Blocking retinal capillary regression to prevent retinopathy

Abstract

Provided herein are compositions and methods for preventing ocular vascular regression, preserving visual function, or both, comprising: identifying a subject in need of treatment for ocular vascular regression; and providing the subject with an effective amount of an agent that is: an agonist that increases expression of at least one gene selected from: ETS-Related Gene (ERG), Fli-1 Proto-Oncogene; ETS Transcription Factor (FLU); KEF transcription factor 2 (KLF2); KEF transcription factor 4 (KLF4); or a Piezo-Type Mechanosensitive Ion Channel Component 1 (PIEZO1) agonist, or both; wherein the agent is provided in an amount sufficient to prevent the ocular vascular regression, preserving visual function, or both.

Description

BLOCKING RETINAL CAPILLARY REGRESSION TO PREVENT RETINOPATHYCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is a PCT International patent application of and claims priority to U.S. provisional patent application serial number 63 / 666,397 filed on July 1, 2024 and U.S. provisional patent application serial number 63 / 737,194 on December 20, 2024, the contents of which are incorporated by reference in its entirety..TECHNICAL FIELD OF THE INVENTION

[0002] The present invention relates in general to the field of retinopathies, and more particularly, to novel compositions and methods for blocking retinal capillary regression to prevent retinopathy.STATEMENT OF FEDERALLY FUNDED RESEARCH

[0003] This invention was made with government support under R35HL144605 awarded by the National Institutes of Health. The government has certain rights in the invention.INCORPORATION-BY-REFERENCE OF MATERIALS FILED ON COMPACT DISC

[0004] None.BACKGROUND OF THE INVENTION

[0005] Without limiting the scope of the invention, its background is described in connection with retinopathies.

[0006] Retinopathy of prematurity (ROP) is an eye disease associated with infants delivered at early gestational ages and with low birth weights. Approximately 600 premature infants born in the U.S. become legally blind each year due to ROP, and at least 50,000 infants around the world are diagnosed with the disease annually. ROP fundamentally stems from dysregulated blood vessel growth in the retina. In humans, retinal vasculature develops between 16 and 40 weeks of gestation, so this vascular network is immature when infants are born preterm. Retinal blood vessels would normally continue growing after a premature birth to supply energetically demanding photoreceptors with oxygen and nutrients. However, very premature infants are at risk of death due to their underdeveloped lungs, so they frequently receive critical oxygen supplementation from mechanical ventilators in neonatal intensive care units (NICUs). In terms of the eye, this treatment temporarily satisfies the oxygen demands of the growing retina and thus aberrantly arrests normal retinal vascular growth. Moreover, high oxygen levels can ablate retinal blood vessels in humans and mice. Altogether, this oxygen-induced decline in retinal vasculaturecharacterizes the first phase of ROP. When premature infants are eventually removed from ventilators, the diminished retinal vasculature cannot supply sufficient oxygen and nutrients to the eye, which becomes hypoxic. Hypoxia stabilizes transcription factors that drive expression of vascular endothelial growth factor A (VEGFA) — a potent trigger of angiogenesis and vascular permeability. Excessive VEGFA, which is produced by multiple retinal cell types in the hypoxic eye, stimulates a disorganized and pathological angiogenic response during the second phase of ROP. The dysfunctional and leaky blood vessels that arise during this stage can drive scar tissue formation, which in the most severe cases can cause traction on the retina and lead to partial or total retinal detachment. Even if these pathological vessels regress spontaneously and the eye revascularizes in a normal pattern, neural deficits can remain, likely due to death of photoreceptors and other retinal neurons during the hypoxic stage of the disease when the diminished vasculature network cannot supply necessary oxygen and nutrients to the retina.

[0007] Since hyperoxia has long been recognized as a trigger for ROP, clinicians have focused on careful utilization of mechanical ventilators and on monitoring oxygen saturation levels in NICUs to prevent the disease. Nevertheless, inadequate oxygen control persists in developing countries, and the increasing survival of “micropremature” infants (gestational ages of 24 weeks or less or birth weights less than 750 g) is elevating the demand for prolonged oxygen supplementation in countries like the U.S. that had previously seen diminishing ROP rates. Therefore, ROP continues to be one of the leading causes of childhood blindness worldwide. Current ROP treatments primarily focus on the second phase of the disease — when pathological angiogenesis and vessel leakiness occurs. One therapeutic option involves laser treatments to ablate the retinal cells that secrete VEGFA in response to hypoxia. However, laser treatments can cause long-term damage to peripheral and night vision and can exacerbate the myopia that is already associated with ROP. Another common therapy for ROP involves off-label intravitreal injections of anti-VEGF drugs (e.g., bevacizumab, ranibizumab, aflibercept, and conbercept), which can block the growth and leakiness of pathological retinal vasculature. Such drugs can be quite effective, but one concerning aspect of their use in premature infants is that even when injected into the eye, they can be detected in systemic circulation for up to eight weeks. This is undesirable since antiangiogenic drugs could block critical blood vessel expansion in growing organs.

