHIBITORS FOR THE TREATMENT OF RETINAL

NEOVASCULARIZATION

FIELD OF THE INVENTION:

The present invention relates to use of Fzd7 inhibitors for the treatment of retinal neovascularization.

BACKGROUND OF THE INVENTION:

Diabetic retinopathy (DR), one of the major complications of diabetes, and retinopathy of prematurity (ROP) that primarily affects premature infants, are the most common leading causes of blindness in many parts of the world, with an increasing prevalence for both of these two prominent neovascular eye diseases. ROP occurs when newborns are placed in an incubator under controlled oxygenation. Stopping oxygen therapy reduces vascular density responsible for inadequate retinal perfusion resulting in a relative hypoxia of retinal tissue. Hypoxia then stimulates intravitreal vascular proliferation and permeability (Schulenburg et al, Br J Ophthalmol, 2004; Sapieha P et al, J Clin Invest, 2010). In DR, hyperglycemia gradually induces progressive alterations in the retinal microvasculature with a loss of pericyte coverage leading to areas of retinal non-perfusion. In response to retinal ischemia, pathologic intraocular proliferation of retinal vessels increases as well as vasopermeability (Lobo CL et al, J Cataract Refract Surg, 2004; Cheung N et al, Lancet, 2010). With an improved understanding of the disorder from clinical examination and through the use of relevant animal models, it appeared that ROP and DR are characterized by a biphasic process: phase 1 involves delayed physiologic retinal vascular development with a vasoregression and phase 2 involves vasoproliferation with development of abnormal vessels (Hartnett ME et al,N Engl J Med 367, 2012; Hammes HP et al, Diabetes, 2011). Current therapeutics development focused on anti-VEGF therapy has brought new approaches for the treatment of eyes diseases with a significant efficacy in treating ocular neovascularization. However some studies have described adverse effects for intravitreal injection of anti-VEGFA antibody such as local toxicity, short-term effect and drug resistance, with reported persistent avascular retina and recurrent intravitreal neovascularization (NV), suggesting that there is a need for alternative strategies to better control impaired angiogenesis in these diseases. One way is to unraveled new signaling pathways of vessel growth and potential drug targets.

Growing evidence has shown that Wnt/Frizzled (Fzd) are directly involved in blood vessel development by regulating endothelial cell (EC) proliferation, polarity, apoptosis and vascular branching. The secreted Wnt proteins (19 members) activate canonical and non- canonical signaling pathways by binding to the cystein-rich domain (CRD) of Frizzled (Fzd) receptors (10 homologous). Genetic or pharmacological inhibition of elements of the Wnt/Fzd pathway (Fzd4, LRP-5, Dishevelled and b-catenin) suppresses retinal neovascularization and vascular permeability, processes observed in ROP and DR. Moreover, genetic studies have linked FEVR, Norrie disease and Coats' disease with mutations in genes encoding Fzd4, Norrin, or LRP-5 due to impairment in vessel formation/regression. In DR, retinal levels of b-catenin are increased and anti-LRP6 (Fzd co-receptor) inhibits canonical Wnt signaling, vascular leakage and inflammation in the retina of rat diabetic retinopathy models. A recent study has reported the role of microRNA-l84 in modulating canonical Wnt signaling through the regulation of Fzd7 expression in the retina with ischemia-induced neovascularization. Previously it was demonstrated that Fzd7 control vessel formation in the retina via the activation of b-catenin pathway and the regulation of Jagguedl and D114. (Peghaire C et al, ATVB, 2016), two major ligands of Notch pathway, which appears to be an important regulator in several angiogenic steps.

SUMMARY OF THE INVENTION:

The present invention relates to use of Fzd7 inhibitors for the treatment of retinal neovascularization. In particular, the present invention is defined by the claims.

DETAILED DESCRIPTION OF THE INVENTION:

Retinal ischemia and abnormal blood vessels growth are major determinants in the pathogenesis of retinopathy of prematurity (ROP) and proliferative diabetic retinopathy (DR). Understanding the mechanisms involved in aberrant angiogenesis observed in these blinding diseases is necessary to develop therapeutic drugs against new molecular targets. Inventor’s previous work has evidenced the crucial role of Frizzled 7 receptor (Fzd7) in the postnatal angiogenesis of retina by controlling endothelial cells (EC) proliferation and migration through a b-catenin canonical pathway; however its involvement in retinal neovascularization remains unknown. The present study used mice model of oxygen-induced retinopathy (OIR) to investigate the role of Fzd7 during initial vaso-obliteration (VO) and subsequent hypoxia- induced neovascularization (NV) phases. By genetic approach using the transgenic Pdgfb-lCrc line, the inventors observed that depletion of fzd7 in the endothelium (Pdgfb- iCre(+); fzd? ^f/f) resulted in increased retinal tissue sensitivity to hyperoxia during the vaso-obliterative phase of the OIR model. Additionally, fzd7 endothelial deletion early in the ischemic phase of the OIR model at P12 protected against formation of aberrant neovessels into the vitreous by inhibiting proliferation of EC in tufts. To evaluate Fzd7 as a new target for therapeutic inhibition of pathological angiogenesis, we performed two blocking strategies: a monoclonal antibody (mAbFzd7) directed against Fzd7 and a soluble Fzd7 receptor (CRD domain). In vitro both of these Fzd7 blocking agents decreased the activity of b-catenin reporter gene (TOP luciferase activity) induced by Wnt3A ligand in EA.hy926 cells. In vivo intravitreal microinjection of mAbFzd7 or CRD receptor in mice after 5 days of exposure to 75% oxygen (P12) resulted in a significant decrease of pathological neovascularization in the treated eye compared to the control eye at P17. Molecular analysis further revealed that Fzd7 may regulate development of neovascular tufts by modulating expression of both canonical b-catenin and Notch signaling partners. Collectively, the results established that Fzd7 acts as an important regulator of retinal neovascularization and offers a promising anti-angiogenic strategy for the treatment of ischemic retinopathies.

The first object of the present invention relates to a method of treating retinal neovascularization in a patient in need thereof comprising administering to the subject a therapeutically effective amount of a Fzd7 inhibitor.

In some embodiments, the patient suffers from an ischemic retinopathy. As used herein, the“ischemic retinopathy” has its general meaning in the art and refers to a group of diseases where progressive irreversible visual loss occurs as a consequence of retinal neovascularization. Examples of ischemic retinopathies include diabetic retinopathy, age-related macular degeneration, neovascular glaucoma, retinopathy of prematurity, sickle-cell retinopathy, retinal vein occlusion, oxygen induced retinopathy, and neovascularization due to ocular insults such as traumatic or surgical injury, or transplantation of eye tissue.

In some embodiments, the subject suffers from diabetic retinopathy. Diabetic retinopathy is the leading cause of blindness among working age adults in the United States. Initially, the high blood glucose levels common to persons with diabetes mellitus cause an increase in growth factor levels in the eyes. This condition is known as the“pre-diabetic retinopathy stage” and can lead to retinopathy if not prophylactically treated. Non-proliferative or early-stage diabetic retinopathy, also known as “background diabetic retinopathy,” is characterized by thickening of the basement membrane, loss of retinal pericytes, microvascular abnormalities, intraretinal microaneurysms, retinal hemorrhages (known as “dot blot” or “cotton wool” spots), retinal edema, capillary closure, and soft and hard exudates. Late-stage or proliferative diabetic retinopathy, which is characterized by neovascularization and fibrovascular growth, i.e., scarring involving glial and fibrous elements, from the retina or optic nerve over the inner surface of the retina or into the vitreous cavity. Retinal detachment may also occur. In some embodiments, the subject suffers from age-related macular degeneration. Age- related macular degeneration is one of the leading causes of blindness in older adults in the United States, and may account for up to 30 percent of all bilateral blindness among Caucasian Americans. This disease is characterized by loss of central vision, usually in both eyes, due to damage to the retinal pigment epithelial (RPE) cells. RPE cells are aligned in the lowest layer of the retina, on the Bruch's membrane, and absorb the light which reaches the retina so as to prevent reflection. RPE cells also constitute the blood-retinal barrier which partitions the visual cells and the vascular layer of choroid together with the Bruch's membrane. In general, RPE cells have important physical and physiological functions, such as sustainment and regeneration of visual cells.

