WO2022086501A1

WO2022086501A1 - Methods and compositions for treating ocular vascular disorders

Info

Publication number: WO2022086501A1
Application number: PCT/US2020/056469
Authority: WO
Inventors: Martin Friedlander; Peter E. Wright; Rebecca B. Berlow BERLOW; Ayumi Usui-ouchi USUI-OUCHI
Original assignee: The Scripps Research Institute
Priority date: 2020-10-20
Filing date: 2020-10-20
Publication date: 2022-04-28

Abstract

This invention provides methods and related compositions for treating and preventing ocular vascular diseases, e.g., hypoxia induced retinal neovascularization, and for promoting revascularization of the ischemic retina. The therapeutic methods of the invention entail administering to a subject in need of treatment a pharmaceutical composition that contains, or is capable of producing, an effective amount of a VEGF antagonist and a CITED2 derived peptide. The pharmaceutical composition is preferably administered by intravitreal injection.

Description

METHODS AND COMPOSITIONS FOR TREATING OCULAR VASCULAR DISORDERS

STATEMENT CONCERNING GOVERNMENT SUPPORT

[0001] This invention was made with government support under grant numbers EY011254, CA096865, and CA229652 awarded by the National Institutes of Health. The government has certain rights in the invention.

FIELD OF THE INVENTION

[0002] The present invention generally relates to methods and compositions for treating ocular vascular disorders and ocular degenerative diseases.

BACKGROUND OF THE INVENTION

[0003] The retina requires a continuous supply of oxygen (O2) and is known as one of the most metabolically active tissues, consuming O2 more rapidly than any other tissue including the brain. Retinal vascular abnormalities are observed in diabetic retinopathy (DR), retinopathy of prematurity (ROP), and retinal vein occlusion (RVO), leading to ischemia and subsequent hypoxia-induced neovascularization (NV) and fibroproliferation, which are major causes of vision loss or blindness.

[0004] A number of studies have demonstrated the critical role of the pro-angiogenic cytokine VEGF in development of pathological NV in the eye. Anti-VEGF drugs have been used clinically to treat choroidal NV and macular edema, as well as hypoxia-induced NV in patients with DR or ROP. While anti-VEGF therapy has dramatically improved our ability to treat these patients, stabilizing or improving visual outcomes in approximately 60% of treated patients, directly targeting VEGF has potential drawbacks. Many patients do not respond to VEGF -targeting therapy, and those who do may experience negative side effects. Furthermore, since the trophic functions of VEGF are critical to maintaining cardiovascular, renal, and nervous tissues including retinal neurons, blood vessels, and the retinal pigment epithelium (RPE), there are safety concerns associated with repeated intravitreal administration of anti-VEGF therapies in ischemic retinal diseases, especially for diabetic patients or premature babies who are particularly vulnerable to disruption of crucial trophic factors.

[0005] There is a critical unmet medical need for additional therapeutic strategies in the treatment of ischemic retinal diseases, and specifically hypoxia-induced NV. The present invention addresses this and other unfulfilled needs in the art.

SUMMARY OF THE INVENTION

[0006] In one aspect, the present invention provides methods for inhibiting retinal neovascularization and promoting revascularization in a subject. The methods involve administering to a subject in need of treatment a pharmaceutical composition containing or capable of expressing a therapeutically effective amount of (a) a VEGF antagonist and (b) a CITED2 peptide that can bind to the TAZ1 domain of the general transcriptional coactivators CBP and p300. A combination of the two agents enables inhibition of hypoxia- induced retinal neovascularization and promoting revascularization in the subject. In some embodiments, the subject to be treated is one afflicted with an ocular disorder associated with retinal neovascularization. For example, the subject can be one who has or is at risk of developing ischemic retinopathy, diabetic retinopathy, retinopathy of prematurity, retinal vein occlusion, neovascular glaucoma, macular edema, familial exudative vitreoretinopathy, retinal angioma, macular degeneration, or retinitis pigmentosa.

[0007] In some embodiments, the involved CITED2 peptide is derived from the disordered C-terminal transactivation domain of CITED2. Typically, the employed CITED2 peptide contains or encompasses a sequence DEEVLMX²⁰LVIX²⁴MGLX²⁸RIX³¹X³² (SEQ ID NO:51) or a conservatively modified variant thereof, wherein X²⁰ is S, K, D, E, A, P, or phosphoSer; X²⁴ is E, K or R; X²⁸ is D, E, I, L, M or W; X³¹ is K or P; and X³² is E or P. In some exemplified embodiments, the CITED2 peptide can have the amino acid shown in any one of SEQ ID NOs:2-23, a substantially identical sequence or a conservatively modified variant thereof. In some embodiments, the involved VEGF antagonist is a VEGF specific inhibitor polypeptide, an antibody, or an aptamer. In some of these embodiments, the VEGF antagonist is Aflibercept, Ranibizumab, Bevacizumab, or Pegaptanib. In some methods, the administered pharmaceutical composition contains or is capable of expressing (1) VEGF antagonist is Aflibercept or a functional derivative thereof, and (2) a CITED2 peptide with an amino acid sequence shown in any one of SEQ ID NOs:2-23 or a conservatively modified variant thereof.

[0008] In some embodiments, the administered pharmaceutical composition contains an engineered cell that stably or transiently expresses the VEGF antagonist and/or the CITED2 peptide. In some of these methods, the engineered cell expresses both the VEGF antagonist and the CITED2 peptide. In some embodiments, the pharmaceutical composition contains the CITED2 peptide and an engineered cell that expresses the VEGF antagonist. In some other embodiments, the pharmaceutical composition contains the VEGF antagonist and an engineered cell that expresses the CITED2 peptide. In various embodiments, the engineered cell in the pharmaceutical composition is derived from retinal pigment epithelium. In some embodiments, the engineered cell in the pharmaceutical composition is encapsulated in porous material prior to administration to the subject. In some embodiments, the pharmaceutical composition is administered to the subject via intravitreal injection. In some preferred embodiments, the subject to be administered the pharmaceutical composition is a human patient.

[0009] In a related aspect, the invention provides methods for treating or preventing an ocular vascular disorder associated with hypoxia-induced neovascularization in a subject. These methods entail administering to the subject a pharmaceutical composition containing or capable of expressing a therapeutically effective amount of (a) a VEGF antagonist and (b) a CITED2 peptide that can bind to the TAZ1 domain of the general transcriptional coactivators CBP and p300. The presence of a combination of the two agents enables treatment and/or prevention of the ocular vascular disorder in the subject. Some of the methods are directed to treating ocular vascular disorders that are manifested by retinal neovascularization and/or vaso-obliteration. In various embodiments, the ocular vascular disorder to be treated is ischemic retinopathy, diabetic retinopathy, retinopathy of prematurity, retinal vein occlusion, neovascular glaucoma, macular edema, familial exudative vitreoretinopathy, retinal angioma, macular degeneration or retinitis pigmentosa. In some embodiments, the subject to be treated is one who is afflicted with the ocular disorder. In some other embodiments, the subject to be treated is one who is at risk of developing, or predisposed to develop, the ocular disorder.

[0010] In another aspect, the invention provides pharmaceutical compositions or kits that can be used in the therapeutic methods described herein. In various embodiments, the pharmaceutical compositions or kits can contain (a) a VEGF antagonist and a CITED2 derived peptide that specifically binds to the TAZ1 domain of transcriptional coactivators CBP/p300; (b) a CITED2 derived peptide that specifically binds to the TAZ1 domain of transcriptional coactivators CBP/p300, and an engineered cell expressing a VEGF antagonist; (c) a VEGF antagonist, and an engineered cell expressing a CITED2 derived peptide that specifically binds to the TAZ1 domain of transcriptional coactivators CBP/p300; or (d) an engineered cell expressing a VEGF antagonist and a CITED2 derived peptide that specifically binds to the TAZ1 domain of transcriptional coactivators CBP and p300. In some embodiments, the VEGF antagonist is Aflibercept or a conservatively modified variant thereof. The employed CITED2 peptide contains or encompasses a sequence DEEVLMX²⁰LVIX²⁴MGLX²⁸RIX³¹X³² (SEQ ID NO:51) or a conservatively modified variant thereof, wherein X²⁰ is S, K, D, E, A, P, or phosphoSer; X²⁴ is E, K or R; X²⁸ is D, E, I, L, M or W; X³¹ is K or P; and X³² is E or P. In some exemplified embodiments, the CITED2 peptide can have the amino acid shown in any one of SEQ ID NOs:2-23, or a conservatively modified variant thereof.

[0011] In still another aspect, the invention provides polynucleotide sequences encoding or vectors expressing both (a) a VEGF antagonist polypeptide and (b) a CITED2 derived peptide that specifically binds to the TAZ1 domain of the general transcriptional coactivators CBP and p300. In some embodiments, the VEGF antagonist is Aflibercept or a conservatively modified variant thereof. In some embodiments, the CITED2 derived peptide has an amino acid sequence shown in any one of SEQ ID NOs:2-23 or a conservatively modified variant thereof.

[0012] In another related aspect, the invention provides engineered cells that express a VEGF antagonist polypeptide and a CITED2 peptide that can bind to the TAZ1 domain of the general transcriptional coactivators CBP and p300. In some of these embodiments, the VEGF antagonist polypeptide and/or the CITED2 peptide are stably expressed. In some other embodiments, the VEGF antagonist polypeptide and/or the CITED2 peptide are transiently expressed.

[0013] A further understanding of the nature and advantages of the present invention may be realized by reference to the remaining portions of the specification and claims.

DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 shows that combination therapy with a CITED2 peptide and Aflibercept rescues retinal neovascularization and vaso-obliteration in OIR. (A) Immunofluorescent staining of OIR retinas at Pl 7 after intravitreal injection of Aflibercept (200 ng), Aflibercept and CITED2 peptide (200 ng Aflibercept and 3.4 ng CITED2 peptide), or CITED2 peptide (3.4 ng) at P12. Retinal whole mounts were stained with GS-lectin. Representative images for each treatment are shown in the upper panel and the same images are shown in the lower panel with NV highlighted and VO highlighted as used for quantification. Scale bars: 1 mm. (B) Quantification of the percentage of VO area in whole retinas. (C) Quantification of the percentage of NV area in whole retinas. For (B) and (C), n > 7 per group. -values were calculated using one-way ANOVA with Tukey’s multiple comparisons test. *P < 0.05, **P < 0.01, ***P < 0.001, ****/> < 0.0001. (D) Validation of expression of HIF target genes in CITED2 and Aflibercept treated OIR retinas. Total RNA was isolated 24 hours after intravitreal injection of CITED2 peptide (3.4 ng) or Aflibercept (10 pg) on P12 (n=6 per group). -values were calculated using multiple t-tests. **P < 0.01, *** P < 0.001, **** P < 0.0001. (E) Quantification of VEGF protein levels in P15 retinas by ELISA (n=6 per group). P- values were calculated using one-way ANOVA with Tukey’s multiple comparisons test. *P < 0.05, **** P < 0.0001. For panels (B), (C), (D), and (E), the mean and SEM are shown.