[0008] Despite these advances, and based on these limitations and risks associated with current ROP treatments, a need remains for new approaches that prevent the disease and its associated vision loss.SUMMARY OF THE INVENTION

[0009] As embodied and broadly described herein, an aspect of the present disclosure relates to a method of preventing ocular vascular regression, preserving visual function, or both, comprising: identifying a subject in need of treatment to prevent ocular vascular regression; and providing the subject with an effective amount of an agent that is: an agonist that increases expression of at least one gene selected from: erythroblast transformation-specific (ETS)-Related Gene (ERG), Fli-1 Proto-Oncogene, ETS Transcription Factor (FLU); Kriippel-like factor (KLF) transcription factor 2 (KLF2); KLF transcription factor 4 (KLF4); or a Piezo-Type Mechanosensitive Ion Channel Component 1 (PIEZO1) agonist, or both; wherein the agent is provided in an amount sufficient to prevent the ocular vascular regression, preserving visual function, or both. In one aspect, the subject is in need of treatment for retinopathy of prematurity (ROP), diabetic retinopathy (DR), capillary regression in heart or kidney. In another aspect, the agent that increases the expression of at least one of ERG, FLU, KLF2, or KLF4, is a nucleic acid-based therapeutic. In another aspect, the nucleic acid-based therapeutic is selected from a DNA, an RNA, an mRNA, a siRNA, RNAi, promoter RNA (pRNAs), enhancer RNA (eRNAs), or superenhancer RNA (seRNAs). In another aspect, the agent is a small molecule agonist that increases the expression of ERG, FLU, KLF2, or KLF4 mRNA, stabilized an ERG, FLU, KLF2, or KLF4 mRNA, or both. In another aspect, the ocular vascular regression is retinal capillary regression. In another aspect, the PIEZO 1 agonist is Yoda-1. In another aspect, the agent is administered topically, subconjunctivally, intracamerally, subtenonally, subretinally, subchoroidally, suprachoroidally, supraorbitally, retrobulbarlly, as an ocular implant, or intravitreally, or wherein the agent is formulated into an eye drop, gel, ointment, spray, a reservoir, or mist. In another aspect, the agent prevents least one of: decrease retinal neovessels by at least 40% or a retinal avascular area by at least 60% compared to a vehicle-injected contralateral eye. In another aspect, the agent does not inhibit vascular endothelial growth factor (VEGF).

[0010] As embodied and broadly described herein, an aspect of the present disclosure relates to a method for preventing or reducing a retinopathy of prematurity (ROP) or diabetic retinopathy (DR)in a patient comprising the steps of: identifying the patient in need of prevention or treatment for ocular vascular regression; and providing the patient with an effective amount of an agent that: increases expression of at least one gene selected from at least one of: erythroblast transformationspecific (ETS)-Related Gene (ERG), Fli-1 Proto-Oncogene, ETS Transcription Factor (FLU); Kriippel-like factor (KLF) transcription factor 2 (KLF2); KLF transcription factor 4 (KLF4); or is a Piezo-Type Mechanosensitive Ion Channel Component 1 (PIEZO1) agonist, or both; wherein the agent is provided in an amount effective to prevent or reduce the ocular vascular regression, preserving visual function, or both. In one aspect, the agent that increases the expression of at leastone of ERG, FLU, KLF2, or KLF4, is a nucleic acid-based therapeutic. In another aspect, the nucleic acid-based therapeutic is selected from a DNA, an RNA, an mRNA, a siRNA, RNAi, promoter RNA (pRNAs), enhancer RNA (eRNAs), or superenhancer RNA (seRNAs). In another aspect, the agent is a small molecule agonist that increases the expression of ERG, FLU, KLF2, or KLF4 mRNA, stabilized an ERG, FLU, KLF2, or KLF4 mRNA, or both. In another aspect, the ocular vascular regression is retinal capillary regression. In another aspect, the PIEZO 1 agonist is Yoda-1. In another aspect, the agent is administered topically, subconjunctivally, intracamerally, subtenonally, subretinally, subchoroidally, suprachoroidally, supraorbitally, retrobulbarlly, as an ocular implant, or intravitreally, or wherein the agent is formulated into an eye drop, gel, ointment, spray, a reservoir, or mist. In another aspect, the agent prevents least one of: decrease retinal neovessels by at least 40% or a retinal avascular area by at least 60% compared to a vehicle- injected contralateral eye. In another aspect, the agent does not inhibit vascular endothelial growth factor (VEGF).