In some embodiments, the subject suffers from retinopathy of prematurity. Retinopathy of prematurity (ROP) is a common cause of blindness in children in the United States. Premature infants are exposed to hyperoxic conditions after birth even without the administration of supplemental oxygen due to the higher partial pressure of oxygen in the atmosphere as compared to in utero conditions. This relative hyperoxia is necessary for the survival of premature infants yet may result in ROP. The hyperoxic atmosphere causes retinal blood vessels to stop developing into the peripheral retina, resulting in ischemia and localized hypoxic conditions as the metabolic demands of the developing retina increase. The resulting localized hypoxia stimulates retinal neovascularization. The neovascularization usually regresses, but may lead to irreversible vision loss. There are at least 10,000 new cases per year of ROP with a worldwide estimate of 10 million total cases.

As used herein, the term "treatment" or "treat" refer to both prophylactic or preventive treatment as well as curative or disease modifying treatment, including treatment of patient at risk of contracting the disease or suspected to have contracted the disease as well as patients who are ill or have been diagnosed as suffering from a disease or medical condition, and includes suppression of clinical relapse. The treatment may be administered to a subject having a medical disorder or who ultimately may acquire the disorder, in order to prevent, cure, delay the onset of, reduce the severity of, or ameliorate one or more symptoms of a disorder or recurring disorder, or in order to prolong the survival of a subject beyond that expected in the absence of such treatment. By "therapeutic regimen" is meant the pattern of treatment of an illness, e.g., the pattern of dosing used during therapy. A therapeutic regimen may include an induction regimen and a maintenance regimen. The phrase "induction regimen" or "induction period" refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the initial treatment of a disease. The general goal of an induction regimen is to provide a high level of drug to a patient during the initial period of a treatment regimen. An induction regimen may employ (in part or in whole) a "loading regimen", which may include administering a greater dose of the drug than a physician would employ during a maintenance regimen, administering a drug more frequently than a physician would administer the drug during a maintenance regimen, or both. The phrase "maintenance regimen" or "maintenance period" refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the maintenance of a patient during treatment of an illness, e.g., to keep the patient in remission for long periods of time (months or years). A maintenance regimen may employ continuous therapy (e.g., administering a drug at a regular intervals, e.g., weekly, monthly, yearly, etc.) or intermittent therapy (e.g., interrupted treatment, intermittent treatment, treatment at relapse, or treatment upon achievement of a particular predetermined criteria [e.g., disease manifestation, etc.]).

As used herein, the term“Frizzled 7 receptor” or“Fzd7” has its general meaning in the art and refers to the frizzled class receptor 7. Members of the 'frizzled' gene family encode 7- transmembrane domain proteins that are receptors for Wnt signaling proteins. The Fzd7 protein contains an N-terminal signal sequence, 10 cysteine residues typical of the cysteine-rich extracellular domain of Fz family members, 7 putative transmembrane domains, and an intracellular C-terminal tail with a PDZ domain-binding motif. An exemplary human amino acid sequence is represented by SEQ ID NO: 1. The main extracellular domain ranges from the amino acid residue at position 33 to the amino acid residue at position 256 in SEQ ID NO:l .

Fzd7 binds to Wnt molecules that include WNT1, WNT2, WNT2B, WNT3, WNT3A, WNT4,

WNT5A, WNT5B, WNT6, WNT7A, WNT7B, WNT8A, WNT8B, WNT9A, WNT9B,

WNT 10 A, WNT 10B, WNT11, and WNT16. In particular, Fzd7 binds to WNT3A.

SEQ ID NO: 1 >sp | 075084 | Fzd7_HUMAN Frizzled-7 OS=Homo sapiens OX=9606 GN=Fzd7 PE=1 SV=2); the main extracellular domain is underlined in the sequence

MRDPGAAAPLSSLGLCALVLALLGALSAGAGAQPYHGEKGISVPDHGFCQPISIPLOT

DIAYNQTILPNLLGHTNQEDAGLEVHQFYPLVKVQCSPELRFFLCSMYAPVCTVLDQA

IPPCRSLCERARQGCEALMNKFGFQWPERLRCENFPVHGAGEICVGQNTSDGSGGPGG

GPTAYPTAPYLPDLPFTALPPGASDGRGRPAFPFSCPRQLKVPPYLGYRFLGERDCGA

PCEPGRANGLMYFKEEERRFARLWVGVWSVLCCASTLFTVLTYLVDMRRFSYPERPI I

FLSGCYFMVAVAHVAGFLLEDRAVCVERFSDDGYRTVAQGTKKEGCTILFMVLYFFGM

ASSIWWVILSLTWFLAAGMKWGHEAIEANSQYFHLAAWAVPAVKTITILAMGQVDGDL

LSGVCYVGLSSVDALRGFVLAPLFVYLFIGTSFLLAGFVSLFRIRTIMKHDGTKTEKL

EKLMVRIGVFSVLYTVPATIVLACYFYEQAFREHWERTWLLQTCKSYAVPCPPGHFPP

MSPDFTVFMIKYLMTMIVGITTGFWIWSGKTLQSWRRFYHRLSHSSKGETAV

As used herein, the term“Fzd7 inhibitor” has its general meaning in the art and refers to a compound that inhibits the activity or expression of Fzd7. In particular, the inhibitor selectively blocks or inactivates Fzd7. In particular, the inhibitor selectively inhibits the binding of Fzd7 to a Wnt molecule. The term“Fzd7 inhibitor” also refers to a compound that selectively blocks the binding of Fzd7 to its downstream effectors. As used herein, the term“selectively blocks or inactivates” refers to a compound that preferentially binds to and blocks or inactivates Fzd7 with a greater affinity and potency, respectively, than its interaction with the other sub- types of the Frizzled receptor family (e.g. Fzdl, Fzd2, Fzd3, Fzd4, Fzd5, Fzd6, Fzd8, Fzd9 or FzdlO). Typically, an Fzd7 inhibitor compound is a small organic molecule, a polypeptide, an aptamer, an antibody, an oligonucleotide or a ribozyme.

In some embodiments, the Fzd7 of the present invention is an antibody, in particular an antibody having specificity for Fzd7. In some embodiments, the antibody of the present invention binds to an extracellular domain of Fzd7. In some embodiments, the antibody of the present invention binds to an epitope located in the extracellular domain that ranges from the amino acid residue at position 33 to the amino acid residue at position 256 in SEQ ID NO: 1. In some embodiments, the antibody inhibits the binding of Fzd7 to a Wnt molecule.

As used herein, the term "antibody" is thus used to refer to any antibody-like molecule that has an antigen binding region, and this term includes antibody fragments that comprise an antigen binding domain such as Fab', Fab, F(ab')2, single domain antibodies (DABs), TandAbs dimer, Fv, scFv (single chain Fv), dsFv, ds-scFv, Fd, linear antibodies, minibodies, diabodies, bispecific antibody fragments, bibody, tribody (scFv-Fab fusions, bispecific or trispecific, respectively); sc-diabody; kappa(lamda) bodies (scFv-CL fusions); BiTE (Bispecific T-cell Engager, scFv-scFv tandems to attract T cells); DVD-Ig (dual variable domain antibody, bispecific format); SIP (small immunoprotein, a kind of minibody); SMIP ("small modular immunopharmaceutical" scFv-Fc dimer; DART (ds-stabilized diabody "Dual Affinity ReTargeting"); small antibody mimetics comprising one or more CDRs and the like. The techniques for preparing and using various antibody-based constructs and fragments are well known in the art (see Rabat et ah, 1991, specifically incorporated herein by reference). Diabodies, in particular, are further described in EP 404, 097 and WO 93/11161; whereas linear antibodies are further described in Zapata et al. (1995). Antibodies can be fragmented using conventional techniques. For example, F(ab’)2 fragments can be generated by treating the antibody with pepsin. The resulting F(ab')2 fragment can be treated to reduce disulfide bridges to produce Fab’ fragments. Papain digestion can lead to the formation of Fab fragments. Fab, Fab' and F(ab')2, scFv, Fv, dsFv, Fd, dAbs, TandAbs, ds-scFv, dimers, minibodies, diabodies, bispecific antibody fragments and other fragments can also be synthesized by recombinant techniques or can be chemically synthesized. Techniques for producing antibody fragments are well known and described in the art. For example, each of Beckman et ah, 2006; Holliger & Hudson, 2005; Le Gall et ah, 2004; Reff & Heard, 2001 ; Reiter et ah, 1996; and Young et ah, 1995 further describe and enable the production of effective antibody fragments.