DETAILED DESCRIPTION

I. Overview

[0015] The invention disclosed herein is related to methods and compositions for inhibiting or preventing hypoxia-induced retinal neovascularization and for promoting revascularization of ischemic retina. The invention is derived in part from the studies undertaken by the present inventors to target molecules upstream of the VEGF signaling cascade, such as the hypoxia inducible transcription factor HIF-la. In the ischemic retina, HIF-la promotes the expression of VEGF and other angiogenic factors. HIF-la also modulates tissue adaptation to hypoxia by directing the expression of genes involved in glycolysis, homeostasis, angiogenesis, and vasculogenesis. One such gene encodes the intrinsically disordered protein CITED2, which functions as a negative feedback regulator of HIF-la by competing for binding to the TAZ1 domain of the general transcriptional coactivators CBP and p300. The oxygen-induced retinopathy (OIR) mouse model of hypoxia-induced NV is a well-established model for investigating putative anti-angiogenic compounds. As detailed herein, the inventors observed that intravitreal injection of a 59- amino acid CITED2 peptide (SEQ ID NO:4) in OIR mice dramatically rescues both hypoxia-induced retinal NV and VO while also promoting revascularization of the damaged retina. In contrast, anti-VEGF therapies such as Aflibercept only rescue NV, but not VO, in OIR mice. Importantly, when combined with the CITED2 peptide, a significant reduction of both NV and VO can be observed with greater than 50-fold lower doses of the VEGF antagonist (Figure 1). In addition to the native CITED2 peptide, a number of derivative peptides (e.g., SEQ ID NOs:5-23) were also examined for their activities in competing against HIF-la. These include CITED2 derivative peptides that contain, relative to the exemplified native peptide, terminal truncations, internal substitutions, and/or terminal addition of non-native residues (e.g., a nuclear localization signal sequence).

[0016] Results from the studies demonstrate that targeting the interaction between HIF- la and CBP/p300 as well as the circulating levels of VEGF in the ischemic retina is beneficial not only for inhibiting pathological neovascularization, but also for promoting the normalization of disrupted vasculature. These data indicate that combinations of the CITED2 peptide with VEGF antagonists or other modulators of pathological angiogenesis could be useful as novel therapeutic agents for the treatment of neovascular eye diseases.

[0017] In accordance with these findings, the invention provides combination therapies for treating and/or preventing ocular vascular disorders. In particular, the invention is suitable for treating hypoxia-induced retinal neovascularization and for promoting revascularization of the ischemic retina. By dual targeting of HIF-la and VEGF with, e.g., combinations of CITED2 peptides and VEGF antagonists (e.g., Aflibercept, ranibizumab, and bevacizumab), the therapeutic methods of the invention could have broad applications in treatment of diseases and disorders in which hypoxia plays a role. These include, but are not limited to, blinding eye diseases (including diabetic retinopathy, macular telangiectasia, age- related macular degeneration, and retinopathy of prematurity), cancer, cardiovascular disease, inflammation, and developmental disorders.

[0018] There are a number of surprising and unexpected advantages associated with the methods of the invention. VEGF antagonists are a popular class of therapeutics for angiogenesis-related diseases. Directly targeting VEGF with VEGF antagonists for angiogenesis-related diseases has significant drawbacks. Many patients do not respond to VEGF targeting therapy, and those that do may experience negative side effects. Additionally, complete inhibition or knockdown of VEGF itself can lead to major neurovascular defects since VEGF is a potent neurovascular agent. Further, known anti- VEGF therapies for retinal neovascular diseases such as Aflibercept, rescued NV but not VO in OIR. In contrast, the combination therapy described herein, e.g., combining a CITED2 peptide and Aflibercept, significantly decreased both NV and VO. Importantly, the therapeutic effect of the combination therapy can be achieved with a significantly lower (> 50-fold lower) dose of Aflibercept. Such a synergistic effect produced by the combination therapy can be very beneficial to minimize the off-target effects of VEGF antagonists. Intravitreal injection, typically used in anti -VEGF therapies, has the advantage of limiting off-target systemic effects. However, the high doses of VEGF antagonists administered by intravitreal injection are known to reduce VEGF levels outside of the eye, which introduces additional risks especially for patients with diabetic retinopathy and retinopathy of prematurity. With the combination therapies of the invention, a comparable reduction of pathological retinal neovascularization can be obtained with a substantially lower dosage of VEGF antagonists. This can also lead to a reduction of the frequency of injection to produce a desired response, or to minimize side effects or improve efficacy of alternative treatments. [0019] The following sections provide more detailed guidance for practicing the invention.

II. Definitions

[0020] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which this invention pertains. The following references provide one of skill with a general definition of many of the terms used in this invention: Academic Press Dictionary of Science and Technology, Morris (Ed.), Academic Press (1^st ed., 1992); Illustrated Dictionary of Immunology, Cruse (Ed.), CRC Pr I Lie (2^nd ed., 2002); Oxford Dictionary of Biochemistry and Molecular Biology, Smith et al. (Eds.), Oxford University Press (revised ed., 2000); Encyclopaedic Dictionary of Chemistry, Kumar (Ed.), Anmol Publications Pvt. Ltd. (2002); Dictionary of Microbiology and Molecular Biology, Singleton et al. (Eds.), John Wiley & Sons (3^rd ed., 2002); Dictionary of Chemistry, Hunt (Ed.), Routledge (1^st ed., 1999);

Dictionary of Pharmaceutical Medicine, Nahler (Ed.), Springer-Verlag Telos (1994); Dictionary of Organic Chemistry, Kumar and Anandand (Eds.), Anmol Publications Pvt. Ltd. (2002); and A Dictionary of Biology (Oxford Paperback Reference) , Martin and Hine (Eds.), Oxford University Press (4^th ed., 2000). In addition, the following definitions are provided to assist the reader in the practice of the invention.

[0021] The term “agent” or “test agent” includes any substance, molecule, element, compound, entity, or a combination thereof. It includes, but is not limited to, e.g., protein, polypeptide, peptide or mimetic, small organic molecule, polysaccharide, polynucleotide, and the like. It can be a natural product, a synthetic compound, or a chemical compound, or a combination of two or more substances. Unless otherwise specified, the terms “agent”, “substance”, and “compound” are used interchangeably herein.

[0022] The term “variant” or “functional derivative” is used herein to refer to a molecule that structurally resembles a reference molecule (e.g., a known VEGF antagonist or CITED2 peptide described herein) but which has been modified in a targeted and controlled manner, by replacing a specific substituent of the reference molecule with an alternate substituent. Compared to the reference molecule, the variant or functional derivative would be expected, by one skilled in the art, to exhibit the same, similar, or improved utility. Generation and screening of variants of a known polypeptide agent to identify derivatives having improved characteristics (such as higher binding affinity for a target molecule) is an approach that is well known in pharmaceutical chemistry.

[0023] CITED2 (“Cbp/p300-interacting trans activator 2” or “Cbp/p300 interacting transactivator with Glu/Asp rich carboxy -terminal domain 2”) is an intrinsically disordered protein. The protein is a negative feedback regulator of HIF-la by inhibiting transcription of HIF- la-regulated target genes. It competes with HIF-la for binding to the TAZ1 domain of the general transcriptional coactivators CBP and p300. In humans, CITED2 is encoded by the CITED2 gene. A human CITED2 protein amino acid sequence is shown in UniProt ID Q99967 (SEQ ID NO: 1).

[0024] Neovascularization refers to the abnormal formation of new and fragile blood vessels, usually in response to ischemia following an occlusion (blockage) of an existing blood vessel. Neovascularization often occurs in the retina. It is widely considered to be highly undesirable as these fragile vessels are prone to hemorrhaging, thereby starving surrounding tissue of blood nutrients. This condition is traditionally treated by photocoagulation which is the use of lasers to cauterise these new vessels.

Neovascularization differs from angiogenesis in that angiogenesis is mainly characterized by the protrusion and outgrowth of capillary buds and sprouts from pre-existing blood vessels. In ophthalmology, choroidal neovascularization is the formation of a microvasculature within the innermost layer of the choroid of the eye.

[0025] Revascularization or physiological revascularization refers to restoration or growth of the existing retinal network of blood vessels. [0026] The term "conservatively modified variant" applies to both amino acid and nucleic acid sequences. With respect to particular nucleic acid sequences, conservatively modified variants refer to those nucleic acids which encode identical or essentially identical amino acid sequences, or where the nucleic acid does not encode an amino acid sequence, to essentially identical sequences. Because of the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given protein. For instance, the codons GCA, GCC, GCG and GCU all encode the amino acid alanine. Thus, at every position where an alanine is specified by a codon, the codon can be altered to any of the corresponding codons described without altering the encoded polypeptide. Such nucleic acid variations are “silent variations,” which are one species of conservatively modified variants. Every nucleic acid sequence herein which encodes a polypeptide also describes every possible silent variation of the nucleic acid. One of skill will recognize that each codon in a nucleic acid (except AUG, which is ordinarily the only codon for methionine, and TGG, which is ordinarily the only codon for tryptophan) can be modified to yield a functionally identical molecule. Accordingly, each silent variation of a nucleic acid that encodes a polypeptide is implicit in each described sequence.

[0027] For polypeptide sequences, “conservatively modified variants” refer to a variant which has conservative amino acid substitutions, amino acid residues replaced with other amino acid residue having a side chain with a similar charge. Families of amino acid residues having side chains with similar charges have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), betabranched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).

[0028] The terms “subject” and "patient" are used interchangeably and refer to mammals such as human patients and non-human primates, as well as experimental animals such as rabbits, rats, and mice, and other animals. Animals include all vertebrates, e.g., mammals and non-mammals, such as dogs, cats, sheeps, cows, pigs, rabbits, chickens, and etc. Preferred subjects for practicing the therapeutic methods of the present invention are human. Subjects in need of treatment include patients already suffering from an ocular vascular disease or disorder as well as those prone to developing the disorder. [0029] As used herein, "treating" or "ameliorating" includes (i) preventing a pathologic condition (e.g., neovascularization or ischemic retinopathy) from occurring (e.g. prophylaxis); (ii) inhibiting the pathologic condition or arresting its development; and (iii) relieving symptoms associated with the pathologic condition. Thus, “treatment” includes the administration of therapeutic agents or compositions described herein to prevent or delay the onset of the symptoms, complications, or biochemical indicia of an ocular disease described herein, alleviating or ameliorating the symptoms or arresting or inhibiting further development of the disease, condition, or disorder. “Treatment” further refers to any indicia of success in the treatment or amelioration or prevention of the ocular disease, condition, or disorder described herein, including any objective or subjective parameter such as abatement; remission; diminishing of symptoms or making the disease condition more tolerable to the patient; slowing in the rate of degeneration or decline; or making the final point of degeneration less debilitating. Detailed procedures for the treatment or amelioration of an ocular disorder or symptoms thereof can be based on objective or subjective parameters, including the results of an examination by a physician.

III. CITED2 peptides and VEGF antagonists

[0030] The invention provides methods and compositions for treating (or inhibiting) hypoxia-induced retinal neovascularization and also for promoting revascularization of the ischemic retina. In a related aspect, the invention provides therapies for treating ocular disorders or ischemic retinal diseases that are associated with or manifested by hypoxia- induced retinal neovascularization. Typically, the novel therapeutic methods of the invention involve administering to a subject in need of treatment a VEGF antagonist, and also a CITED2 derived peptide or peptide mimetic capable of binding to transcriptional coactivators CBP and p300.

[0031] The CITED2 derived peptides to be used in the invention are capable of binding to the TAZ1 domain of transcriptional coactivators CBP and/or p300, in competition with HIF-la. They include native CITED2 fragment peptides (i.e., CITED2 peptides), as well as variants or derivatives that contain modifications of a native CITED2 fragment sequence. The TAZ1 domain of CBP and p300 is highly conserved in both sequence and structure. While CITED2 peptides that specifically bind to the TAZ1 domain of CBP are expected to also similarly bind to the TAZ1 domain of p300, CITED2 derived peptides suitable for the invention also include variants that have different binding activities for the CBP and p300, e.g., just bind to one of the two proteins. Typically, the CITED2 derived peptide for use in the invention contains, at the minimum, a fragment or motif corresponding to a portion or the entirety of the disordered C-terminal transactivation domain (TAD) of CITED2 where the needed binding activity resides. The TAD domain of CITED2 was described and defined in the literature, e.g., Shioda et al., Gene 204: 235-241, 1997; and Bhattacharya et al., Genes. Dev. 13: 64-75, 1999. Any CITED2 derived peptide or mimetic possessing such an activity can be used in the invention.