[0011] As embodied and broadly described herein, an aspect of the present disclosure relates to a method of preventing or treating retinopathy of prematurity (ROP), diabetic retinopathy (DR), or both, in a subject exposed to high levels of oxygen comprising providing the subject with an effective amount of an agent that: increases expression of at least one of: erythroblast transformation-specific (ETS)-Related Gene (ERG), Fli-1 Proto-Oncogene, ETS Transcription Factor (FLU); Kriippel-like factor (KLF) transcription factor 2 (KLF2); KLF transcription factor 4 (KLF4); or is a Piezo-Type Mechanosensitive Ion Channel Component 1 (PIEZO1) agonist, or both; wherein the agent is provided in an amount sufficient to prevent or treat the ROP, DR, or both. In one aspect, the agent that increases the expression of at least one of ERG, FLU, KLF2, or KLF4, is a nucleic acid-based therapeutic. In another aspect, the nucleic acid-based therapeutic is selected from a DNA, an RNA, an mRNA, a siRNA, RNAi, promoter RNA (pRNAs), enhancer RNA (eRNAs), or superenhancer RNA (seRNAs). In another aspect, the PIEZO1 agonist is Yoda- 1. In another aspect, the agent is administered topically, subconjunctivally, intracamerally, subtenonally, subretinally, subchoroidally, suprachoroidally, supraorbitally, retrobulbarlly, as an ocular implant, or intravitreally, and wherein the agent s formulated into an eye drop, gel, ointment, spray, a reservoir, or mist.

[0012] As embodied and broadly described herein, an aspect of the present disclosure relates to a method of preventing or treating retinal disease in a subject with diabetes comprising: identifying a subject with diabetes in need of treatment to prevent or treat ocular vascular regression; and providing the subject with diabetes with an effective amount of an agent that is: an agonist that increases expression of at least one gene selected from: erythroblast transformation-specific(ETS)-Related Gene (ERG), Fli-1 Proto-Oncogene, ETS Transcription Factor (FLU); Kriippel- like factor (KLF) transcription factor 2 (KLF2); KLF transcription factor 4 (KLF4); or a PiezoType Mechanosensitive Ion Channel Component 1 (PIEZO 1) agonist, or both; wherein the agent is provided in an amount sufficient to prevent the ocular vascular regression, preserving visual function, or both in the patient with diabetes. In one aspect, the subject is in need of treatment for capillary regression in heart or kidney. In another aspect, the agent that increases the expression of at least one of ERG, FLU, KLF2, or KLF4, is a nucleic acid-based therapeutic. In another aspect, the nucleic acid-based therapeutic is selected from a DNA, an RNA, an mRNA, a siRNA, RNAi, promoter RNA (pRNAs), enhancer RNA (eRNAs), or superenhancer RNA (seRNAs). In another aspect, the agent is a small molecule agonist that increases the expression of ERG, FLU, KLF2, or KLF4 mRNA, stabilized an ERG, FLU, KLF2, or KLF4 mRNA, or both. In another aspect, the ocular vascular regression is retinal capillary regression. In another aspect, the PIEZO 1 agonist is Yoda-1. In another aspect, the agent is administered topically, subconjunctivally, intracamerally, subtenonally, subretinally, subchoroidally, suprachoroidally, supraorbitally, retrobulbarlly, as an ocular implant, or intravitreally, or wherein the agent is formulated into an eye drop, gel, ointment, spray, a reservoir, or mist. In another aspect, the agent prevents least one of: decrease retinal neovessels by at least 40% or a retinal avascular area by at least 60% compared to a vehicle-injected contralateral eye. In another aspect, the agent does not inhibit vascular endothelial growth factor (VEGF). In another aspect, the agent unblocks blood flow in diabetic capillaries clogged with adherent leukocytes by blocking the binding of lymphocyte function- associated antigen 1 (LFA-1) and intercellular adhesion molecule 1 (ICAM-1). In another aspect, the agent that unblocks blood flow is lifitegrast.BRIEF DESCRIPTION OF THE DRAWINGS

[0013] For a more complete understanding of the features and advantages of the present invention, reference is now made to the detailed description of the invention along with the accompanying figures and in which:

[0014] FIG. 1 is a schematic of the oxygen-induced retinopathy (OIR) protocol in mice, with respective phases of oxygen exposure and retinal vascular effects. Note that the formation of neovascular (NV) tufts peaks at P17, and the tufts undergo a spontaneous regression process (i.e., resolution) by P25.