In some embodiments, the antibody of the present invention is a single chain antibody. As used herein the term“single domain antibody” has its general meaning in the art and refers to the single heavy chain variable domain of antibodies of the type that can be found in Camelid mammals which are naturally devoid of light chains. Such single domain antibody are also “nanobody®”. For a general description of (single) domain antibodies, reference is also made to the prior art cited above, as well as to EP 0 368 684, Ward et al. (Nature 1989 Oct 12; 341 (6242): 544-6), Holt et al, Trends Biotechnol., 2003, 21(11):484-490; and WO 06/030220, WO 06/003388.

In some embodiments, the antibody is a“chimeric” antibody in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity (see, e.g., U.S. Pat. No. 4,816,567; and Morrison et al, Proc. Natl. Acad. Sci. USA 81 :6851-6855 (1984)). Chimeric antibodies include PRIMATTZED® antibodies wherein the antigen-binding region of the antibody is derived from an antibody produced by, e.g., immunizing macaque monkeys with the antigen of interest.

In some embodiments, the antibody is a humanized antibody. As used herein, "humanized" describes antibodies wherein some, most or all of the amino acids outside the CDR regions are replaced with corresponding amino acids derived from human immunoglobulin molecules. Methods of humanization include, but are not limited to, those described in U.S. Pat. Nos. 4,816,567, 5,225,539, 5,585,089, 5,693,761, 5,693,762 and 5,859,205, which are hereby incorporated by reference.

In some embodiments, the antibody is a fully human antibody. Fully human monoclonal antibodies also can be prepared by immunizing mice transgenic for large portions of human immunoglobulin heavy and light chain loci. See, e.g., U.S. Pat. Nos. 5,591,669, 5,598,369, 5,545,806, 5,545,807, 6,150,584, and references cited therein, the contents of which are incorporated herein by reference.

In some embodiments, the antibody of the present invention does not mediate antibody- dependent cell-mediated cytotoxicity and thus does not comprise an Fc portion that induces antibody dependent cellular cytotoxicity (ADCC). In some embodiments, the neutralizing antibody does not comprise an Fc domain capable of substantially binding to a FcgRIIIA (CD 16) polypeptide. In some embodiments, the neutralizing antibody lacks an Fc domain (e.g. lacks a CH2 and/or CH3 domain) or comprises an Fc domain of IgG2 or IgG4 isotype. In some embodiments, the neutralizing antibody consists of or comprises a Fab, Fab', Fab'-SH, F (ab') 2, Fv, a diabody, single-chain antibody fragment, or a multispecific antibody comprising multiple different antibody fragments. In some embodiments, the neutralizing antibody is not linked to a toxic moiety. In some embodiments, one or more amino acids selected from amino acid residues can be replaced with a different amino acid residue such that the antibody has altered C2q binding and/or reduced or abolished complement dependent cytotoxicity (CDC). This approach is described in further detail in U.S. Patent Nos. 6,194,551 by ldusogie et al.

In some embodiments, the Fzd7 inhibitor is a polypeptide comprising a functional equivalent of Fzd7 respectively. As used herein, a“functional equivalent of Fzd7” is a polypeptide which is capable of binding to a Wnt molecule, thereby preventing its interaction with Fzd7. The term "functional equivalent" includes fragments, mutants, and muteins of Fzd7. The term "functionally equivalent" thus includes any equivalent of Fzd7 obtained by altering the amino acid sequence, for example by one or more amino acid deletions, substitutions or additions such that the protein analogue retains the ability to bind to a Wnt molecule. Amino acid substitutions may be made, for example, by point mutation of the DNA encoding the amino acid sequence. Functional equivalents include molecules that bind a Wnt molecule and comprise all or a portion of the extracellular domains of Fzd7 so as to form a soluble receptor that is capable to trap a Wnt molecule. Thus the functional equivalents include soluble forms of the Fzd7. A suitable soluble form of these proteins, or functional equivalents thereof, might comprise, for example, a truncated form of the protein from which the transmembrane domain has been removed by chemical, proteolytic or recombinant methods. Typically, the functional equivalent is at least 80% identical to the corresponding protein. In a preferred embodiment, the functional equivalent is at least 90% (i.e. 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100%) identical as assessed by any conventional analysis algorithm. In some embodiments, the functional equivalent comprises an amino acid sequence having at least 90% of identity with the amino acid sequence ranging from the amino acid residue at position 33 to the amino acid 256 in SEQ ID NO: l . The term "a functionally equivalent fragment" as used herein also may mean any fragment or assembly of fragments of Fzd7 that binds to a Wnt molecule. Accordingly the present invention provides a polypeptide capable of inhibiting binding of Fzd7 to a Wnt molecule, which polypeptide comprises consecutive amino acids having a sequence which corresponds to the sequence of at least a portion of an extracellular domain of Fzd7, which portion binds to a Wnt molecule. In some embodiments, the polypeptide comprises an extracellular domain of Fzd7. In some embodiments, the polypeptide comprises the amino acid sequence which ranges from the amino acid residue at position 33 to the amino acid residue at position 256 in SEQ ID NO: l . In some embodiments, the polypeptide comprises a functional equivalent of Fzd7 which is fused to an immunoglobulin constant domain (Fc region) to form an immunoadhesin. Immunoadhesins can possess many of the valuable chemical and biological properties of human antibodies. Since immunoadhesins can be constructed from a human protein sequence with a desired specificity linked to an appropriate human immunoglobulin hinge and constant domain (Fc) sequence, the binding specificity of interest can be achieved using entirely human components. The immunoglobulin sequence typically, but not necessarily, is an immunoglobulin constant domain. The immunoglobulin moiety in the chimeras of the present invention may be obtained from IgGl, IgG2, IgG3 or IgG4 subtypes, IgA, IgE, IgD or IgM, but typically IgGl or IgG3. In some embodiments, the functional equivalent of the PD-l or Fzd7 and the immunoglobulin sequence portion of the immunoadhesin are linked by a minimal linker. As used herein, the term“linker” refers to a sequence of at least one amino acid that links the polypeptide of the invention and the immunoglobulin sequence portion. Such a linker may be useful to prevent steric hindrances. In some embodiments, the linker has 4; 5; 6; 7; 8; 9; 10; 11 ; 12; 13; 14; 15; 16; 17; 18; 19; 20; 21; 22; 23; 24; 25; 26; 27; 28; 29; 30 amino acid residues. One useful group of linker sequences are linkers derived from the hinge region of heavy chain antibodies as described in WO 96/34103 and WO 94/04678. Other examples are poly-alanine linker sequences.

In some embodiments, the Fzd7 inhibitor is an inhibitor of Fzd7 expression. An “inhibitor of expression” refers to a natural or synthetic compound that has a biological effect to inhibit the expression of a gene. In some embodiments, said inhibitor of gene expression is a siRNA, an endonuclease, an antisense oligonucleotide or a ribozyme.

In some embodiments, the inhibitor of expression is a siRNA. Small inhibitory RNAs (siRNAs) can also function as inhibitors of expression for use in the present invention. Fzd7 gene expression can be reduced by contacting a patient or cell with a small double stranded RNA (dsRNA), or a vector or construct causing the production of a small double stranded RNA, such that Fzd7 gene expression is specifically inhibited (i.e. RNA interference or RNAi).