[0032] In general, the CITED2 derived peptides suitable for the invention comprise a sequence that is encompassed by DEEVLMX²⁰LVIX²⁴MGLX²⁸RIX³¹X³² (SEQ ID NO:51) or a substantially identical or conservatively modified variant thereof, wherein X²⁰ is S, K, D, E, A, P, or phosphoSer; X²⁴ is E, K or R; X²⁸ is D, E, I, L, M or W; X³¹ is K or P; and X³² is E or P. Specific examples of such CITED2 derived peptides are shown in SEQ ID NOs:2- 23. In addition to these specific peptides, peptides with a substantially identical amino acid sequence or a conservatively modified sequence can also be employed in the practice of the invention.

[0033] In addition to the sequence shown in SEQ ID NO:51, some employed CITED2 derivative peptides for the invention can contain one or more additional residues at the N- terminus and/or the C-terminus. The additional residues can be the native residues in the wildtype CITED2 sequence. They can also be non-native residues (e.g., a nuclear localization signal sequence as detailed below) or conservative substitutions of the native residues. In some of these embodiments, the CITED2 derived peptides comprise X¹²X¹³DEEVLMX²⁰LVIX²⁴MGLX²⁸RIX³¹X³² (SEQ ID NO:52), wherein X¹² is F, Y, W, E or D; X¹³ is I, F, W or Y; and X²⁰, X²⁴, X²⁸, X³¹ and X³² are respectively as noted above for SEQ ID NO:51.

[0034] Relative to SEQ ID NO:51 or 52, CITED2 derived peptides suitable for the invention can include extra native amino acid residues (or conservatively substituting residues thereol) at the N-terminus and/or C-terminus. For example, they can contain 1, 2, 3, 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 or more extra native amino acid residues at one terminus or independently at both termini. In various embodiments, the employed CITED2 derivative peptides comprise an amino acid sequence that is encompassed by sequence formula X¹X²X³X⁴X⁵X⁶X⁷X⁸X⁹X¹¹X¹²X¹³DEEVLMX²⁰LVIX²⁴MGLX²⁸RIX³¹X³²X³³X³⁴X³⁵X³⁶X³⁷ _X38_X39_X40_X4i_X42_X43_X44_X45 x⁴⁶X⁴⁷X⁴⁸X⁴⁹X⁵⁰X⁵¹X⁵²X⁵³X⁵⁴X⁵⁵X⁵⁶X⁵⁷X⁵⁸X⁵⁹, wherein X¹ is G or absent; X² is S or absent; X³ is H or absent; X⁴ is M or absent; X⁵ is S or absent; X⁶ is N or absent; X⁷ is V or absent; X⁸ is I or absent; X⁹ is D or absent; X¹⁰ is T or absent; X¹¹ is D or absent; X¹², X¹³, X²⁰, X²⁴, X²⁸, X³¹ and X³² are respectively as noted above for SEQ ID NO:52; X³³ is L, A or absent; X³⁴ is P or absent; X³⁵ is E, A or absent; X³⁶ is L, A or absent; X³⁷ is W, Y, F, P or absent; X³⁸ is L, W, A, V, I, P, F, M or absent; X³⁹ is G or absent; X⁴⁰ is Q or absent; X⁴¹ is N or absent; X⁴² is E or absent; X⁴³ is F or absent; X⁴⁴ is A or absent; X⁴⁵ is F or absent; X⁴⁶ is M or absent; X⁴⁷ is T or absent; X⁴⁸ is A or absent; X⁴⁹ is F or absent; X⁵⁰ is V or absent; X⁵¹ is C or absent; X⁵² is K or absent; X⁵³ is Q or absent; X⁵⁴ is Q or absent; X⁵⁵ is P or absent; X⁵⁶ is S or absent; X⁵⁷ is R or absent; X⁵⁸ is V or absent; and X⁵⁹ is S or absent.

[0035] In some embodiments, the employed CITED2 peptides for practicing methods of the invention encompass or contain a specific sequence motif that falls under SEQ ID NO:51, DEEVLMSLVIEMGLDRI KE (SEQ ID NO:53). This sequence corresponds to residues 224-242 of the wildtype or native sequence of a human CITED2 protein (SEQ ID NO: 1). Activities of some specific CITED2 peptides encompassing this sequence motif are exemplified herein, e.g., peptides with a sequence shown in any one of SEQ ID NOs:2-8, 13 and 15-23.

[0036] In some embodiments, the employed CITED2 peptide encompasses or corresponds to residues 224-255 (SEQ ID NO:2) of the wildtype or native sequence of a human CITED2 protein (SEQ ID NO: 1). This CITED2 peptide sequence was shown to be able to disrupt a HIF-la-p300 complex in vitro and in vivo (Bhattacharya et al., Genes. Dev. 13: 64-75, 1999). In some embodiments, the CITED2 peptide to be used in the invention is a truncated fragment of SEQ ID NO:2. Relative to SEQ ID NO:2, the fragment can have truncation of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more residues at the N-terminus, and/or truncation of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more residues at the C-terminus.

[0037] In some embodiments, the employed CITED2 peptide can encompass or correspond to C-terminal residues 216-269 (SEQ ID NO: 3) of a wildtype human CITED2 protein, as exemplified herein. In various embodiments, the employed peptide can contain one or more additional residues at the N-terminus and/or C-terminus of the exemplified sequences. Thus, relative to SEQ ID NO:2, the CITED2 derived peptide for use in the invention can include at least 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 extra N-terminal residues of the native CITED2 sequence. Additionally or alternatively, relative to SEQ ID NO:2, the CITED2 derived peptide for use in the invention can include at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 extra C-terminal residues of the native CITED2 sequence.

[0038] Other than residues from the native CITED2 sequences (e.g., any one of SEQ ID NO:2, 3 and 53, or a fragment thereof), the employed CITED2 peptide can also contain at the N-terminus and/or the C-terminus one or more extra residues that are not residues native to CITED2 or adjacent to a native CITED2 fragment sequence in the wildtype or native CITED2 sequence. In some of these embodiments, the extra residues are heterologous to the native CITED2 sequence. One example of such a CITED2 peptide contains 5 extra nonnative residues as shown in SEQ ID NO:4. Other specific examples are shown in SEQ ID NOs: 15-22.

Human CITED2 sequence (SEQ ID NO: 1): UniProt ID Q99967

MADHMMAMNH GRFPDGTNGL HHHPAHRMGM GQFPSPHHHQ QQQPQHAFNA LMGEHIHYGA GNMNATSGIR HAMGPGTVNG GHPPSALAPA ARFNNSQFMG PPVASQGGSL PASMQLQKLN NQYFNHHPYP HNHYMPDLHP AAGHQMNGTN QHFRDCNPKH SGGSSTPGGS GGSSTPGGSG SSSGGGAGSS NSGGGSGSGN MPASVAHVPA AMLPPNVIDT DFIDEEVLMS LVIEMGLDRI KELPELWLGQ NEFDFMTDFV CKQQPSRVSC

32 aa CITED2 peptide (224-255) (SEQ ID NO: 2) DEEVLMS LVIEMGLDRI KELPELWLGQ NEFDF

54 aa CITED2 peptide (216-269) (SEQ ID NO: 3)

NVIDT DFIDEEVLMS LVIEMGLDRI KELPELWLGQ NEFDFMTDFV CKQQPSRVS

59 aa CITED2 peptide with 5 N-terminal non-native residues (SEQ ID NO:4) GSHMS - NVIDT DFIDEEVLMS LVIEMGLDRI KELPELWLGQ NEFDFMTDFV CKQQPSRVS

[0039] In addition to CITED2 derived peptides containing the native sequence of a CITED2 protein (e.g., human CITED2 shown in SEQ ID NO:1) or its fragments (e.g., SEQ ID NOs:2 and 3), suitable CITED2 derived peptides for use in the invention also include variants or functional derivatives that can be generated from the native sequence. In various embodiments, the variants or derivatives of a peptide or a polypeptide agent can have amino acid deletions and/or insertions and/or substitutions while maintaining one or more of the bioactivities (e.g., binding to CBP/p300) and therefore can also be used in practicing the methods of the present invention. Some specific variants are exemplified herein, e.g., peptides shown in SEQ ID NOs:5-14 and 23. Relative to SEQ ID NO:3 or 4, some of these variants contain deletions of one or more of the terminal residues. Some other variants are functional derivatives having substitution of one or more residues of a native CITED2 peptide sequence shown in SEQ ID NO:2, 3 or 53. In some embodiments, the employed variant or derivative has a sequence that is substantially identical to the native CITED2 sequence. The substantially identical variants should contain a sequence that is, e.g., at least 80%, 90%, 95% or 99% identical to the native peptide sequence. In some embodiments, the employed variant or derivative has a sequence that is a conservatively modified variant of the native peptide sequence. In some embodiments, the employed variant or derivative is produced by non-conservative substitutions to the extent that that they substantially retain the activities of the native CITED2 peptide (e.g., binding to the TAZ1 domain of CBP/p300).

[0040] In various embodiments, CITED2 peptides suitable for the invention include variants generated by, e.g., incorporation of non-canonical amino acids, backbone modifications, introduction of post-translational modifications, and incorporation of D- amino acids. In some embodiments, the employed CITED2 variant or derivative contains one or more unnatural amino acids designed to enhance efficacy, stability and lifetime in the cell. In some embodiments, the employed CITED2 variant or derivative can contain mutations that could stabilize secondary structural elements of the native CITED2 peptide. Such variants could have stronger ability to compete with HIF-la for binding of the TAZ1 domain of CBP/p300, thus with enhanced potency as HIF-la inhibitors. In some embodiments, the CITED2 derived peptides can be designed as fusions with well- characterized cell penetrating peptides, nuclear localization signals, and/or small molecule tags to improve bio-availability.

[0041] As exemplification, some specific CITED2 variant peptides containing a terminal nuclear localization signal sequence are described herein, e.g., SEQ ID NOs: 15-22. In these embodiments, the nuclear localization signal sequence can be located at either the N- terminus (e.g., SEQ ID NOs: 15, 17, 19 and 21) or the C-terminus (e.g., SEQ ID NOs: 16, 18, 20, and 22). The nuclear localization signal sequence can be obtained from various sources. Examples include a nuclear localization sequence from the SV40 large T-antigen, PKKKRKV (SEQ ID NO:54) as present in SEQ ID NOs: 15 and 16; a nuclear localization sequence from the p50 subunit of NF-kB, QRKRQK (SEQ ID NO:55) as present in SEQ ID NOs: 17 and 18; a nuclear localization sequence from the p65 subunit of NF-kB, EEKRKR (SEQ ID NO:56) as present in SEQ ID NOs: 19 and 20; and a nuclear localization sequence from p53, PQPKKKPLDG (SEQ ID NO:57) as present in SEQ ID NOs:21 and 22. To ensure that the nuclear localization signal sequence would not interfere with binding of the variant peptide to the TAZ1 domain of CBP/p300, a short linker sequence can be inserted between the nuclear localization signal sequence and the CITED2 derived sequence. For example, the peptides shown in SEQ ID NOs: 15, 17, 19, and 21 contain a SSGS linker (SEQ ID NO:58) to connect the N-terminal nuclear localization signal sequence to the CITED2 derived sequence. Similarly, the peptides shown in SEQ ID NOs: 16, 18, 20, and 22 contain a SG linker to connect the C-terminal nuclear localization signal sequence to the CITED2 derived sequence.