[0015] FIG. 2: NV tufts undergo regression in Flil / Er^ECkomice exposed to OIR. Littermate control and FlH / ErECkopups were subjected to OIR and induced with tamoxifen at P16-P17. Retinas were immunostained at Pl 8 for CD31+ vasculature and were quantified with the Image Jplugin SWIFT_NV. Numbers of NV tufts were comparable in controls and mutants (not shown), but mutant tufts were smaller, resulting in decreased NV area (n=6-10). Stats: unpaired two-tailed t-test.

[0016] FIG. 3: Endothelial Erg overexpression reduces hyperoxia-induced retinal capillary regression. Littermate control and ErgECoepups were induced with tamoxifen at P5-P7 and subjected to OIR. Retinas were immunostained at P8 for CD31+ vasculature, which was analyzed with the ImageJ plugin SWIFT NV to quantify avascular area (n=2-5).

[0017] FIG. 4: Reduced hyperoxia-induced retinal capillary regression m ErECoemice correlates with reduced formation of neovascular tufts. Littermate control and ErgECoepups were induced with tamoxifen at P5-P7 and subjected to OIR. Retinas were immunostained at P17 for CD31+ vasculature, which was analyzed with the ImageJ plugin SWIFT NV to quantify avascular area (n=4-8). Stats: unpaired two-tailed t-test.

[0018] FIG. 5: The PIEZO1 agonist Yodal reduces hyperoxia-induced retinal capillary regression. Littermate C57B1 / 6J pups were injected (s.c. to back) with vehicle (PBS) or Yodal (600nmol / kg / day) from P5-P7. Pups were exposed to hyperoxia (75% O2) for 24 hr starting at P7. Retinas were subsequently immunostained at P8 for CD31+vasculature, which was analyzed with the ImageJ plugin SWIFT NV to quantify avascular area (n=5-6). Stats: unpaired two-tailed t- test.

[0019] FIG. 6: Capillary regression is elevated in STZ-challenged mice. 8-week-old male C57B1 / 6J mice were injected with STZ (50 mg / kg) for 5 consecutive days. Hyperglycemia (>250 mg / dl) was confirmed 2 weeks after the last STZ dose. At 6, 10, and 14 weeks after STZ treatment, retinas were harvested and immunostained for CD31 and the basement membrane component ColIV. Empty collagen sleeves (CD31- / ColIV+; arrows) — which are indicative of capillary regression — were manually counted in 20X confocal fields (3 per eye) in the deep retinal layer and were compared against eyes from age-matched, vehicle-treated controls (“No STZ”). Stats: one-way ANOVA compared to controls; each dot = one mouse.

[0020] FIGS. 7A and 7B show evidence of low flow in regressed retinal vessels after STZ challenge. Mice were treated with STZ (as in FIG. 6), and 8 weeks later were perfused with FITC- conjugated conA for visualization of patent vessels. Retinas were also immunostained for CD31 and collagen IV (ColIV). (FIG. 7A) White arrows indicate unperfused conA- / CD31- / ColIV+ collagen sleeves (i.e., regressed capillaries). (FIG. 7B) conA+ cells (gray arrows) can be found at the junction of some regressed capillaries (white arrow), where they potentially interfered with blood flow prior to regression.