In some embodiments, the inhibitor of expression is an endonuclease. The term “endonuclease” refers to enzymes that cleave the phosphodiester bond within a polynucleotide chain. Some, such as Deoxyribonuclease I, cut DNA relatively nonspecifically (without regard to sequence), while many, typically called restriction endonucleases or restriction enzymes, cleave only at very specific nucleotide sequences. The mechanism behind endonuclease-based genome inactivating generally requires a first step of DNA single or double strand break, which can then trigger two distinct cellular mechanisms for DNA repair, which can be exploited for DNA inactivating: the error prone non-homologous end-joining (NHEJ) and the high-fidelity homology-directed repair (HDR). In a particular embodiment, the endonuclease is CRISPR- Cas. As used herein, the term“CRISPR-Cas” has its general meaning in the art and refers to clustered regularly interspaced short palindromic repeats associated which are the segments of prokaryotic DNA containing short repetitions of base sequences. In some embodiment, the endonuclease is CRISPR-cas9, which is from Streptococcus pyogenes. The CRISPR/Cas9 system has been described in US 8697359 Bl and US 2014/0068797. In some embodiment, the endonuclease is CRISPR-Cpfl which is the more recently characterized CRISPR from Provote lla and Francisella 1 (Cpfl) in Zetsche et al. (“Cpfl is a Single RNA-guided Endonuclease of a Class 2 CRISPR-Cas System (2015); Cell; 163, 1-13).

In some embodiments, the inhibitor of expression is an antisense oligonucleotide. The term“antisense oligonucleotide” refers to an oligonucleotide sequence that is inverted relative to its normal orientation for transcription and so expresses an RNA transcript that is complementary to a target gene mRNA molecule expressed within the host cell (e.g., it can hybridize to the target gene mRNA molecule through Watson-Crick base pairing). An antisense strand may be constructed in a number of different ways, provided that it is capable of interfering with the expression of a target gene. For example, the antisense strand can be constructed by inverting the coding region (or a portion thereof) of the target gene relative to its normal orientation for transcription to allow the transcription of its complement, (e.g., RNAs encoded by the antisense and sense gene may be complementary). Furthermore, the antisense oligonucleotide strand need not have the same intron or exon pattern as the target gene, and noncoding segments of the target gene may be equally effective in achieving antisense suppression of target gene expression as coding segments. As used herein, the term "oligonucleotide" refers to a nucleic acid sequence, 3'-5' or 5'-3' oriented, which may be single- or double-stranded. The antisense oligonucleotide used in the context of the invention may in particular be DNA or RNA. According to the invention, the antisense oligonucleotide of the present invention targets an mRNA encoding Fzd7, and is capable of reducing the amount of Fzd7 in cells, in particular in endothelial cells. As used herein, an oligonucleotide that“targets” an mRNA refers to an oligonucleotide that is capable of specifically binding to said mRNA. That is to say, the antisense oligonucleotide comprises a sequence that is at least partially complementary, preferably perfectly complementary, to a region of the sequence of said mRNA, said complementarity being sufficient to yield specific binding under intra-cellular conditions. As immediately apparent to the skilled in the art, by a sequence that is“perfectly complementary to” a second sequence is meant the reverse complement counterpart of the second sequence, either under the form of a DNA molecule or under the form of a RNA molecule. A sequence is“partially complementary to” a second sequence if there are one or more mismatches. The antisense oligonucleotide of the present invention that target an mRNA encoding Fzd7 may be designed by using the sequence of said mRNA as a basis, e.g. using bioinformatic tools. Methods for determining whether an oligonucleotide is capable of reducing the amount of Fzd7 in cells are known to the skilled in the art. This may for example be done by analyzing Fzd7 protein expression by Western blot, and by comparing Fzd7 protein expression in the presence and in the absence of the antisense oligonucleotide to be tested. In some embodiments, the antisense oligonucleotide of the present invention has a length of from 12 to 50 nucleotides, e.g. 12 to 35 nucleotides, from 12 to 30, from 12 to 25, from 12 to 22, from 15 to 35, from 15 to 30, from 15 to 25, from 15 to 22, from 18 to 22, or about 19, 20 or 21 nucleotides. The antisense oligonucleotide according to the invention may for example comprise or consist of 12 to 50 consecutive nucleotides, e.g. 12 to 35, from 12 to 30, from 12 to 25, from 12 to 22, from 15 to 35, from 15 to 30, from 15 to 25, from 15 to 22, from 18 to 22, or about 19, 20 or 21 consecutive nucleotides of a sequence complementary to the mRNA of SEQ ID NO: 2. In some embodiments, the antisense oligonucleotide of the present invention is further modified, preferably chemically modified, in order to increase the stability and/or therapeutic efficiency of the antisense oligonucleotide in vivo. In particular, the antisense oligonucleotide used in the context of the invention may comprise modified nucleotides. Chemical modifications may occur at three different sites: (i) at phosphate groups, (ii) on the sugar moiety, and/or (iii) on the entire backbone structure of the antisense oligonucleotide. For example, the antisense oligonucleotide may be employed as phosphorothioate derivatives (replacement of a non-bridging phosphoryl oxygen atom with a sulfur atom) which have increased resistance to nuclease digestion. 2’-methoxyethyl (MOE) modification (such as the modified backbone commercialized by ISIS Pharmaceuticals) is also effective. Additionally or alternatively, the antisense oligonucleotide of the present invention may comprise completely, partially or in combination, modified nucleotides which are derivatives with substitutions at the 2' position of the sugar, in particular with the following chemical modifications: O-methyl group (2'-0-Me) substitution, 2-methoxyethyl group (2'-0-MOE) substitution, fluoro group (2 - fluoro) substitution, chloro group (2'-Cl) substitution, bromo group (2'-Br) substitution, cyanide group (2'-CN) substitution, trifluoromethyl group (2'-CF3) substitution, OCF3 group (2'-OCF3) substitution, OCN group (2'-OCN) substitution, O-alkyl group (2'-0-alkyl) substitution, S-alkyl group (2'-S-alkyl) substitution, N-alkyl group (2'-N-akyl) substitution, O-alkenyl group (2'-0- alkenyl) substitution, S-alkenyl group (2'-S-alkenyl) substitution, N-alkenyl group (2'-N- alkenyl) substitution, SOCH3 group (2'-SOCH3) substitution, S02CH3 group (2'-S02CH3) substitution, 0N02 group (2'-0N02) substitution, N02 group (2 -N02) substitution, N3 group (2 -N3) substitution and/or NH2 group (2 -NH2) substitution. Additionally or alternatively, the antisense oligonucleotide of the present invention may comprise completely or partially modified nucleotides wherein the ribose moiety is used to produce locked nucleic acid (LNA), in which a covalent bridge is formed between the 2’ oxygen and the 4’ carbon of the ribose, fixing it in the 3’-endo configuration. These constructs are extremely stable in biological medium, able to activate RNase H and form tight hybrids with complementary RNA and DNA. Accordingly, in a preferred embodiment, the antisense oligonucleotide used in the context of the invention comprises modified nucleotides selected from the group consisting of LNA, 2’- OMe analogs, 2’-phosphorothioate analogs, 2’-fluoro analogs, 2’-Cl analogs, 2’-Br analogs, 2’- CN analogs, 2’-CF3 analogs, 2’-OCF3 analogs, 2’-OCN analogs, 2’-0-alkyl analogs, 2’-S- alkyl analogs, 2’-N-alkyl analogs, 2’-0-alkenyl analogs, 2’-S-alkenyl analogs, 2’-N-alkenyl analogs, 2’-SOCH3 analogs, 2’-S02CH3 analogs, 2’-0N02 analogs, 2’-N02 analogs, 2’-N3 analogs, 2’-NH2 analogs and combinations thereof. More preferably, the modified nucleotides are selected from the group consisting of LNA, 2’-OMe analogs, 2’-phosphorothioate analogs and 2’-fluoro analogs. In some embodiments, the antisense is a Tricyclo-DNA antisense. The term“tricyclo-DNA (tc-DNA)” refers to a class of constrained oligodeoxyribo nucleotide analogs in which each nucleotide is modified by the introduction of a cyclopropane ring to restrict conformational flexibility of the backbone and to optimize the backbone geometry of the torsion angle g as (Ittig D, et al., Nucleic Acids Res, 2004, 32:346-353; Ittig D, et al., Prague, Academy of Sciences of the Czech Republic. 1 :21-26 (Coll. Symp. Series, Hocec, M., 2005); Ivanova et al., Oligonucleotides 2007, 17:54-65; Renneberg D, et al., Nucleic Acids Res, 2002, 15 30:2751-2757; Renneberg D, et al., Chembiochem, 2004, 5: 1114-1118; and Renneberg D, et al., JACS, 2002, 124:5993-6002). In detail, the tc-DNA differs structurally from DNA by an additional ethylene bridge between the centers C(3') and C(5') of the nucleosides, to which a cyclopropane unit is fused for further enhancement of structural rigidity. See e.g. WO2010115993 for examples of tricyclo- DNA (tc-DNA) antisense oligonucleotides. The advantage of the tricyclo-DNA chemistry is that the structural properties of its backbone allow a reduction in the length of an AON while retaining high affinity and highly specific hybridization with a complementary nucleotide sequence.