[0042] The various CITED2 variants or derivatives described herein for use in the invention can be produced via, e.g., recombinant means and chemical synthesis. Standard techniques for introducing modifications to a polynucleotide encoding a polypeptide of interest are well known and routinely practiced in the art. The CITED2 peptides for use in the invention can be derived from any known CITED2 proteins. Preferably, they are derived from a human CITED2 protein, e.g., the protein with sequence shown in SEQ ID NO: 1. Due to a substantial degree of sequence homology in the transactivation domains of CITED2 proteins from different species, non-human CITED2 proteins may also be used for generating the CITED2 peptides suitable for the methods of the invention. Similarly, other CITED proteins, e.g., CITED1, CITED3 and CITED4, all share a very high degree of sequence homology in their transactivation domains with that of CITED2. Therefore, these other CITED proteins may also be used for obtaining “CITED2 derived peptides” in the practice of the invention. Sequences of a number of CITED2 proteins from non-human species and other CITED proteins are all known in the art. See, e.g., Freedman et al., Nat. Struct. & Mol. Biol. 10: 504-12, 2003.

[0043] The methods of the invention rely on the combination of a CITED2 derived peptide described herein and also a VEGF antagonist agent. Any VEGF antagonist agents or compounds can be used in the methods of the invention. These include agents that inhibit VEGF or VEGF receptor expression or cellular level, agents that antagonize or inhibit VEGF, and agents that target the signaling cascade. Suitable agents that can be employed for practicing the present invention include peptides, peptide mimetics, antibodies, nucleic acid agents, and small molecule chemical compounds. In some embodiments, the employed VEGF antagonist can be an agent that downregulates expression or cellular level of VEGF, e.g., siRNA agent such as Bevasiranib. See, e.g., Mousa et al., BioDrugs 24: 183-94, 2010. In some embodiments, the employed VEGF antagonist is a siRNA agent that targets VEGF receptor, e.g., AGN211745. See, e.g., Ambati, Invest. Ophthalmol. Vis Sci. 52: 2166-69, 2011. In some other embodiments, the employed VEGF antagonist can be a compound that inhibits the VEGF signaling pathway, e.g., targeting the tyrosine kinase cascade. A number of such agents are known in the art, including, e.g., Vatalanib, TGI 00801, TG101095, Pazopanib, AG013958, and AL39324. See, e.g., Dragovich et al., Cancer Chemother Pharmacol. 74: 379-87, 2014; Freeman et al., Investigative Ophthalmology & Visual Science 49: 3771, 2011; Danis et al., Br. J. Ophthalmol. 98: 172-8., 2014; Barakat et al., Expert Opin. Investig. Drugs 18: 637-46, 2009; and Avitabile, BMC Geriatr 10: L4, 2010.

[0044] In some other embodiments, the employed VEGF antagonist targets VEGF by inhibiting its binding to and activation of the receptor. In some of these embodiments, the VEGF antagonist used in these embodiments can be polypeptide agents known as VEGF receptor decoy. A well-known example of such agents is Aflibercept, which is a recombinant fusion protein consisting of VEGF-binding regions of the extracellular domains of the human VEGF-receptor fused to the Fc portion of human IgG. See, e.g., Fraser et al., Endocrinology 149: 4413-20, 2008; and Duncan et al., Endocrinology 149: 3313-20, 2008. Some other embodiments of the invention can employ antibody agents that specifically bind to VEGF. These include, e.g., Ranibizumab (Lucentis), which is a small anti-VEGF Fab protein which was affinity-improved and made in prokaryotic E. coli, and Bevacizumab (Avastin), which is a humanized monoclonal antibody (mAb) against VEGF produced in CHO cells. In still some other embodiments, the employed VEGF -targeting agent can be an aptamer molecule such as Pegaptanib (Macugen). See, e.g., Trujillo et al., Clin Ophthalmol. 1: 393-402, 2007. In addition to these well-known VEGF antagonists, their variants or derivatives with similar activities can also be used in the methods of the invention. For example, Aflibercept derivatives having an amino acid sequence that is substantially identical to, or a conservatively modified variant of, Aflibercept can be employed in the practice of the invention.

[0045] To determine appropriateness for use in the methods of the invention, activities of various CITED2 derived peptides and their combinations with a VEGF antagonist can be examined via the experimental protocols exemplified herein or methods well known in the art. For example, localization and cell type specificity of the CITED2 peptides (alone and in combination with available VEGF antagonists) can be assessed by intravitreal injection of fluorescently-labeled peptides. Concentration and stability of the CITED2 peptides can be examined ex vivo in the vitreous humor as well as the retina by reversed-phase HPLC and mass spectrometry to establish a pharmacokinetic profile for the CITED2 peptides in various formulations. For safety studies, CITED2 peptides can be injected intravitreally and examined in laboratory animals, e.g., adult wild-type mice. Changes to overall retinal morphology and neuronal function can be assessed using in vivo imaging modalities (e.g., Heidelberg fundus imaging and optical coherence tomography) and electroretinography. Vascular integrity and neuronal toxicity can be assessed using indocyanine green Heidelberg imaging and TUNEL staining, respectively.

IV. Engineered cells expressing CITED2 peptides and/or VEGF antagonists

[0046] In addition to compositions containing a combination of an isolated or purified CITED2 peptide and a VEGF antagonist, the invention also provides vectors and engineered cells that express such a combination of therapeutic agents or just one of the agents. Such engineered cells are also suitable for administering to a subject for treatment of ocular disorders as detailed below. In some embodiments, the engineered cells are derived from a cell line from human retina, e.g., a human retinal pigment epithelium cell line. The cell line can be modified so as to stably or transiently express a VEGF antagonist and/or a CITED2 peptide described herein. In some of these embodiments, the cell line can be transfected with a vector harboring a polynucleotide that encodes both the VEGF antagonist and the CITED2 peptide. In some other embodiments, the cell line is transfected with two vectors that respectively express the VEGF antagonist and the CITED2 peptide. In some preferred embodiments, the cells are engineered with vectors that allows one (e.g., the VEGF antagonist) or both of the therapeutic agents to be stably expressed.

[0047] The engineered cells can contain and express additional genes which are therapeutically useful for treatment of ocular disorders. In some embodiments, the engineered cells can harbor a gene operably encoding an antiangiogenic agent. Examples of such agents include fragments derived from the carboxyl-terminal fragment of tryptophan tRNA synthetase (T2-TrpRS), angiopoietin 2, endostatin, angiostatin, PEX, IL-12, IFN-a, prolactin and thrombospondin TSP-1 and TSP-2.

[0048] Various vectors can be used for introducing into the engineered cells polynucleotide sequences expressing the VEGF antagonist and/or the CITED2 peptide. The employed vector should allow introduction of the exogenous sequences into the cells and also subsequent expression (transient or stable) of the sequences once transfected into the cells. In some embodiments, retroviral vectors and corresponding packaging cell lines well known in the art can be employed. Particularly suitable for the present invention are lentiviral vectors. Lentiviral vectors are retroviral vectors that are able to transduce or infect non-dividing cells and typically produce high viral titers.

[0049] Retroviral vectors are comprised of cv.s'-acting long terminal repeats with packaging capacity for up to 6-10 kb of foreign sequence. The minimum c/.s'-acting LTRs are sufficient for replication and packaging of the vectors, which are then used to integrate the therapeutic gene into the target cell to provide permanent transgene expression. Widely used retroviral vectors include those based upon murine leukemia virus (MuLV), gibbon ape leukemia virus (GaLV), simian immunodeficiency virus (SIV), human immunodeficiency virus (HIV), and combinations thereof (see, e.g., Buchscher et al., J. Virol. 66:2731-2739, 1992; Johann etal., J. Virol. 66:1635-1640, 1992; Sommerfelt et al., Virol. 176:58-59, 1990; Wilson et al., J. Virol. 63-.231 -23Ti, 1989; Miller et al., J. Virol. 65:2220-2224, 1991; and PCT/US94/05700). Any of these vectors may be employed in the present invention. In particular, a number of retroviral vectors have been used for gene transfer in clinical applications in the art. These include pLASN and MFG-S, retroviral vectors that have been used in clinical trials (Dunbar et al., Blood 85:3048-305 (1995); Kohn et al., Nat. Med.

1:1017-102 (1995); Malech et al., Proc. Natl. Acad. Sci. U.S.A. 94:22 12133-12138 (1997)). Another vector is PA317/pLASN, which was the first therapeutic vector used in a gene therapy trial. (Blaese et al., Science 270:475-480, 1995). Transduction efficiencies of 50% or greater have been observed for MFG-S packaged vectors (Ellem et al., Immunol Immunother. 44:10-20, 1997; Dranoff et al., Hum. Gene Ther. 1:111-2, 1997).

[0050] Vectors suitable for the present invention can be constructed using standard recombinant techniques widely available to one skilled in the art. Such techniques can be found in common molecular biology references such as Sambrook, et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press (1989), D. Goeddel, ed., Gene Expression Technology, Methods in Enzymology series, Vol. 185, Academic Press, San Diego, Calif (1991), and Innis, et al. PCR Protocols: A Guide to Methods and Applications, Academic Press, San Diego, Calif (1990). In order to obtain transcription of the gene encoding one or two of the therapeutic agents described herein within an engineered cell (e.g., a RPE cell or cell line), a transcriptional regulatory region capable of driving gene expression in the target cell is utilized. The transcriptional regulatory region can comprise a promoter, enhancer, silencer or repressor element and is functionally associated with a nucleic acid of the present invention. Preferably, the transcriptional regulatory region drives high level gene expression in the target cell. Transcriptional regulatory regions suitable for use in the present invention include but are not limited to the human cytomegalovirus (CMV) immediate-early enhancer/promoter, the SV40 early enhancer/promoter, the JC polyomavirus promoter, the albumin promoter, PGK and the a-actin promoter coupled to the CMV enhancer.

[0051] Many producer cell lines or packaging cell lines for transfecting retroviral vectors and producing viral particles are also known in the art. The producer cell to be used in the invention needs not to be derived from the same species as that of the engineered cells expressing a VEGF antagonist and/or a CITED2 derived peptide (e.g., human retinal pigment epithelium cells). For example, producer or packaging cell lines suitable for the present invention include cell lines derived from human (e.g., HEK 293 cells), monkey (e.g., COS-1 cells), mouse (e.g., NIH 3T3 cells) or other species (e.g., canine). Additional examples of retroviral vectors and compatible packaging cell lines for producing recombinant retroviruses in gene transfers are reported in, e.g., Markowitz et al., Virol. 167:400-6, 1988; Meyers et al., Arch. Virol. 119:257-64, 1991 (for spleen necrosis virus (SNV)-based vectors such as vSNO21); Davis et al., Hum. Gene. Ther. 8:1459-67, 1997 (the “293-SPA” cell line); Povey et al., Blood 92:4080-9, 1998 (the “1MI-SCF” cell line); Bauer et al., Biol. Blood Marrow Transplant. 4: 119-27, 1998 (canine packaging cell line “DA”); Gerin et al., Hum. Gene Ther. 10:1965-74, 1999; Sehgal et al., Gene Ther. 6: 1084-91, 1999; Gerin et al., Biotechnol. Prog. 15:941-8, 1999; McTaggart et al., Biotechnol. Prog. 16:859- 65, 2000; Reeves et al., Hum. Gene. Ther. 11:2093-103, 2000; Chan et al., Gene Ther. 8:697-703, 2001; Thaler et al., Mol. Ther. 4:273-9, 2001; Martinet et al., Eur. J. Surg. Oncol. 29:351-7, 2003; and Lemoine et al., I .Gene Med. 6:374-86, 2004. Any of these and other retroviral vectors and packaging or producer cell lines can be used in the practice of the present invention.