[0021] FIGS. 8 A to 8M show EC-specific deletion of both Erg and Flil (Erg / FlHiECko') promotes NV tuft regression. (FIG. 8 A) Schematic of subjecting single knockout (KO) and littermate flox control mice (ErgEf kovs. Er^,' FlHlECkovs. FH1FF) and double KO and littermate flox control mice (Erg / FliliECkovs. Erg / FliPP12) to the oxygen-induced retinopathy (OIR) model. Pups were housed in room air (21% O2) from birth to P7 and were then exposed to hyperoxia (75% O2) for 5 days from P7 to P12, when pathological retinal vascular regression occurs. Pups were returned to room air from P12 to P18, and P17 was the time of peak neovascular (NV) tuft formation. To induce Cdh5(PAC)-CreERT2activation, tamoxifen was administered orally at P16 and P17. Retinas were harvested at P18 for flatmount staining of CD31+retinal vessels. (FIG. 8B, FIG. 8C) Quantification of NV areas from ErgKf'komice and controls (n = 6-9 mice) and from FlHlECkomice and controls (n = 6-7 mice). (FIG. 8D) Representative images of CD31 -stained retinal flatmounts from Pl 8 Erg / FlillECkoand littermate control mice. The Imaged plugin SWIFT NV (77) was used to label and quantify NV tuft areas (white in the whole flatmounts). The inset monochrome images correspond to the white squares in the images at the far left. Scale bar: 500 pm in whole flatmounts and 50 pm in the magnified inset images. (FIG. 8E) Quantification of NV areas from Erg / FliliECkoand control mice (n = 6-10 mice). (FIG. 8F) Quantification of average tuft sizes from Erg / FlillECkoand control mice (n = 6-10 mice). Data are presented as mean ± SD. Erg‘krkoEr^lox / E°x;Cdh5(PAC)-CreERT2, FliPECko'. Fli lflox / fl°x;Cdh5(PAC)-CreERT2, NV: Neovascular, OIR: Oxygen-induced retinopathy. EC-specific deletion of either Erg ox Flil does not promote NV tuft regression. (FIG. 8G) Representative images of CD31 -stained retinal flatmounts from Pl 8 Er fi kand ErgiECkomice, and littermate control mice. The Imaged plugin SWIFT_NV (77) was used to label and quantify NV areas (white in the whole flatmounts) and avascular (AV) areas (gray layer). The inset monochrome images correspond to the white squares in the images at the far left. Scale bar: 500 pm in whole flatmounts and 50 pm in the magnified inset images. (FIG. 8H) Quantification of AV area from Ergl,2 k' FliPECko, Erg / FliPECko, and littermate control mice (n = 6-10 mice). (FIG. 81) Quantification of total tuft number from Ergi2'k,FliliECko, Erg / FlHiECko, and littermate control mice (n = 6-10 mice). (FIG. 8d) Representative images of ERG, FLU, and CD31 staining from Pl 8 Erg / FlUlECkoand control mice after tamoxifen administration and OIR challenge, as shown in FIG. 8A. Scale bar: 50 pm. (FIG. 8K) Representative immunoblots of ERG and FLU in retinal lysates from Pl 8 Erg / FliPECkoand control mice after tamoxifen administration and OIR challenge, as shown in FIG. 8A. (FIG. 8L) Representative images of empty basement membrane sleeves (arrows; CD31 ColIV+) from Pl 8 Erg / FliliECkomice and littermate control mice that were raised in room air. Scale bar is 50 pm. (FIG. 8M) Quantification of empty basement membrane sleeves in (FIG. 8L) (n = 6 mice). Data are presented as mean ± SD. AV: Avascular; OIR: Oxygen-induced retinopathy.

[0022] FIGS. 9A to 9N show NV tuft regression in Erg / FliliECkomice does not improve OIR- induced visual defects. (FIG. 9A) Schematic of testing the effects of the OIR model on visual function in Erg / FliliECkoand littermate control (Erg / FliFF) mice. Note that age-matched mice of both genotypes that were raised in room air (RA) served as negative controls for the OIR model. Tamoxifen was administered orally to mice of both genotypes at P16 and P17 to induce Cre activation. Visual function was measured by electroretinogram and optokinetic tracking (OKT) at P21. (FIG. 9B) Representative curves of scotopic and photopic amplitudes from the four different groups. Scale bar labels are shown at the bottom right. (FIG. 9C- FIG. 9F) Quantification of scotopic a-wave (FIG. 9C), scotopic b-wave (FIG. 9D), photopic a-wave (FIG. 9E), and photopic b-wave (FIG. 9F) readings from the four different groups (n = 6 mice). (FIG. 9G) Quantification of spatial frequency threshold (c / d) measured by OKT (n = 8-16 mice). Data are presented as mean ± SD. OIR: Oxygen-induced retinopathy, OKT: Optokinetic tracking, RA: Room air. Visual function is still compromised in wild type mice at P30 after OIR challenge. WT C57BL / 6J mice were challenged with the OIR model. Electroretinogram and optokinetic tracking (OKT) were measured at P30, and retinas were subsequently harvested for whole flatmount immunostaining. (FIG. 9H) Representative images of CD31 -stained retinal vasculature from littermate mice challenged with OIR or raised in room air (RA) as controls. Scale bar: 500 pm in whole flatmounts and 200 pm in the magnified images. (FIG. 91) Representative curves of scotopic and photopic amplitudes from P30 OIR and RA mice. Scale bar labels are shown at the bottom right. (FIG. 9J- 9M) Quantification of scotopic a-wave (FIG. 9J), scotopic b-wave (FIG. 9K), photopic a-wave (FIG. 9L), and photopic b-wave (FIG. 9M) in (FIG. 91) (n = 6 mice). (FIG. 9N) Quantification of spatial frequency threshold (c / d) measured by OKT (n = 6 mice). Data are presented as mean ± SD. OIR: Oxygen-induced retinopathy, OKT: Optokinetic tracking, RA: Room air.