The inhibitor of expression may be delivered in vivo alone or in association with a vector. In its broadest sense, a "vector" is any vehicle capable of facilitating the transfer of the inhibitor of expression and preferably in endothelial cells expressing Fzd7, and more particularly in endothelial cells. In general, the vectors useful in the invention include, but are not limited to, plasmids, phagemids, viruses, other vehicles derived from viral or bacterial sources. Viral vectors are a preferred type of vector. In some embodiments, the vector is an adeno-associated virus (AAV) vector. As used herein, the term "AAV vector" means a vector derived from an adeno- associated virus serotype, including without limitation, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, and mutated forms thereof. AAV vectors can have one or more of the AAV wild-type genes deleted in whole or part, preferably the rep and/or cap genes, but retain functional flanking ITR sequences.

By a "therapeutically effective amount" of the inhibitor of the present invention as above described is meant a sufficient amount of the inhibitor for inhibiting retinal neovascularization at a reasonable benefit/risk ratio applicable to any medical treatment. It will be understood, however, that the total daily usage of the inhibitors and compositions of the present invention will be decided by the attending physician within the scope of sound medical judgment. The specific therapeutically effective dose level for any particular subject will depend upon a variety of factors including the disorder being treated and the severity of the disorder; activity of the specific inhibitor employed; the specific composition employed, the age, body weight, general health, sex and diet of the subject; the time of administration, route of administration, and rate of excretion of the specific inhibitor employed; the duration of the treatment; drugs used in combination or coincidental with the specific inhibitor employed; and like factors well known in the medical arts. For example, it is well within the skill of the art to start doses of the inhibitor at levels lower than those required to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved. However, the daily dosage of the products may be varied over a wide range from 0.01 to 1,000 mg per adult per day. Typically, the compositions contain 0.01, 0.05, 0.1, 0.5, 1.0, 2.5, 5.0, 10.0, 15.0, 25.0, 50.0, 100, 250 and 500 mg of the inhibitor of the present invention for the symptomatic adjustment of the dosage to the subject to be treated. A medicament typically contains from about 0.01 mg to about 500 mg of the inhibitor of the present invention, preferably from 1 mg to about 100 mg of the inhibitor of the present invention. An effective amount of the drug is ordinarily supplied at a dosage level from 0.0002 mg/kg to about 20 mg/kg of body weight per day, especially from about 0.001 mg/kg to 7 mg/kg of body weight per day.

The inhibitor of the invention may be used or prepared in a pharmaceutical composition. Typically, the inhibitor of the invention may be combined with pharmaceutically acceptable excipients, and optionally sustained-release matrices, such as biodegradable polymers, to form therapeutic compositions. "Pharmaceutically" or "pharmaceutically acceptable" refer to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to a mammal, especially a human, as appropriate. A pharmaceutically acceptable carrier or excipient refers to a non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type. In the pharmaceutical compositions of the present invention for oral, sublingual, intramuscular, intravenous, local or rectal administration, the active principle, alone or in combination with another active principle, can be administered in a unit administration form, as a mixture with conventional pharmaceutical supports, to animals and human beings. Suitable unit administration forms comprise oral-route forms such as tablets, gel capsules, powders, granules and oral suspensions or solutions, sublingual and buccal administration forms, aerosols, implants, intraperitoneal, intramuscular, intravenous and intranasal administration forms and rectal administration forms. Preferably, the pharmaceutical compositions contain vehicles which are pharmaceutically acceptable for a formulation capable of being injected. These may be in particular isotonic, sterile, saline solutions (monosodium or disodium phosphate, sodium, calcium or magnesium chloride and the like or mixtures of such salts), or dry, especially freeze-dried compositions which upon addition, depending on the case, of sterilized water or physiological saline, permit the constitution of injectable solutions. The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions; formulations including sesame oil, peanut oil or aqueous propylene glycol; and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases, the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi. Solutions comprising inhibitors of the invention as free base or pharmacologically acceptable salts can be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms. The inhibitor of the invention can be formulated into a composition in a neutral or salt form. Pharmaceutically acceptable salts include the acid addition salts (formed with the free amino groups of the protein) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, histidine, procaine and the like. The carrier can also be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetables oils. The proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminium monostearate and gelatine. Sterile injectable solutions are prepared by incorporating the active inhibitors in the required amount in the appropriate solvent with several of the other ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile- filtered solution thereof. Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective. The formulations are easily administered in a variety of dosage forms, such as the type of injectable solutions described above, but drug release capsules and the like can also be employed. For parenteral administration in an aqueous solution, for example, the solution should be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose. These particular aqueous solutions are especially suitable for intravenous, intramuscular and intraperitoneal administration. In this connection, sterile aqueous media which can be employed will be known to those of skill in the art in light of the present disclosure. Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject. In addition to the inhibitors of the invention formulated for parenteral administration, such as intravenous or intramuscular injection, other pharmaceutically acceptable forms include, e.g. tablets or other solids for oral administration; liposomal formulations; time release capsules; and any other form currently used.

The invention will be further illustrated by the following figures and examples. However, these examples and figures should not be interpreted in any way as limiting the scope of the present invention.

FIGURES:

Figure 1. Blocking Fzd7 signaling by antibody or soluble receptor prevents the development of aberrant neovascularization. A and B represents luciferase reporter assay to assess b-catenin activity. EA.hy926 human endothelial cells were transiently co-transduced with a lentivirus encoding with TOP-flash luciferase reporter gene and a lentivirus encoding with murine Fzd7 and were cultured in the presence of a control supernatant (CT sup) or Wnt3a conditioned media. After 24h treatment with Fzd7 soluble receptor (CRD) (A) or an anti-mouse Fzd7 antibody (anti-Fzd7 mAh) versus goat immunoglobulin G as a control (B), luciferase activity was measured. RFU indicated relative light units. Error bars represented standard error of mean from triplicates. (C and E) Schematic representations of Fzd7 pharmacological blocking in retinas from C57BF/6 mice at P12 after OIR. Immediately upon mice returned to room air, the right eye was injected intravitreally with 0.7 mg/mP of CRD (C) or 0.4 mg/mP of monoclonal anti-Fzd7 antibody (E), while the left eye was injected with equivalent doses of either PBS (C) or goat IgG control (E), respectively. At P17, retinas from intravitreally injected mice were collected, dissected as flat-mounts and stained with Iso lectin B4 (C) or CD31 (E). (D and F) Results of quantification of the % of avascular area and neovascular tufts area in P17 OIR retinas. In retinas treated with CRD, the relative % of both the avascular areas and those of neovascular areas significantly decreased compared with the control OIR eye; *p<0.05; paired Student’s / test (n=7) (D). In the retinas injected with anti-Fzd7 mAh, the % of avascular area was equivalent to those measured in the IgG injected contralateral eye but neovascular tufts significantly decreased; **p<0.0l; paired Student’s t test (n=l 1) (F).