[0052] Many of the retroviral vectors and packaging cell lines used for gene transfer in the art can be obtained commercially. For example, a number of retroviral vectors and compatible packing cell lines are available from Clontech (Mountain View, CA). Examples of lentiviral based vectors include, e.g., pLVX-Puro, pLVX-IRES-Neo, pLVX-IRES-Hyg, and pLVX-IRES-Puro. Corresponding packaging cell lines are also available, e.g., Lenti-X 293T cell line. In addition to lentiviral based vectors and packaging systems, other retroviral based vectors and packaging systems are also commercially available. These include MMLV based vectors pQCXIN, pQCXIQ and pQCXIH, and compatible producer cell lines such as HEK 293 based packaging cell lines GP2-293, EcoPack 2-293 and AmphoPack 293, as well as the NIH/3T3-based packaging cell line RetroPack PT67. Any of these and other retroviral vectors and producer cell lines can be employed in the practice of the present invention.

[0053] Once recombinant retroviruses expressing a VEGF antagonist and/or a CITED2 peptide are produced, they can be readily used to infect a retinal derived cell population, e.g., human RPE cells. Methods for infecting primary cells with a recombinant retrovirus are well known in the art. For example, the recombinant viruses can be transfected into the cells in accordance with methods well known in the art of gene therapy (see, e.g., Mulligan et al., Hum. Gene Ther. 5:543-563, 1993). Following the transfection, the engineered cells can be administered or implanted into a subject as described below. In some preferred embodiments, the engineered cells to be transfected with the recombinant virus are isolated from a subject to be treated. In some other embodiments, the transfected cells are not autologous to the subject to whom the cells are ultimately administered.

V Pharmaceutical compositions and administration

[0054] The invention provides novel pharmaceutical compositions or kits for treating ocular disorders that are associated with or manifested by retinal neovascularization or ischemic retina, e.g., ocular vascular diseases or ocular degenerative disorders. These compositions or kits contain or are capable of expressing a combination of a VEGF antagonist and a CITED2 derived peptide described herein. Specific examples of diseases that can be treated with the compositions of the invention include ischemic retinopathy, diabetic retinopathy, retinopathy of prematurity, retinal vein occlusion, neovascular glaucoma, macular edema, familial exudative vitreoretinopathy, retinal angioma, macular degeneration and retinitis pigmentosa. In addition to the two active ingredients, the pharmaceutical compositions typically also contain one or more pharmaceutically acceptable carriers or salts.

[0055] The active ingredients, the VEGF antagonist and the CITED2 derived peptide, can be provided in the pharmaceutical compositions or kits as substantially purified polypeptides or peptides. For example, the pharmaceutical composition can contain a therapeutically effective amount of a CITED2 derived peptide (e.g., any one of SEQ ID NOs:2-23) and a VEGF antagonist agent (e.g., Aflibercept). Alternatively, the therapeutic agents can be provided in the pharmaceutical compositions or kits in the form of engineered cells that are capable of expressing the polypeptide or peptide agents. The engineered cells in such pharmaceutical compositions can either express just one of the two therapeutic agents or express both of the agents. Thus, in some of these embodiments, the pharmaceutical compositions contain a CITED2 peptide and a population of engineered cells expressing a VEGF antagonist (e.g., Aflibercept or a VEGF antibody fragment). In some of these embodiments, the pharmaceutical compositions contain a VEGF antagonist (e.g., Aflibercept or a VEGF antibody fragment) and a population of engineered cells expressing a CITED2 peptide. In still some other embodiments, the pharmaceutical compositions contain a population of engineered cells expressing both the CITED2 peptide and the VEGF antagonist. In these embodiments, the CITED2 peptide and/or the VEGF antagonist can be either stably or transiently expressed from the encapsulated cells. The two agents can be expressed from one expression vector introduced into the encapsulated cells. Alternatively, they can be expressed separately from two vectors.

[0056] Ocular administration of therapeutic agents via engineered cells can be performed in accordance with the well-known encapsulated cell technology (ECT). In these embodiments, the therapeutic agents are administered to subjects in need of treatment via the use of encapsulated cells for the production of the agents locally within the eye. This delivery method utilizes cells encapsulated in porous material that allows the exchange of nutrients and waste products, but prevents the efflux or influx of cells. The cells are genetically modified to secrete the therapeutic agents of choice. See, e.g., Tao, Expert Opinion on Biological Therapy 6: 717-26, 2006; Annamalai et al., Translational Vision Science & Technology 7: 3, 2018; and Kauper et al., Investigative Ophthalmology & Visual Science 54: 3295, 2013. In the practice of the invention, engineered cells that express or overexpress the VEGF antagonist and/or the CITED2 peptide can be readily modified via ECT and then administered to subjects in need of treatment. As described above, the therapeutic agents can be stably expressed from the engineered and encapsulated cells. In some embodiments, the therapeutic agents can be transiently expressed from the engineered and encapsulated cells. In some embodiments, one of the therapeutic agents (e.g., the VEGF antagonist) can be stably expressed from the cells, and the other agent (e.g., the CITED2 peptide) can be transiently expressed via an expression vector transfected into the cells. Encapsulation and administration of engineered cells (e.g., human RPE cells) stably or transiently expressing a transgene for ocular administration can be prepared in accordance with methods known in the art. See, e.g., Annamalai et al., Translational Vision Science & Technology 7: 3, 2018. For administration, the engineered cells are first encapsulated before being injected (e.g., intravitreally) into the eyes of the subjects. Cell encapsulation, e.g., with sodium alginate, can be readily performed as described in the art. See, e.g., Moore et al., Microsc. Microanal. 19: 213-26, 2013.

[0057] The pharmaceutical compositions that contain the therapeutic agents or encapsulated cells expressing the therapeutic agents can be prepared in various forms. Suitable solid or liquid pharmaceutical preparation forms are, e.g., granules, powders, tablets, coated tablets, (micro)capsules, suppositories, syrups, emulsions, suspensions, creams, aerosols, drops or injectable solution in ampule form and also preparations with protracted release of active compounds. The pharmaceutical compositions of the invention can be prepared in accordance with the standard protocols well known in the art, e.g., Remington: The Science and Practice of Pharmacy, Gennaro (ed.), Lippincott Williams & Wilkins (20^th ed., 2003). The pharmaceutical compositions typically contain an effective amount of the therapeutic agents or encapsulated cells expressing the agents that is sufficient to lessen or ameliorate symptoms of an ocular vascular disease or ocular degenerative disorder. In addition to the CITED2 peptide and the VEGF antagonist or cells expressing the agents, the pharmaceutical compositions can also contain certain pharmaceutically acceptable carriers which enhance or stabilize the composition, or facilitate preparation of the composition. For example, the CITED2 peptide and the VEGF antagonist can be complexed with carrier proteins such as ovalbumin or serum albumin prior to their administration in order to enhance stability or pharmacological properties. Pharmaceutically acceptable carriers are determined in part by the particular composition being administered as well as by the particular method used to administer the composition. They should also be both pharmaceutically and physiologically acceptable in the sense of being compatible with the other ingredients and not injurious to the subject. The carrier may take a wide variety of forms depending on the form of preparation desired for administration. In addition to the carriers, the various forms of pharmaceutical compositions can also contain excipients and additives and/or auxiliaries such as disintegrants, binders, coating agents, swelling agents, lubricants, flavorings, sweeteners and elixirs containing inert diluents commonly used in the art, such as purified water. [0058] For therapeutic or prophylactic applications, a pharmaceutical composition of the invention can be administered locally or systemically in a therapeutically effective amount or dose. For example, they may be administered parenterally, enterically, by injection, rapid infusion, nasopharyngeal absorption, dermal absorption, and orally. However, in preferred embodiments, local administration of the composition is desired in order to achieve the intended therapeutic effect. In these embodiments, the compositions are typically administered to the subject in need of treatment via intravitreal injection. This can be performed in accordance with standard procedures known in the art. See, e.g., Ritter et al., J. Clin. Invest. 116:3266-76, 2006; Russelakis-Cameiro et al., Neuropathol. Appl. Neurobiol. 25:196-206, 1999; and Wray et al., Arch. Neurol. 33:183-5, 1976.

[0059] A therapeutically effective amount means an amount that that is sufficient to reduce or inhibit the symptoms of the disorder or condition to be treated in a subject. In the practice of the present invention, the amount of the administered agents or cells expressing the agents should be effective for repairing retinal damage of the eye, stabilizing retinal neovasculature, maturing retinal neovasculature, and preventing or repairing vascular leakage and vascular hemorrhage. Such effective amount will vary from subject to subject depending on the ocular disorder afflicted by the subject, stage and severity of the disorder, the subject’s general conditions (such as height, weight, age, and health), the particular compound administered, and other factors. One skilled in the art can easily identify the effective amount of the compound by using routinely practiced pharmaceutical methods. Typically, dosages used in vitro may provide useful guidance in the amounts useful for in situ administration of the pharmaceutical composition, and animal models may be used to determine effective dosages for treatment of particular disorders in human subjects (see Examples below). More often, a suitable therapeutic dose can be determined by clinical studies on mammalian species to determine maximum tolerable dose and on normal human subjects to determine safe dosage.

[0060] In general, except under certain circumstances when higher dosages may be required, the preferred dosage of a CITED2 peptide or a VEGF antagonist lies within the range of from about 0.001 to about 1000 mg, more usually from about 0.01 to about 500 mg per day. As a general rule, the quantity of administered agent is the smallest dosage which effectively and reliably prevents or minimizes the conditions of the subjects. Also, the dosages to be administered and frequency of administration can vary depending on whether the treatment is prophylactic or therapeutic. In prophylactic applications, a relatively lower dosage may be administered at relatively infrequent intervals over a long period of time. Some subjects may continue to receive treatment for the rest of their lives. In therapeutic applications, a relatively higher dosage at relatively short intervals may be required until progression of the disease is reduced or terminated, and preferably until the subject shows partial or complete amelioration of symptoms of the ocular vascular disease. Thereafter, the subject can be administered a prophylactic regime.

[0061] As is readily apparent, the above dosage ranges are intended to provide general guidance and support for the teachings herein, but are not intended to limit the scope of the invention. Additional guidance for preparation and administration of the pharmaceutical compositions of the invention has also been described in the art. See, e.g., Goodman & Gilman's The Pharmacological Bases of Therapeutics, Hardman et al., eds., McGraw-Hill Professional (10^th ed., 2001); Remington: The Science and Practice of Pharmacy, Gennaro, ed., Lippincott Williams & Wilkins (20^th ed., 2003); and Pharmaceutical Dosage Forms and Drug Delivery Systems, Ansel et al. (eds.), Lippincott Williams & Wilkins (7^th ed., 1999). Further, as demonstrated herein, the use of a CITED2 peptide with the VEGF antagonist (e.g., Aflibercept) leads to a synergistic effect, which allows a significant reduction of the dosage of the VEGF antagonist. Thus, the amount of the VEGF antagonist (e.g., Aflibercept) provided or expressed in the pharmaceutical composition of the invention can be substantially lower than the dosage of the VEGF antagonist that would normally be required for treatment with the VEGF antagonist alone. Such a reduced dosage of VEGF antagonist can be readily adjusted in pharmaceutical compositions containing the VEGF antagonist. For compositions with encapsulated cells expressing the therapeutic agents, expression levels of the agents can be controlled via the use of different expression vectors and/or transcription regulatory elements (e.g., inducible promoters).