[0023] FIGS. 10A to 10O show that hyperoxia downregulates ERG in wild type retinal ECs in vivo and in vitro. (FIG. 10 A) Representative images of CD31 -stained retinal flatmounts from wild type C57B1 / 6J pups exposed to 75% O2 for the indicated times. Central gray layer indicates avascular (AV) area. Scale bar: 500 pm. (FIG. 10B) Quantification of AV areas in (A) (n = 6 mice). (FIG. 10C) Representative immunostaining of ERG, CD31, and DAPI in retinas from P8 wild type C57B1 / 6J pups after exposure to 75% O2 for 24 hr. Arrows indicate EC nuclei with low ERG signal at the capillary regression front. Scale bar: 100 pm in the large image and 20 pm in the inset. (FIG. 10D) Representative immunoblots from whole retinas of P7 littermate wild type pups exposed to room air (RA) or to 75% O2 for 12 hr. (FIG. 10E) Densitometry quantification of ERG normalized to endomucin (EMCN) (n = 6 mice). (FIG. 10F) Representative immunoblots from primary human retinal microvascular endothelial cells (HREC) cultured under RA for 24 hror 75% O2 for the indicated time. (FIG. 10G) Densitometry quantification of normalized ERG in (FIG. 10F) (n = 3 technical replicates). Data are presented as mean ± SD. AV: Avascular Area, EMCN: Endomucin, HREC: Human retinal microvascular endothelial cells, RA: Room air, VE- Cad: Vascular endothelial cadherin. Hyperoxia potentially degrades ERG but not FLU via upregulation of the ubiquitin ligase TRIM25. (FIG. 10H) Representative immunoblots from whole retinas of P7 littermate wild type pups exposed to room air (RA) or to 75% O2 for 12 hr. (FIG.IOI) Densitometry quantification of FLU normalized to endomucin (EMCN) (n = 6 mice). (FIG.IOJ) Representative immunoblots from primary human retinal microvascular endothelial cells (HREC) cultured under RA for 24 hr or 75% O2 for the indicated time. (FIG. 10K) Densitometry quantification of normalized FLI1 in (FIG. 10 J) (n = 3 technical replicates). (FIG. 10L) Immunoblot for TRIM25 protein expression in HRECs cultured under 1% O2 or 20% O2 for 24 or 48h. (FIGS. 10M-10O) Primary Human Umbilical Vein ECs (HUVECs) were treated with nonspecific (NS) or / 77 / / A725-targeting siRNAs for 72h followed by quantification of ERG protein and transcript expression by immunoblot (FIG. 10M and FIG. ION) and qPCR (FIG. 100), respectively (n = 3 technical replicates). Data are presented as mean ± SD. EMCN: Endomucin, HREC: Human retinal microvascular endothelial cells, HUVEC: Human umbilical vein endothelial cells, RA: Room air, VE-Cad, Vascular endothelial cadherin.

[0024] FIGS. 11A to 11 J show EC-specific ERG overexpression (ErECoereduces hyperoxia- induced regression and OIR-induced neovascularization. (FIG. 11 A) Schematic of testing the effects of inducible endothelial ERG overexpression (Eigll-I oe) in the OIR model. Tamoxifen was administered orally or by eyedrops to ErgiECoeand (7v / / ' / 2-negative littermate control pups from P5 to P7. Vascular regression was measured at P8, and NV tufts were measured at Pl 7. Neuronal damage was measured at P13 by TUNEL staining, and visual function was measured at P21 by electroretinograms and optokinetic tracking. (FIG. 1 IB) Representative immunoblots from whole retinas of P8 littermate controls and ErgiECoepups after tamoxifen administration from P5 to P7. (FIG. 11C) Densitometry quantification of normalized ERG in (FIG. 11B) (n = 6-7 mice). (FIG. 1 ID) Representative immunostaining of CD31 and ERG on retinal flatmounts at P8 after 24 hr of hyperoxia exposure. Avascular (AV) areas are labeled with gray layers. The inset magnified images correspond to the white squares in the images at the far left. Scale bar: 500 pm in the whole flatmount images and 150 pm in the magnified inset images. (FIG. HE) Quantification of the central AV area in (FIG. 11D) (n = 6 mice). (FIG. 1 IF) Representative images of CD31-stained retinas from ErgiECoepups and littermate controls at P17 after OIR challenge. SWIFT NV was used to mark AV (gray) and NV tuft (white) areas. Scale bar: 500 pm in the whole flatmount images and 250 pm in the magnified insets. (FIG. 11G, FIG. 11H) Quantification of theneovascular (NV) areas (G) and AV areas (H) shown in (FIG. 1 IF) (n = 6-8 mice). Data are presented as mean ± SD. (FIG. 1 II, FIG. 11 J) ERG overexpression is comparable in ErECoepups after oral or eyedrop routes of tamoxifen administration. (FIG. I ll) Representative immunoblots from whole retinas of P8 ErECoepups and littermate controls. Tamoxifen was administered via oral or eyedrop routes from P5 to P7. (FIG. 11 J) Densitometry quantification of ERG normalized to Actin in (FIG. 1 II) (n = 6 mice). Data are presented as mean ± SD.