EXAMPLE:

Material & Methods

Mutant mice

Floxed Fzd7 mice were generated previously in our laboratory by Ferreira et al (Ferreira-Tojais et al, Cardiovasc Res, 2014). The neomycin cassette was subsequently removed via flippase-mediated recombination by breeding with Flp mice. Floxcd mice were subsequently backcrossed on C57BL6/J background. Interbred, homozygous fzd7^F/F mice exhibit apparently normal development, and are viable and fertile. For EC-specific deletion, Platelet-derived growth factor b (Pdgfb)-iCre transgenic mice were bred with homozygote floxed Fzd7^f/f females to generate Pdgf-iCre(+); Fzd ^f/f and their corresponding wild-type littermates, Pdgf-iCre(-); Fzd ^F . Endothelial deletion of Fzd7 was obtained after intragastric or intraperitoneal administration of 50mg/kg Tamoxifen either at P1-P2 or at P12-P13 respectively, depending on the protocol. The number of backcrosses were > than 10. All mice used in this study were bred and maintained at the institute. This study was conducted in accordance with Bordeaux University institutional committee guidelines (committee CEEA50) and those in force in the European community for experimental animal use (L358-86/609/EEC). Tails of pups were genotyped by PCR using the P3/P4 primer set for the Pdgfb-iCre allele.

Oxygen-Induced Retinopathy Model

The experiments on the OIR model were carried out as described in detail previously (Smith et al, IOVS, 1994). Briefly, neonatal mice and their nursing mother were exposed to 75% oxygen at postnatal day 7 (P7) for 5 days. At P12, they were returned to normal room air. Animals were euthanized at P12, P14 or P17 to assess the degree of vascular regression and to determine the rate of retinal revascularization and preretinal neovascularization. As postnatal weight gain has been shown to affect outcome in the OIR model (Stahl et al, Am J Pathol, 2010) only the pups weighing > 5g at P17 were included in the study.

Tissue immunofluorescence

Immunostaining in whole-mounted retinas was conducted on eyes from pups as previously described (Dufourcq et al, Am J Pathol, 2008; Descamps et al., Circ Res, 2012). Mice were euthanized on P12, P14 and P17 and eyes were enuclated and fixed with 4% paraformaldehyde for 20 min. Corneas were removed with scissors along the limbus and intact retinas were dissected. Blocking/permeabilization was performed using a blocking buffer adapted from protocol by Franco et al (Franco et al, Development, 2013), consisting of 10% Donkey serum (Interchim, San Diego, USA), 0.5% triton XI 00 (Euromedex, Souffelweyersheim, France), 0.01% Na deoxycholate (Fisher Scientific, Hampton, USA), in PBS for lh at room temperature on a rocking platform. Primary and secondary antibodies were incubated at the desired concentration in 1 : 1 blocking buffer:PBS at 4°C overnight in a rocking platform. Primary antibodies used for staining retinas were: Hypoxyprobe plus Kit (Hypoxyprobe, Burlington, USA), anti-BrdU (Abeam, Cambridge, UK), anti-CD3 l (BMA, Augst, Switzerland), Isolectin-B4-FITC (Sigma, Saint-Louis, USA), anti-Ki67 (Abeam), anti- Jaggedl (R&D, Minneapolis, USA) and anti-DH4 (R&D). Retinas were mounted on slides using Vectashield mounting medium (Vector Laboratories, Burlingame, USA). For retinal imaging we used a Carl Zeiss VI 6 Axiozoom and a Carl Zeiss LSM700 scanning confocal microscope (Carl Zeiss, Jena, Germany).

Analysis of retinal vasobliteration, neovascularization and proliferation

The two main readouts in OIR are vasobliteration (VO) and subsequent neovascularization (NV). VO develops during the hyperoxic phase of the model, and NV follows from P14 onward. Percentages of VO and retinal NV were calculated by comparing the central avascular area or the vitreoretinal neovascular area respectively to the total retinal area, using a computer-aided technique allowing semi-automated quantification based on ImageJ software (National Institutes of Health, Bethesda, USA) together with appropriate homemade plugins and macros.

For fluorescence analysis done by confocal microscopy, 3D images were analyzed and quantified by Bitplane Imaris software (Bitplane AG, Zurich, Switzerland).

For in vivo proliferation assays, intraperitoneal (IP) injection of 50m1 of 5-Bromo-2’- desoxyuridine (BrdU) (Sigma) at lOmg/ml was performed and mice sacrificed after 4 hours at P17. After PFA fixation and dissection, anti-BrdU (Abeam) and IB4 (Sigma) staining was performed. To assess vessel proliferation, the ratio of BrdU and IB4 positive cells/volume of tufts (pm³) was analyzed using Bitplane Imaris software. Proliferation was also examined after Ki67 staining on flat-mounted retinas and evaluated by calculating the ratio of Ki67 positive cells/volume of tufts (pm³).

Mouse intravitreal injection and treatment

For intravitreal injection, mice were anesthetized with Isoflurane (Aerrane®, Baxter, Mississauga, Ontario, USA) and with a topical anesthetic of Oxybuprocaine chlorhydrate at 1.6 mg/0.4 mL (Thea, Clermont-Ferrand, France). A volume of 0.5pL delivered with UltraMicroPomp III (World Precision Instruments, Sarasota, USA) was used to inject the different solutions of Fzd7 soluble receptor CRD (Biotem, Aprieu, France), anti-mouse Fzd7 monoclonal antibody (R&D, Minneapolis, USA), goat anti-mouse Immunoglobulin G (Sigma) and PBS. For intravitreal injection, mice were injected in the right eye at P12 with CRD at 0.7 mg/mL or anti-Fzd7 mAb at 0.40 mg/ml. PBS or goat IgG control was injected respectively in the contra-lateral eye of the same animal to account for biological variation of retinal vascular development, and animals were sacrificed at P17 for analysis.

Cell culture and reporter gene assay EA.hy926 cells were cultured in DMEM (Dulbecco’s Modified Eagle Medium, Life Technologies, Carlsbad, USA) supplemented with 10% FBS (Fetal Bovine Serum, Life Technologies) and penicillin-streptomycin (Life Technologies).

For reporter gene assay, EA.hy926 cells were transiently co-transduced with a lentivirus encoding with TOP-flash luciferase reporter gene (#468 7xTcf-Ffluc LV 24306) and a lentivirus encoding with murine Fzd7 (#422 MNDEGFP mFZ7) as described previously (Ferreira-Tojais et al, Cardiovasc Res, 2014; Sewduth et al, Nat Commun, 2014). Lentivirus preparations were produced at the Bordeaux University lentivirus platform.

Cells were incubated with a control supernatant or Wnt3A conditioned media (CM). Wnt3 A CM was obtained from cell lines expressing Wnt3 A (L Wnt3A CRL 2647) as described in the American Type Culture Collection (ATCC) protocol. Luciferase activity was determined as previously described (Descamps et al., Circ Res, 2012) after 24h of Wnt3A activation and soluble receptor CRD (Biotem) or anti-mouse Fzd7 antibody (R&D) treatment.

MTT assay

MTT assay (Thermofisher Scientific, Waltham, USA) was employed to observe the growth of EA.hy926 cells transiently transduced with the lentivirus encoding with murine Fzd7 after Wnt3A activation and anti-Fzd7 antibody (R&D) treatment. MTT assay was performed following the experimental protocol described by Thermofisher Scientific. Absorbance of samples was measured at wavelength of 570nm 0, 24, 48 and 72h after blocking treatment with anti-Fzd7 antibody.

RNA preparation and qPCR

Mouse tissues or cells were homogenized in TRI-REAGENT™ (Euromedex) and RNA extracted according to the manufacturer’s instructions. qPCR was performed as previously (Ferreira Tojais N et al, Cardiovasc Res. 2014). All experiments were performed in duplicate and differences in cDNA input were compensated by normalization to expression of b-actin.