VI. Inhibiting retinal neovascularization and treating related ocular disorders

[0062] The present invention provides methods for inhibiting or suppressing retinal neovascularization, and for promoting revascularization of ischemic retina. In a related aspect, the invention also provides methods for treating or preventing ocular disorders and neuronal degeneration that are associated with or mediated by neovascularization. The therapeutic methods of the invention entail administering to a subject in need of treatment or prevention a combination of a VEGF antagonist and a CITED2 derived peptide described herein. As described above, the combination of agents is contained in or expressed from a pharmaceutical composition that is administered to the retina of the subject, e.g., by intravitreal injection.

[0063] The subjects suitable for treatment with methods of the invention can be neonatal, juvenile or fully mature adults. In some embodiments, the subject to be treated with methods of the invention is one suffering from an ocular degenerative disease or ocular vascular disease, e.g., one at an early stage of the ocular disease. In some other embodiments, the subject is one who is otherwise healthy but known to be predisposed to the development of an ocular degenerative disease (i.e. , through genetic predisposition). In some embodiments, the subjects to be treated are neonatal subjects suffering from ocular disorders such as oxygen induced retinopathy or retinopathy of prematurity. In some preferred embodiments, the subjects are human.

[0064] Typically, the ocular diseases or disorders to be treated with methods of the invention are associated with or mediated by retinal neovascularization and/or vaso- obliteration. Subjects suffering from various ocular vascular diseases or ocular degenerative disorders are suitable for treatment with the methods of the invention. These include ocular diseases such as retinal degenerative diseases, retinal vascular degenerative diseases, retinal edema (including macular edema), ischemic retinopathies, vascular hemorrhages, vascular leakage, choroidopathies, retinal injuries and retinal defects involving an interruption in or degradation of the retinal vasculature. As used herein, ocular vascular disorder or ocular neovascular disease refers to any pathological conditions characterized by altered or unregulated proliferation and invasion of new blood vessels into the structures of ocular tissues such as the retina or cornea. Examples of such ocular diseases include ischemic retinopathy, diabetic retinopathy (including non-proliferative diabetic retinopathy), retinopathy of prematurity, macular degeneration including age-related macular degeneration, retinitis pigmentosa, glaucoma, retinal degeneration, iris neovascularization, intraocular neovascularization, comeal neovascularization, retinal neovascularization, choroidal neovascularization, and diabetic retinal ischemia.

[0065] In some embodiments, therapeutic methods of the invention are directed to treating diseases associated with retinal/ choroidal neovascularization. These include, but are not limited to, diabetic retinopathy, macular degeneration, sickle cell anemia, sarcoid, syphilis, pseudoxanthoma elasticum, Pagets disease, vein occlusion, artery occlusion, carotid obstructive disease, chronic uveitis/vitritis, mycobacterial infections, Lyme's disease, systemic lupus erythematosis, retinopathy of prematurity, retinitis pigmentosa, retinal edema (macular edema), Von Hippel Lindau disease, familial exudative vitreoretinopathy, Eales disease, Bechets disease, infections causing a retinitis or choroiditis, presumed ocular histoplasmosis, Bests disease, myopia, optic pits, Stargardts disease, pars planitis, herpetic keratitis, pinguecula, pterygium, chronic retinal detachment, hyperviscosity syndromes, toxoplasmosis, trauma and post-laser complications. Other diseases include, but are not limited to, diseases associated with rubeosis (neovascularization of the angle) and diseases caused by the abnormal proliferation of fibrovascular or fibrous tissue including all forms of proliferative vitreoretinopathy.

[0066] Some embodiments of the invention are directed to treating subjects afflicted with disorders associated with vaso-obliteration but not manifested by neovascularization. These include subjects suffering from retinal vein occlusion who do not normally develop retinal neovascularization, as well as subjects with early stage of diabetic retinopathy, early stage of severe non proliferative diabetic retinopathy (severe NPDR), or stage 1-2 of retinopathy of prematurity (ROP).

[0067] In some embodiments, therapeutic methods of the invention are directed to treating other ocular vascular disorder or diseases associated with comeal neovascularization. These include, but are not limited to, epidemic keratoconjunctivitis, Vitamin A deficiency, contact lens overwear, atopic keratitis, superior limbic keratitis, pterygium keratitis sicca, Sjogrens, acne rosacea, phylectenulosis, syphilis, Mycobacteria infections, lipid degeneration, chemical bums, bacterial ulcers, fungal ulcers, Herpes simplex infections, Herpes zoster infections, protozoan infections, Kaposi sarcoma, Mooren ulcer, Terrien's marginal degeneration, mariginal keratolysis, rheumatoid arthritis, systemic lupus, polyarteritis, trauma, Wegeners sarcoidosis, scleritis, Steven's Johnson disease, periphigoid radial keratotomy, herpetic keratitis, pinguecula, pterygium, and comeal graph rejection.

EXAMPLES

[0068] The following examples are offered to illustrate, but not to limit the present invention.

Example 1. CITED2 peptide rescues both NV and VO in OIR

[0069] To investigate whether the CITED2 peptide is able to inhibit HIF-la in vivo, we used the oxygen-induced retinopathy (OIR) mouse model of hypoxia-induced retinal NV (Smith et al., Invest Ophthalmol Vis Sci. 35: 101-11, 1994). In the OIR model, the hyperoxic phase from P7 to P12 induces regression of retinal vessels, resulting in an avascular area in the central retina. Returning OIR mice to room air at P12 results in hypoxia relative to the previous five days; HIF stabilization in the avascular retina leads to hypoxia-induced retinal NV in the area surrounding the avascular zone, with maximal NV observed at P17. OIR mice were injected intravitreally on P12 with three different doses of the CITED2 peptide (0.68 ng, 3.4 ng, or 34 ng), the same amounts of a negative control peptide (CITED2 APAA) that is deficient in binding to the TAZ1 domain of CBP/p300 and competing with HIF-la for CBP/p300 binding, or equivalent volumes of vehicle. Retinas from OIR mice injected with the CITED2 peptide had significantly decreased retinal NV and VO compared to both vehicle and CITED2 APAA peptide controls at Pl 7. The expression of pro-angiogenic genes such as Vegfa and Epo and pro-inflammatory cytokines and chemokines such as Illb, Tnfa, Ccl2 and Ccl3 were significantly upregulated in OIR retinas compared to normoxic retinas. These same genes were significantly downregulated in OIR retinas after intravitreal injection of the CITED2 peptide, suggesting that the CITED2 peptide can regulate the hypoxic response and modulate activation of angiogenic molecules in the retina.

Example 2. Distribution of CITED2 peptide in retina after intravitreal injection [0070] We hypothesized that the CITED2 peptide rescued NV and VO in the OIR model by inhibiting HIF-mediated transcription. To confirm the effect of the CITED2 peptide on HIF transcriptional activity, we performed a luciferase reporter assay using HEK293 cells transfected with a plasmid containing three copies of the hypoxia response element (HRE) upstream of the firefly luciferase gene. Luciferase activity was significantly reduced in CITED2 peptide treated cells compared to CITED2 APAA peptide treated cells. These data indicate that the CITED2 peptide inhibits the transcriptional activity of HIF in vitro, and suggest that the reduction in NV and VO observed in the OIR experiments is due to HIF inhibition by the CITED2 peptide.

[0071] To further assess the mechanism underlying HIF inhibition by the CITED2 peptides in vivo, we generated Alexa488-conjugated peptides to identify cellular uptake and tissue localization of the CITED2 peptides. P12 OIR mice were injected with Alexa488- CITED2 peptide, Alexa488-CITED2 APAA peptide, or an equivalent volume of non- reactive Alexa488 dye and retinas were harvested 12 hours after intravitreal injection. The Alexa488-conjugated CITED2 peptides were widely distributed throughout the retina, whereas we did not observe fluorescence in retinas from eyes injected with Alexa488 dye alone. Cryosections of Alexa488-CITED2 peptide treated retinas show that the CITED2 peptide is taken up by cells in the inner plexiform layer (IPL), inner nuclear layer (INL), and outer plexiform layer (OPL), and that the CITED2 peptide localizes to the nuclei of cells in the INL. In control experiments with the Alexa488-CITED APAA peptide (where we did not observe an inhibitory effect on NV, VO, or transcriptional activity), the peptide localized to the same regions.

[0072] To confirm which specific types of retinal cells are affected by the CITED2 peptide, we prepared a single cell suspension from OIR retinas and sorted for cells with Alexa488 fluorescence, indicating that they had taken up the Alexa488-labeled CITED2 peptides. This cell population was then probed for expression of representative retinal cell type specific marker genes by RT-PCR. Gfap, Vim, Glul, and Pax6 were highly expressed, indicating that the Alexa488-CITED2 peptide localized to astrocytes (Gfap), Muller glia (Vim/Glul), and ganglion cells and/or amacrine cells (Pax6). On the other hand, Cdllb, which is a marker for microglia, and Tie2, which is a marker for endothelial cells, were only weakly expressed in the cells that contained the Alexa488-CITED2 peptide, suggesting that the CITED2 peptide does not selectively localize to these cell types in OIR retinas. We then used RT-qPCR to assess the differences in gene expression in Alexa488-CITED2 peptide and Alexa488-CITED2 APAA peptide-containing cells. These data show that the expression of known HIF target genes such as Vegfa, Epo, Ldha, and Ndufa4l2 were significantly downregulated in CITED2 peptide-containing retinal cells compared to CITED2 APAA peptide-containing retinal cells, confirming that the CITED2 peptide specifically inhibits HIF transcriptional activity in the retina.

Example 3. In vivo inhibition of vascular development by CITED2 peptide

[0073] Given the striking effect of the CITED2 peptide in preventing pathological NV in the OIR model, we sought to characterize the general anti-angiogenic potential of the CITED2 peptide. We performed an in vivo Matrigel plug assay, in which FGF2 and VEGF were used as proangiogenic factors to induce blood vessel formation and invasion of the Matrigel plug. Both FGF2 and VEGF induced formation of neovessels in Matrigel plugs 5 days after injection. When the CITED2 peptide was added to the Matrigel in combination with FGF2 prior to injection, FGF2-induced NV was visibly reduced and the percentage of CD45-negative/CD31-positive endothelial cells was also significantly decreased. In contrast, the CITED2 peptide did not significantly reduce VEGF-induced NV in VEGF and CITED2 peptide containing Matrigel plugs, suggesting that VEGF-driven vascular development in this assay is less dependent on HIF activation than FGF2-mediated angiogenesis.

[0074] We next observed whether the CITED2 peptides have effects on physiological retinal vascular development of normal mice. The mouse retinal vasculature develops during the first three weeks following birth. Vessels develop first in the superficial (inner) plexus from PO to P8 using the astrocytes as a scaffold, then in the deep (outer) plexus from P7 to Pl 2, and finally in the intermediate plexus from P14 to P21 (24). To determine whether the CITED2 peptides have an effect on formation of the vasculature of the superficial plexus, we intravitreally injected the CITED2 peptide, CITED2 APAA peptide, and vehicle into normoxic C57/B6J mice on P2 and evaluated the formation of the superficial plexus at P6. The vascularized area and the number of branching points in the superficial plexus were slightly, but significantly, decreased in CITED2 peptide injected eyes compared to CITED2 APAA peptide or vehicle injected eyes. On the other hand, when we injected the CITED2 peptide into the eyes of P4 and P7 mice and analyzed retinal vasculature at P10 and P14, respectively, the number of vessel branching points of all layers were similar in CITED2 peptide and vehicle injected retinas suggesting that the CITED2 peptide has an effect on very early retinal vascular development of the superficial plexus but does not affect later stages of retinal vascular development.