[0025] FIGS. 12A to 12H show OIR-induced neural cell death and visual defects are reduced in ErECoemice. (FIG. 12 A) Following the OIR challenge schematic shown in FIG. 12 A, TUNEL staining was performed on ocular cryosections of P13 ErgiECoeand littermate Cre^^-negative pups. DAPI was used as a counterstain. Scale bar: 250 pm in whole retinal sections and 25 pm in the magnified insets. (FIG. 12B) Quantification of TUNEL+cells per whole retinal section (n = 6- 8 mice). (FIG. 12C) Representative curves of scotopic and photopic amplitudes from both genotypes exposed to the OIR model or to room air (RA) as a negative control for the OIR model. Scale bar labels are shown at the bottom right. (FIG. 12D- FIG. 12G) Quantification of scotopic a- wave (FIG. 12D), scotopic b-wave (FIG. 12E), photopic a-wave (FIG. 12F), and photopic b- wave (FIG. 12G) readings as in (FIG. 12C) (n = 6-12 mice). (FIG. 12H) Quantification of spatial frequency threshold (c / d) by optokinetic tracking (OKT) (n = 8-9 mice). Data are presented as mean ± SD. C: Central, GCL: Ganglion cell layer, INL: Inner nuclear layer, OIR: Oxygen-induced retinopathy, ONL: Outer nuclear layer, P: Peripheral, RA: Room air.

[0026] FIGS. 13 A to 13E show that ERG and FLU are downregulated in retinal capillaries of patients with non-proliferative diabetic retinopathy (NPDR). Paraffin sections of postmortem human eyes were acquired from NPDR patients and age-matched individuals without DR (control). (FIG. 13 A) Representative immunostaining of ERG, FLU, CD31, and DAPI in human eye sections. Scale bar: 25 pm. White arrows indicate retinal capillaries; gray arrows indicate choroidal capillaries (CD31+DAPI+). (FIG. 13B) Quantification of ERG intensity in (FIG. 13 A) (n = 5 individuals per group). (FIG. 13C) Quantification of FLU intensity in (FIG. 13 A) (n = 5 individuals per group). (FIG. 13D) Ages of NPDR patients and age-matched controls. (FIG. 13E) The time from death to harvest of eyes from NPDR patients and age-matched controls. Data are presented as mean ± SD. GCL: Ganglion cell layer, INL: Inner nuclear layer, ONL: Outer nuclear layer, RPE: Retinal pigmented epithelium.DETAILED DESCRIPTION OF THE INVENTION

[0027] While the making and using of various embodiments of the present invention are discussed in detail below, it should be appreciated that the present invention provides many applicable inventive concepts that can be embodied in a wide variety of specific contexts. The specificembodiments discussed herein are merely illustrative of specific ways to make and use the invention and do not delimit the scope of the invention.

[0028] To facilitate the understanding of this invention, a number of terms are defined below. Terms defined herein have meanings as commonly understood by a person of ordinary skill in the areas relevant to the present invention. Terms such as “a”, “an” and “the” are not intended to refer to only a singular entity, but include the general class of which a specific example may be used for illustration. The terminology herein is used to describe specific embodiments of the invention, but their usage does not delimit the invention, except as outlined in the claims.

[0029] The present invention provides compositions and methods for preventing the first phase of retinopathy of prematurity (ROP), when hyperoxia causes retinal vascular regression. The inventors were able to prevent hyperoxia-induced retinal vascular regression, thereby providing a novel way to prevent, block, and / or treat the progression of ROP. Targeting prevention of ROP and related retinopathies has not been possible because fundamental mechanisms underlying the process of vessel regression were poorly understood. The present invention is based at least in part on a new understanding of the mechanisms underlying ROP, and more specifically, providing ways to inhibit the hyperoxia-induced capillary regression associated with ROP. The present invention can also be used to treat diabetic retinopathy (DR).

[0030] The present invention uses a completely new approach to tackle ROP based on a novel understanding of ocular vascular regression. In doing so, it shifts the status quo of ROP therapeutic treatments away from the second phase of the disease — in which pathological angiogenesis and neuronal cell death has already begun — toward a focus on the first phase of the disease when vascular dropout occurs under hyperoxia. In other words, it attempts to move clinical practice beyond current management-based ROP therapies toward preventative ones. Secondly, we previously managed to drive regression of pathological retinal vasculature (i.e., neovascular tufts) in a mouse model of ROP, which is strategically like current clinical approaches of treating ROP patients with laser ablation or anti-VEGF drugs. Since we now appreciate that long-term visual dysfunction can still occur in ROP patients after these treatments, the inventors took advantage of the opportunity to adapt the current understanding of ocular vascular regression toward the development of novel therapeutic approaches that target the first phase of ROP. By way of explanation but not a limitation of the present invention, it is hypothesized that the loss of vessels in this stage not only drives subsequent angiogenesis but also hampers growth of neurons that support visual function. Therefore, it is important to protect existing vasculature to protect the health of the eye. Finally, by shifting our focus from promoting ocular vascular regression to preventing the process, many more therapeutic options become available.