Statistical analysis

Results were expressed as mean ± SD or mean ± min to max. Statistical significance was determined by two-tailed Student's /-test. Multiple group comparison was performed with a one-way ANOVA followed by Bonferroni’s post-test correction. A value of / <0.05 was considered to be statistically significant.

Results

Fzd7 expression is up regulated after OIR and its endothelial deletion modulates response to hyperoxia/hypoxia To study the involvement of Fzd7 in a context of retinal ischemia, we used the well characterized oxygen- induced retinopathy (OIR) model (Smith et ah, 1994) (Data not shown) which recapitulates pathologies of both ROP and proliferative DR and is commonly used as a studying model of retinopathy (Stahl A et al. OVS, 2010). Mice neonates and their nursing mother were exposed to 75% oxygen for five days from ages P7-P12. This high oxygen environment suppresses oxygen-regulated growth factors causing inhibition of vessel growth and a central vessel loss (vasobliteration). Stopping oxygen therapy at P12 leads to an inadequate retinal perfusion resulting in a relative hypoxia of retinal tissue. Hypoxia then stimulates intravitreal vascular proliferation and triggers both normal vessel regrowth and a pathologic formation of extra-retinal neovascularization (NV) with a maximum severity at P17.

To determine the molecular determinants induced by OIR, real-time PCR were performed on retinas from non-OIR and OIR mice, both at P12 and P17. As expected, retinal mRNA levels of Vegf a major pro-angiogenic factor, were substantially higher in OIR mice than in room-air controls, with a maximum of 2.5 fold increase at P17 (p<0,00l; Data not shown). We observed that Fzd7 was expressed at the two stages of OIR, with a specific induction of Fzd7 transcripts at P17, corresponding to maximal NV in OIR retinas as compared to controls (p<0.05, Data not shown). By comparison, mRNA expression levels of Fzdl and Fzd2 were not modulated in OIR retinas, whereas these two receptors share a strong structural homology to Fzd7 (Data not shown). Consistent with the literature, our data confirmed that b- catenin mRNA increased during OIR (Data not shown). Temporally, expression profiles of retinal b-catenin and Fzd7 mRNA were strongly correlated during the phase 2 of OIR, suggesting a close relationship between Fzd7/Wnt-canonical pathway and the vasoproliferative phase of retinopathy.

To explore the role of Fzd7 in retinal pathological angiogenesis, we used mice with an inducible endothelium-restricted deletion of the Fzd7 gene using the Platelet-derived growth factor b (Pdgfb)-iCre/loxP system, as previously described (Peghaire et al, ATVB, 2016). Deletion of Fzd7 was induced at P1/P2 by Tamoxifen intragastric injections and the retinal vascular phenotype was characterized at P 12 to study phase 1 or at P 14 and P17 to study phase 1+2 of the OIR model.

At P12, mice had just returned from a hyper-oxygen environment, leading to vasobliteration, vessel regression and subsequent hypoxia of the retinal tissue as confirmed by double-labeling retinal vasculature and hypoxic zones with CD31 and Pimonidazole (Hypoxyprobe) in both Fzd7 EC-WT mice (Pdgf-iCre(-); Fzd ^f//) and Fzd7 EC-deleted mice (Pdgf-iCre(+); Fzd ' ) (Data not shown). Phenotype quantification at P12 revealed that Fzd7 deletion in EC at P1/P2 was associated with larger hypoxic and avascular zones in retinas than control (respectively 44% versus 36%, 29% versus 24%, p<0.0l; Data not shown).

During the second phase of OIR, coverage of the avascular area by newly formed vessels was evaluated by measuring the avascular area decreased between P12 and P17. No difference in avascular area reduction was observed between control and Fzd7 EC-deleted mice (14% versus 15%, Data not shown), revealing that normal revascularization of the retina takes place at the same speed between the two groups of mice, as a Fzd7-independent process.

Moreover, examination of the area of pre-retinal tufts in IB4-labeled flat-mount retinas at P17 (Data not shown) suggested that Fzd7 EC-deleted mice has developed less severe pathologic neovascular processes than control mice. Quantification of aberrant angiogenesis by relating the area covered by NV to the total retinal area confirmed that in Fzd7 EC-deleted mice, the % of NV decreased compared to Fzd7 EC-WT mice (3.3% versus 7.2% at P14, p<0.00l; 3.8% versus 8.8% at P17, p<0.05; Data not shown).

Neovessels development is strongly correlated to EC proliferation in the tufts. We then investigated proliferation in tufts by evaluating BrdU incorporation in EC. After IB4/BrdU double staining, we showed that the number of BrdU+ endothelial cells in tufts decreased by 50% after specific endothelial cell Fzd7 deletion (p<0.05; Data not shown).

These findings demonstrate that Fzd7 modulates response to OIR in the both phases.

Specific endothelial deletion of Fzd7 during the vasoproliferative phase of OIR decreases the ectopic growth of neovessels by limiting EC proliferation in tufts

To explore how Fzd7 endothelial deletion could specifically affect the NV phase of OIR, gene deletion was obtained after intraperitoneal injections of Tamoxifen at P12 and P13, just after Pdgf-iCre;

pups returned to room air (Data not shown). Vascular phenotype was analyzed at P17 by quantifying the avascular area and abnormal neovascular area with IB4 staining in whole-mounted retinas (Data not shown). At P17 as expected the percentage of avascular area was not modified in the both Fzd7 EC-WT mice (Pdgf-iCre(-); Fzd- ) and Fzd7 EC-deleted mice (Pdgf-iCre(+); Fzd ¹¹) (Data not shown). In contrast, visual appearance of the blood vessel system clearly differed in the two groups of mice. Fzd7 EC-WT retinas appeared more severely affected than in Fzd7 EC-deleted retinas, with a larger number of clusters and disorganized, small-sized vascular tufts (Data not shown). Quantification established that the extent of neovascularization in Fzd7 EC-deleted mice was significantly decreased at P17 as compared to littermates (13.2% versus 7.9%, p<0.05; Data not shown).

Lastly, we studied the expression of Ki67, a marker for cellular proliferation, at P17 of OIR. The anti-proliferative effect of Fzd7 deletion was confirmed on retinas after KΪ67/IB4 double-staining (Data not shown). As seen in Fig. 2G, quantitative analysis revealed that the number of Ki67+ cells in tufts was drastically reduced after Fzd7 deletion (p<0.00l, Data not shown).

All these results corroborate that Fzd7 could promote ECs proliferation in pathological vascularization of the retina and identify Fzd7 as a new target for therapeutic inhibition of pathological angiogenesis in proliferative retinopathy.

Blocking Fzd7 signaling by antibody or soluble receptor prevents the development of aberrant neovascularization

To evaluate Fzd7 as a potential anti-angiogenic target in eye diseases, we have experimented two blocking strategies: a monoclonal antibody (anti-Fzd7 mAb) that recognizes specifically Fzd7 extracellular domain for blocking Fzd7 signaling at the receptor level, and a soluble Fzd7 receptor (CRD domain) that traps Wnt molecules. First, using a TOP-Flash reporter construct for canonical b-catenin signaling (that contain TCF binding sites upstream of the luciferase) and EAHY926 cell overexpressing Fzd7, we measured the effect of Fzd7 blocking agents on luciferase activity. Both Fzd7 CRD and anti-Fzd7 mAb significantly decreased the activity of b-catenin reporter gene induced by Wnt3A in endothelial cells (Fig 1AJ3), when compared to control conditions. Interestingly, the magnitude of inhibitory effects on TOP-Flash activity was concentration-dependent for both CRD and anti-Fzd7 mAb.

After in vitro validation of both pharmacological tools, we then explored the effect of the soluble receptor and Fzd7 antibody on the OIR model by direct intravitreal micro -injections into C57/B16 mice at P12. Immediately upon return to room air, the right eye was injected intravitreally with 0.7 mg/mF of CRD (Fig 1C) or 0.4 mg/mF of monoclonal anti-Fzd7 antibody (Fig I E), while the left eye was injected with equivalent doses of either PBS or goat IgG control, respectively.