Example 4. Combination therapy of the CITED2 peptide and Aflibercept

[0075] Intravitreal injection of the anti-VEGF drugs Aflibercept or Ranibizumab is widely used to treat neovascular retinal diseases such as PDR and ROP as well as AMD, diabetic macular edema (DME), and RVO. However, as targeting VEGF is known to induce off- target effects, we investigated the ability of the CITED2 peptide to function synergistically with a lower dose of Aflibercept in order to minimize the off-target effects of VEGF antagonists. While all doses (200 ng - 20 pg) of intravitreally injected Aflibercept are capable of significantly reducing the area of NV in OIR retinas in a manner comparable to the CITED2 peptide, VO is not rescued with high doses of Aflibercept (10-20 pg) that are comparable to the clinical dose in humans, and decreased VO is only observed for the lowest dose of Aflibercept (200 ng) (Fig. IB). However, in OIR eyes injected with a combination of 10 pg Aflibercept and 3.4 ng of the CITED2 peptide, the area of VO was significantly smaller than observed for Aflibercept monotherapy (Fig. IB). With a lower 200 ng dose of Aflibercept alone, the anti-neovascular effect is comparable to the higher dose of Aflibercept (Fig. 1C). However, combination therapy of 200 ng of Aflibercept and 3.4 ng of the CITED2 peptide significantly rescued both VO and NV (Fig. 1 A-C). Thus, combinations of the CITED2 peptide and Aflibercept can promote revascularization of the avascular retina, an effect not observed for high-dose Aflibercept treatment alone in OIR. Moreover, combined injection of Aflibercept and the CITED2 peptide allows for a reduction in the dose of Aflibercept, suggesting that the CITED2 peptide could be used in combination with anti- VEGF therapies to mitigate the off-target local and systemic effects of high doses of anti- VEGF drugs.

[0076] We then used RT-qPCR to assess the differences in gene expression in CITED2 peptide and Aflibercept treated OIR retinas. These data show that the expression levels of known HIF target genes such as Vegfa, Epo, Ldha, Fgfl 1, and Ndufa4l2 were significantly downregulated in CITED2 peptide treated OIR retinas compared to Aflibercept treated retinas (Fig. ID), confirming that the CITED2 peptide functions as a negative regulator of HIF transcriptional activity, whereas Aflibercept functions by targeting circulating VEGF and thus does not have a similar suppressive effect on transcription of HIF target genes. We additionally observe that the amount of VEGF in P15 OIR retinas is higher for Aflibercept treated retinas than for CITED2 peptide treated retinas (Fig. IE), suggesting that directly targeting the circulating pool of VEGF results in compensatory up-regulation of angiogenic cytokines, a phenomenon that has previously been observed in both the mouse and rat OIR models. See, e.g., McCloskey et al., Invest Ophthalmol Vis Sci. 54:2020-6, 2013; and Dorrell et al., Proc Natl Acad Sci USA 104:967-72, 2007. Taken together, these data highlight the potential therapeutic benefit of combining anti-VEGF therapies like Aflibercept and the CITED2 peptide, a novel inhibitor of HIF-driven transcription, for treatment of pathological NV in patients with ischemic retinal diseases.

Example 5. Activities of additional CITED2 variant peptides

[0077] We further examined the activities of a series of variant peptides that are derived from the CITED2 peptide shown in SEQ ID NO:4. Sequences of these variants are listed in Table 1. Relative to the native CITED2 sequence, some of these peptides contain terminal truncations or substitutions, e.g., peptides shown in SEQ ID NOs:5-14 and 23. Some of the peptides contain a nuclear localization signal sequence at the N- or C-terminus, e.g., peptides shown in SEQ ID NOs: 15-22. Specifically, peptides shown in SEQ ID NOs: 15, 17, 19, and 21 contain aN-terminal nuclear localization signal sequence, which is connected to the CITED2 sequence via a SSGS (SEQ ID NO:58) linker. Peptides shown in SEQ ID NOs:16, 18, 20, and 22 contain a C-terminal nuclear localization signal sequence, which is connected to the CITED2 sequence via a SG linker.

[0078] Relative to the native CITED2 peptide sequence shown in SEQ ID NO:3, the peptide shown in SEQ ID NO:4 contains aN-terminal pentapeptide GSHMS (SEQ ID NO:24) as a result of artifact from cloning and expression. Similarly, actual peptides corresponding to the sequences shown in Table 1 as used in the studies also contain such extra N-terminal residues. Specifically, actual peptides used in the study that correspond to SEQ ID NOs:8, 15, 17, 19, and 21 all have extra N-terminal residues GSHM (SEQ ID NO: 59), while actual peptides corresponding to the other sequences in the Table all have extra N-terminal residues GSHMS (SEQ ID NO:24). The presence or absence of these extra N-terminal residues do not affect activities of the peptides as observed in the study.

Table 1. Sequences of variant CITED2 peptides examined

CITED2 sequence via a SSGS (SEQ ID NO:58) linker. Peptides shown in SEQ ID NOs:16, 18, 20, and 22 contain a C-terminal nuclear localization signal sequence, which is connected to the CITED2 sequence via a SG linker.

[0078] Relative to the native CITED2 peptide sequence shown in SEQ ID NO:3, the peptide shown in SEQ ID NO:4 contains aN-terminal pentapeptide GSHMS (SEQ ID NO:24) as a result of artifact from cloning and expression. Similarly, actual peptides corresponding to the sequences shown in Table 1 as used in the studies also contain such extra N-terminal residues. Specifically, actual peptides used in the study that correspond to SEQ ID NOs:8, 15, 17, 19, and 21 all have extra N-terminal residues GSHM (SEQ ID NO:59), while actual peptides corresponding to the other sequences in the Table all have extra N-terminal residues GSHMS (SEQ ID NO:24). The presence or absence of these extra N-terminal residues do not affect activities of the peptides as observed in the study.

Table 1. Sequences of variant CITED2 peptides examined

[0079] These variant CITED2 peptides were examined for their activities in binding to the TAZ1 domain and competing against HIF- la. They were additionally analyzed for intracellular localization. The TAZ1 binding and HIF- la competition studies were performed as described above in Example 1 and also in Example 6 below. For cellular localization studies, cultured HEK293 or human fetal RPE cells were treated with 0.5-2 pM Alexa488-labeled peptides in the growth medium and incubated for 4-24 hours. Live cells were imaged by fluorescence microscopy.

[0080] Results from these studies are shown in Table 2. These data indicate that, in addition to CITED2 peptides shown in SEQ ID NOs: 2-4, these additional variant CITED2 peptide sequences would similarly function synergistically in combination with VEGF antagonists.

Table 2. TAZl-binding and HIF- la-inhibiting activities of variant peptides

Example 6. Some exemplified methods and materials

[0081] Reagents: Peptides of the CITED2 C-terminal transactivation domain (residues 216-269 of human/mouse CITED2 with the pentapeptide sequence GSHMS (SEQ ID NO:24) at the N-terminus) were expressed in E. coli and purified as described in Berlow et al., Nature 543:447-51, 2017. The L243A/E245A/L246A mutations for the CITED2 APAA mutant peptide were introduced using standard site-directed mutagenesis protocols. The peptides were stored as lyophilized powders and were resuspended and dialyzed into buffer containing 20 mM Tris pH 6.8, 50 mM NaCl, and 2 mM DTT prior to use. The identity and purity of all peptides was confirmed by mass spectrometry.

[0082] The CITED2 peptides contain a single cysteine residue (C261) and were fluorescently labeled with a 3-5 fold excess of AlexaFluor 488 Cs maleimide dye (Invitrogen) in 50 mM Tris pH 7.2. Unreacted dye was removed during buffer exchange into 20 mM Tris pH 6.8, 50 mM NaCl, and 2 mM DTT on a NAP-5 column (GE). For fluorescent dye controls, non-reactive AlexaFluor 488 dye was prepared by dissolving AlexaFluor 488 carboxylic acid, tetrafluorophenyl (TFP) ester (Invitrogen) in water adjusted to pH 9.0 with sodium hydroxide and incubating overnight to hydrolyze the ester and eliminate amine-reactivity.

[0083] Aflibercept (Eylea; Regeneron Pharmaceutical, Tarrytown, NY) was diluted in 20 mM Tris pH 6.8, 50 mM NaCl, and 2 mM DTT. One pg (0.5 pl) of normal human IgG control (R&D systems 1-001 -A) was used as a control.

[0084] Fluorescence anisotropy competition experiments: Fluorescence anisotropy competition assays were carried out in 20 mM Tris pH 6.8, 50 mM NaCl, and 2 mM DTT on aHoriba Fluorolog-3 fluorimeter at 25 °C. Alexa 594-labeled HIF-la and CITED2 peptides were prepared as previously described (Berlow et al., Nature 543:447-51, 2017). For competition assays, unlabeled CITED2 peptides were titrated into a pre-formed complex of 20 nM Alexa594-HIF-la and 250 nM TAZ1 or 20 nM Alexa594-CITED2 and 250 nM TAZ1. Data were analyzed as described previously using Graphpad Prism 8 (GraphPad Software, Inc.).

[0085] Animal experiments: C57BL/6J mice were obtained from the animal facility at The Scripps Research Institute. OIR was induced as previously in Smith et al., supra. Briefly, C57BL/6J mouse pups and their mother were exposed to 75% oxygen from postnatal day 7 (P7) to P12 and subsequently transferred to room air. Retinal NV and VO were analyzed at P17.

[0086] Quantification of OIR: The percentage of the area of NV and VO in OIR retinas was automatically quantified using deep learning segmentation software available at the OIRSEG.org website (Xiao et al., JCI Insight. 2(24), 2017).

[0087] Intravitreal injections: P12 C57BL/6J OIR pups and P2, P4, and P7 normoxic mice were injected in the vitreous cavity with 0.5 pl of CITED2 peptide, CITED2 APAA peptide, Aflibercept or vehicle control using a 10 pl Hamilton syringe with a 34-gage needle.

[0088] Luciferase assay: HEK293 cells were transfected using Lipofectamine 2000 (Invitrogen) with 0.5 pg of pGL2-HRE (Addgene #26731) and were co-transfected with 0.63 pg of CITED2 peptide or 0.63 pg CITED2 APAA mutant peptide according to the manufacturer’s protocol. After 24 hours incubation, cells were washed with PBS and lysed with the luciferase cell culture lysis reagent (Promega), and the luciferase assay was performed using the luciferase assay system (Promega E1500) according to the manufacturer’s protocol.

[0089] Sorting for mouse retinal cells: 2 pM of Alexa488-conjugated CITED2 or CITED2APAA peptides were injected into both eyes of P12 OIR mice. Retinas were collected 16 hours after injection and transferred into cold PBS with Ca^2+/Mg²⁺. A postnatal neural dissociation kit (Miltenyi, 130-092-628) was used to prepare a single cell suspension from mouse retinas. The resuspended cells (in 500 pl of PBS with 1% FBS) were stained with DAPI (1:1000) for exclusion of dead cells. The cells were sorted and analyzed using a FACS Aria flow cytometer (BD) with FlowJo (BD) software. Alexa488 positive and negative cells were sorted from the live cell population. Total RNA was isolated from sorted cells using the RNeasy Micro Kit (QIAGEN) and reverse transcribed using Maxima First Strand cDNA Synthesis Kit for RT-qPCR (Thermo Scientific). For retinal cell profiling, PCR was performed using PrimeSTAR GXL DNA polymerase on a T100 Thermal Cycler (Bio-Rad) and the cycling program was 25 cycles of 94 °C for 30 sec, 58 °C for 30 sec, and 72 °C for 30 sec. qPCR was performed using Power SYBR™ Green PCR Master Mix (Thermo Fisher Scientific) on a Quantstudio 5 Real-Time PCR System (Thermo Fisher Scientific) and 36b4 was used as the reference gene. Primer sequences are listed in Table 3. [0090] Retinal immunofluorescence: Retinas were dissected and prepared for whole mounts or sectioning. For retinal whole mounts, dissected retinas were placed in 4% PFA for 1 hour. After fixation, the vitreous was removed and the retinas were laid flat with 4 radial relaxing incisions and incubated overnight with Al exa568 -conjugated isolectin Griffonia Simplicifolia IB-4 (GS-lectin) (Thermo Fisher Scientific, 121413). Retinas were washed in PBS and mounted with ProLong Diamond Antifade mounting medium (Thermo Fisher Scientific, P36965). For preparation of cross-sections, dissected eyes were incubated in 4% PFA for 3 hours. Retinas were then placed in 20% sucrose at 4 °C for 3 hours and embedded in Tissue-Tek OCT compound (Sakura Finetek; Torrance, CA) for cryo-sectioning. 12-pm retinal sections were washed in PBS and incubated with DAPI to visualize cell nuclei. Images were obtained using a confocal microscope (LSM710, Carl Zeiss; Oberkochen, Germany).

[0091] Real-time PCR analysis: For qPCR, single retinas were collected in 500 pl of Trizol and total RNA was isolated using a PureLink RNA Mini Kit (Thermo Fisher Scientific) according to manufacturer's instructions. 750 ng of RNA was used for RT-qPCR using a high-capacity cDNA reverse transcription kit (Thermo Fisher Scientific). qPCR was performed using Taqman universal master mix (Thermo Fisher Scientific) and Taqman probes or Power SYBR™ Green PCR Master Mix (Thermo Fisher Scientific) and primers on a Quantstudio 5 Real-Time PCR System (Thermo Fisher Scientific). [3-actin (Actb) was used as the reference gene for all experiments. Primer sequences and Taqman assays are listed in Table 3 and Table 4.

Table 3. Primer sequences for PCR.

Table 4. Taqman assays for PCR.

[0092] In vivo Matrigel plug assay: Matrigel Growth Factor Reduced Basement Membrane Matrix, LDEV-free (Coming) was mixed with 1000 ng/ml recombinant VEGF165 (PeproTech #100-20) or 500 ng/ml of FGF2 (PeproTech #450-33) and 1 pM CITED2 peptide or control vehicle on ice. 500 pl of Matrigel was injected subcutaneously on both flanks on the abdominal side after shaving under anesthesia (100 mg/kg ketamine and 10 mg/kg xylazine). After 5 days, mice were euthanized and Matrigel plugs were removed, photographed, fixed with 4% PF A, and embedded in OCT. To visualize endothelial cells that migrated into the Matrigel plug, cryo-sectioned plugs were stained with CD31 antibody (BD #550274) and DAPI. [0093] FACS analysis was used to quantify the neovessels in the plugs as described Adini et al., J Immunol Methods. 342:78-81, 2009. The Matrigel plugs were digested by incubating for 1 hour at 37 °C with an enzymatic mixture containing 25 pg/ml of hyaluronidase (MP Biomedical, Solon, OH), 25 pg/ml of DNase (Sigma- Aldrich, St Louis, MO), 3 u/ml of dispase (Roche, Nutley, NJ), and 3 u/ml of liberase (Roche, Nutley, NJ) dissolved in PBS. The suspension was washed, filtered and stained with CD31-FITC (Biolegend #102405) and CD45-Bv421 (Biolegend #103133). Samples were analyzed with a F ACS LSR II flow cytometer (Becton Dickinson Immunocytometry Systems, San Jose, CA) and data analysis was performed in FlowJo vlO (BD). Endothelial cells were present in the CD31 -positive and CD45-negative population.

[0094] ELISA assays: Retinal tissue was homogenized in 250 pl of PBS and stored overnight at < -20 °C. After two freeze-thaw cycles, the homogenates were centrifuged for 5 minutes at 5000 x g. Total protein was quantified using a BCA assay according to the manufacturer’s protocol (Pierce). Supernatants were then assayed without dilution in duplicate using the Mouse VEGF Quantikine ELISA kit (R&D Systems) according to the manufacturer’s protocol.

[0095] Statistical analysis: All statistical tests were performed in GraphPad Prism 8 (GraphPad Software, Inc). Data comparisons between two groups were performed using unpaired two-tailed Student t-tests or multiple t-tests. Data comparisons between multiple groups were performed with one-way ANOVA with Tukey’s correction. Data are represented as mean ± SEM unless otherwise indicated. A P value < 0.05 was considered significant.

***

[0096] It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are described.

[0097] All publications, GenBank sequences, ATCC deposits, patents and patent applications cited herein are hereby expressly incorporated by reference in their entirety and for all purposes as if each is individually so denoted.

Claims

WE CLAIM:

1. A method for inhibiting retinal neovascularization and promoting revascularization in a subject, comprising administering to a subject a pharmaceutical composition containing or capable of expressing a therapeutically effective amount of (a) a VEGF antagonist and (b) a CITED2 peptide that can bind to the TAZ1 domain of the general transcriptional coactivators CBP and p300, thereby inhibiting hypoxia-induced retinal neovascularization and promoting revascularization in the subject.

2. The method of claim 1, wherein the subject is afflicted with an ocular disorder associated with retinal neovascularization.

3. The method of claim 2, wherein the ocular disorder is ischemic retinopathy, diabetic retinopathy, retinopathy of prematurity, retinal vein occlusion, neovascular glaucoma, macular edema, familial exudative vitreoretinopathy, retinal angioma, macular degeneration or retinitis pigmentosa.

4. The method of claim 1, wherein the CITED2 peptide is derived from the disordered C-terminal transactivation domain of CITED2.

5. The method of claim 4, wherein the CITED2 peptide comprises a sequence DEEVLMX²⁰LVIX²⁴MGLX²⁸RIX³¹X³² (SEQ ID NO:51) or a conservatively modified variant thereof, wherein X²⁰ is S, K, D, E, A, P, or phosphoSer; X²⁴ is E, K or R; X²⁸ is D, E, I, L, M or W; X³¹ is K or P; and X³² is E or P.

6. The method of claim 5, wherein the CITED2 peptide comprises any one of SEQ ID NOs:2-23, a substantially identical sequence or a conservatively modified variant.

7. The method of claim 5, wherein the CITED2 peptide further comprises a N- terminal or C-terminal nuclear localization sequence.

8. The method of claim 1, wherein the VEGF antagonist is a VEGF specific inhibitor polypeptide, an antibody, or an aptamer.

38

9. The method of claim 8, wherein the VEGF antagonist is Aflibercept, Ranibizumab, Bevacizumab, or Pegaptanib.

10. The method of claim 1, wherein the VEGF antagonist is Aflibercept or a functional derivative thereof, and the CITED2 peptide comprises any one of SEQ ID NOs:2- 23 or a conservatively modified variant thereof.

11. The method of claim 1, wherein the pharmaceutical composition comprises an engineered cell that stably or transiently expresses the VEGF antagonist and/or the CITED2 peptide.

12. The method of claim 11, wherein the engineered cell expresses both the VEGF antagonist and the CITED2 peptide.

13. The method of claim 11, wherein the pharmaceutical composition comprises the CITED2 peptide and an engineered cell that expresses the VEGF antagonist.

14. The method of claim 11, wherein the pharmaceutical composition comprises the VEGF antagonist and an engineered cell that expresses the CITED2 peptide.

15. The method of claim 11 , wherein the engineered cell is derived from retinal pigment epithelium.

16. The method of claim 11, wherein the engineered cell is encapsulated in porous material prior to administration to the subject.

17. The method of claim 1, wherein the pharmaceutical composition is administered to the subject via intravitreal injection.

18. The method of claim 1, where the subject is a human.

19. A method for treating or preventing an ocular vascular disorder associated with hypoxia-induced neovascularization in a subject, comprising administering to the subject a pharmaceutical composition containing or capable of expressing a therapeutically effective amount of (a) a VEGF antagonist and (b) a CITED2 peptide that can bind to the TAZ1

39 domain of the general transcriptional coactivators CBP and p300, thereby treating or preventing the ocular vascular disorder in the subject.

20. The method of claim 19, wherein the ocular vascular disorder is manifested by retinal neovascularization and/or vaso-obliteration.

21. The method of claim 19, wherein the ocular disorder is ischemic retinopathy, diabetic retinopathy, retinopathy of prematurity, retinal vein occlusion, neovascular glaucoma, macular edema, familial exudative vitreoretinopathy, retinal angioma, macular degeneration or retinitis pigmentosa.

22. The method of claim 19, wherein the subject is afflicted with the ocular disorder.

23. The method of claim 19, wherein the subject is at risk of developing, or predisposed to develop, the ocular disorder.

24. A pharmaceutical composition, comprising:

(a) a VEGF antagonist and a CITED2 derived peptide that specifically binds to the TAZ1 domain of transcriptional coactivators CBP/p300;

(b) a CITED2 derived peptide that specifically binds to the TAZ1 domain of transcriptional coactivators CBP/p300, and an engineered cell expressing a VEGF antagonist;

(c) a VEGF antagonist, and an engineered cell expressing a CITED2 derived peptide that specifically binds to the TAZ1 domain of transcriptional coactivators CBP/p300; or

(d) an engineered cell expressing a VEGF antagonist and a CITED2 derived peptide that specifically binds to the TAZ1 domain of transcriptional coactivators CBP and p300.

25. The pharmaceutical composition of claim 24, wherein the VEGF antagonist is Aflibercept or a conservatively modified variant thereof.

26. The pharmaceutical composition of claim 24, wherein the CITED2 derived peptide comprises a sequence DEEVLMX²⁰LVIX²⁴MGLX²⁸RIX³¹X³² (SEQ ID NO:51) or a conservatively modified variant thereof, wherein X²⁰ is S, K, D, E, A, P, or phosphoSer; X²⁴ is E, K or R; X²⁸ is D, E, I, L, M or W; X³¹ is K or P; and X³² is E or P.

40

27. The pharmaceutical composition of claim 24, wherein the CITED2 derived peptide comprises any one of SEQ ID NOs:2-23 or a conservatively modified variant thereof.

28. A vector comprising a polynucleotide sequence that encodes both (a) a VEGF antagonist polypeptide and (b) a CITED2 derived peptide that specifically binds to the TAZ1 domain of the general transcriptional coactivators CBP and p300.

29. The vector of claim 28, wherein the VEGF antagonist is Aflibercept or a conservatively modified variant thereof.

30. The vector of claim 28, wherein the CITED2 derived peptide comprises any one of SEQ ID NOs:2-23 or a conservatively modified variant thereof.

31. An engineered cell that expresses a VEGF antagonist polypeptide and a CITED2 peptide that can bind to the TAZ1 domain of the general transcriptional coactivators CBP and p300.

32. The engineered cell of claim 31, wherein the VEGF antagonist polypeptide and/or the CITED2 peptide are stably expressed.

33. The engineered cell of claim 31, wherein the VEGF antagonist polypeptide and/or the CITED2 peptide are transiently expressed.