We demonstrated that CRD treatment was effective in reducing both avascular area (- 6%; p<0.05) and retinal neovascular area (-6.3%; p<0.05) at P17, when comparing treated versus control eye that was injected with PBS (Fig 1 C. D). In addition, analysis of the vascular phenotype after direct injection of the specific antibody (0.40 mg/mF) revealed that Fzd7 inhibition also attenuated neovascularization tufts toward the vitreous of treated compared to contralateral eyes (-3.2%; p<0.0l) whereas no difference in the percentage of vasobliteration was observed at P17 (Fig 1 E, F). Consistent with the results obtained by genetic approaches, these findings demonstrate that blocking Fzd7 with specific antibody or soluble receptor protects from development of aberrant angiogenesis in OIR and strongly suggests that Fzd7 may be an efficient target to impair neovascular phase in eye diseases. Because the mechanisms of action of soluble receptor and Fzd7 antibody are relatively distinct, we proposed that Fzd7 inhibition by specific antibody could be a more selective approach in limiting neovessels formation during proliferative retinopathy. Thus, next experiments were focused on the protective effect of Fzd7 blocking antibody.

Fzd7 blocking antibody inhibits proliferation of EC both in vitro and in vivo

To have proof that pharmacological inhibition of Fzd7 could also control this proliferative process, monoclonal anti-Fzd7 antibody or goat IgG control was delivered into vitreous of pups following previously described protocol /Fig 1E). Then, we analyzed the expression of Ki67 within tufts at P17 of OIR. Three-dimensional image projections of the confocal images displayed the morphological complexity of vascular tufts, as illustrated with disorganized neo vascular network and high vessel density. Anti-Fzd7 mAh treatment apparently decreased the number of Ki67 positive cells in tufts, in comparison to equivalent injection of control IgG in the contralateral eye (Data not shown). Quantification of Ki67 positive endothelial cells confirmed the anti-proliferative effect of anti-Fzd7 versus control antibody ( 1.3.1 O ³ versus 0.48.10³, p<0.0l, Data not shown).

In order to validate the results obtained in mice retinas, we employed MTT assay observing the growth of endothelial cells in vitro. EA.hy926 cells overexpressing Fzd7 receptor were first activated by Wnt3A and then incubated in the presence of either goat IgG control or anti-Fzd7 antibody. Colorimetric determination of MTT incorporation showed a drastic reduction of 47%, 40% and 49% of proliferation in cells treated with Fzd7 antibody compared to control at 24h, 48h or 72h after seeding, respectively (r<0.001, r<0.01, p<0.00l, Data not shown). These results traduce that pharmacological inhibition of Fzd7 is able to abrogate proliferation of endothelial cells both in vitro and in a murine model of retinopathy. Canonical Wnt signaling leads to b-catenin accumulation in the cytoplasm which translocates to the nucleus to modify gene transcription namely genes involved in proliferation. In the present study, we examined mRNA expression of Wnt-target genes, specifically LEF1 and AXIN2, in EA.hy926 cells overexpressing Fzd7. After 48h of anti-Fzd7 antibody treatment, a slight decrease of LEF1 and AXIN2 expression was observed (Data not shown), in agreement with our previous result demonstrating that anti-Fzd7 mAh dose-dependently decreased the activity of b-catenin reporter gene induced by Wnt3A in vitro (Data not shown). Similar findings were observed in eyes of mice that were exposed to OIR and after anti-Fzd7 treatment, indicating that administration of the antibody markedly decreased the expression of b-catenin, Lefl and Axin2 at transcriptional levels (Data not shown). These observations, in line with previous results from our recent study demonstrating that Fzd7 activates the Wnt canonical pathway in EC to control physiologic retinal vascular development (Peghaire et al, ATVB 2016), are consistent with the mode of signaling for the Fzd7 receptor, through a Wnt-canonical signaling.

Fzd7 controls Jaggedl Notch ligand expression in pathological angiogenesis

The Notch pathway is crucial for postnatal vascular development in retina and has recently been described to be markedly dysregulated in retinal endothelial cells of diabetic mice, as illustrated with a high expression of Notch ligand Jaggedl in retinal capillaries (Yoon et al, Circ, 2016). We therefore hypothesized that Notch pathway could be involved in the pathogenesis of OIR. By quantitative PCR, we observed that Jaggedl mRNA expression was upregulated in OIR retinas compared with those in controls, more particularly at P17 corresponding to the proliferative phase of retinopathy (Data not shown). Whole-mounted retinas from OIR mice displayed a strong expression of Jaggedl, specifically localized into the tufts (Data not shown), in contrast to D114, another important Notch ligand, whose expression was increased during OIR (Data not shown) but limited to normal arteries and to intermediate and deep retinal vascular layers under the tufts (Data not shown). Collectively, these results indicated altered Notch signaling in retinopathy.

Notch pathway has been shown to be regulated by b-catenin transactivation activity in embryonic EC or during tumoral angiogenesis (Ref). More recently, we have reported that Fzd7 acts via b-catenin activation, upstream of Notch signaling to control D114 and Jaggedl endothelial expression during retinal vascular development (Peghaire et al, ATVB, 2016). By tridimensional analysis of confocal images, we confirmed that Fzd7 blocking was associated with a less severe appearance of pre-retinal tufts and we observed that Jaggedl expression was strictly correlated to the disorganized and hyperproliferative vascular structures compared to morphologically normal vessels (Data not shown). Fzd7 endothelial deletion led to a significant decrease of Jaggedl transcript in mice retinas (p<0.0l; Data not shown). Similar changes in retinal Jaggedl mRNA expression were found when anti-Fzd7 antibody was administered by intravitreal injections in mice retinas after OIR, highlighting the involvement ofNotch signaling downstream of Fzd7 in the pathogenesis of OIR (p<0.05; Data not shown). These results suggest that Fzd7 may control EC proliferation via the activation of the Wnt^-catenin and Notch signaling regulators, Lefl and Jaggedl respectively.

REFERENCES:

Throughout this application, various references describe the state of the art to which this invention pertains. The disclosures of these references are hereby incorporated by reference into the present disclosure.

Claims

CLAIMS:

1. A method of treating retinal neovascularization in a patient in need thereof comprising administering to the subject a therapeutically effective amount of a Fzd7 inhibitor.

2. The method of claim 1 wherein the patient suffers from an ischemic retinopathy.

3. The method of claim 1 the patient suffers from diabetic retinopathy, age-related macular degeneration, neovascular glaucoma, retinopathy of prematurity, sickle-cell retinopathy, retinal vein occlusion, oxygen induced retinopathy, and neovascularization due to ocular insults such as traumatic or surgical injury, or transplantation of eye tissue.

4. The method of claim 1 wherein the Fzd7 inhibitor is an antibody having specificity for Fzd7.

5. The method of claim 4 wherein the antibody of the present invention binds to an epitope located in the extracellular domain that ranges from the amino acid residue at position 33 to the amino acid residue at position 256 in SEQ ID NO: l.

6. The method of claim 4 wherein the antibody inhibits the binding of Fzd7 to a Wnt molecule.

7. The method of claim 4 wherein the antibody is a single domain antibody.

8. The method of claim 4 wherein the antibody is a chimeric, humanized or human antibody.

9. The method of claim 1 wherein the Fzd7 inhibitor is a polypeptide that bind a Wnt molecule and comprise all or a portion of the extracellular domains of Fzd7 so as to form a soluble receptor that is capable to trap a Wnt molecule.

10. The method of claim 9 wherein the polypeptide has an amino acid sequence comprises an amino acid sequence having at least 90% of identity with the amino acid sequence ranging from the amino acid residue at position 33 to the amino acid 256 in SEQ ID NO: l .

11. The method of claim 9 wherein the polypeptide is fused to an immunoglobulin constant domain (Fc region) to form an immunoadhesin.

12. The method of claim 1 wherein the Fzd7 inhibitor is an inhibitor of Fzd7 gene expression.

13. The method of claim 12 wherein the inhibitor of gene expression is a siR A, an endonuclease, an antisense oligonucleotide or a ribozyme.

14. The method of claim 12 wherein the inhibitor of gene expression is an endonuclease.