[0031] Diabetic retinopathy (DR), sometimes referred to as diabetic eye disease, in which diabetes mellitus leads to damage to the retina and is a leading cause of blindness. Typically, DR affects up to 80 percent of diabetic patients. Importantly, the longer a patient has diabetes, the higher the chances of developing diabetic retinopathy. In the United States, diabetic retinopathy accounts for 12% of all new cases of blindness and is the leading cause of blindness in patients aged 20 to 64.

[0032] The present invention includes compositions and methods of preventing ocular vascular regression, preserving visual function, or both, comprising: identifying a subject in need of treatment for ocular vascular regression; and providing the subject with an effective amount of an agent that is: an agonist that increases expression of at least one gene selected from: ETS-Related Gene (ERG)(HGNC: 3446 NCBI Gene: 2078 Ensembl: ENSG00000157554 OMIM®: 165080 UniProtKB / Swiss-Prot: Pl 1308), Fli-1 Proto-Oncogene (ETS Transcription Factor (FLU) (HGNC: 3749 NCBI Gene: 2313 Ensembl: ENSG00000151702 OMIM®: 193067 UniProtKB / Swiss-Prot: Q01543); KLF transcription factor 2 (KLF2) (HGNC: 6347 NCBI Gene: 10365 Ensembl: ENSG00000127528 OMIM®: 602016 UniProtKB / Swiss-Prot: Q9Y5W3); KLF transcription factor 4 (KLF4) (HGNC: 6348 NCBI Gene: 9314 Ensembl: ENSG00000136826 OMIM®: 602253 UniProtKB / Swiss-Prot: 043474); or a Piezo-Type Mechanosensitive Ion Channel Component 1 (PIEZO1) agonist (HGNC: 28993 NCBI Gene: 9780 Ensembl: ENSG00000103335 OMIM®: 611184 UniProtKB / Swiss-Prot: Q92508). The agent is provided in an amount sufficient to prevent the ocular vascular regression, preserving visual function, or both. The genes described hereinabove can be inserted into a vector for delivery to the eye or a part of the eye. The genes can be delivered as mRNA or in a DNA vector that enters the cells and expresses the genes.

[0033] The subject can be in need of treatment for retinopathy of prematurity (ROP), diabetic retinopathy (DR), or both. In one example, the agent that increases the expression of at least one of ERG, FLU, KLF2, or KLF4, is a nucleic acid-based therapeutic. In another example, the nucleic acid-based therapeutic is selected from a DNA, an RNA, an mRNA, a siRNA, RNAi, promoter RNA (pRNAs), enhancer RNA (eRNAs), or superenhancer RNA (seRNAs). In another example, the agent is a small molecule agonist that increases the expression of ERG, FLU, KLF2, or KLF4 mRNA, stabilized an ERG, FLU, KLF2, or KLF4 mRNA, or both. In another example, the ocular vascular regression is retinal capillary regression. In another example, the PIEZO 1 agonist is Yoda-1.

[0034] Techniques and compositions for making useful dosage forms using the present invention are described in one or more of the following references: Anderson, Philip O.; Knoben, James E.;Troutman, William G, eds., Handbook of Clinical Drug Data, Tenth Edition, McGraw-Hill, 2002; Pratt and Taylor, eds., Principles of Drug Action, Third Edition, Churchill Livingston, New York, 1990; Katzung, ed., Basic and Clinical Pharmacology, Ninth Edition, McGraw Hill, 2007; Goodman and Gilman, eds., The Pharmacological Basis of Therapeutics, Tenth Edition, McGraw Hill, 2001; Remington’s Pharmaceutical Sciences, 20th Ed., Lippincott Williams & Wilkins., 2000, and updates thereto; Martindale, The Extra Pharmacopoeia, Thirty-Second Edition (The Pharmaceutical Press, London, 1999); all of which are incorporated by reference, and the like, relevant portions incorporated herein by reference.

[0035] In one non-limiting example, the agent is administered topically, subconjunctivally, intracamerally, subtenonally, subretinally, subchoroidally, suprachoroidally, supraorbitally, retrobulbarlly, as an ocular implant, or intravitreally, or wherein the agent is formulated into an eye drop, gel, ointment, spray, a reservoir, or mist. In another example, the agent prevents least one of: decrease retinal neovessels by at least 40% or a retinal avascular area by at least 60% compared to a vehicle-injected contralateral eye. In another example, the agent does not inhibit vascular endothelial growth factor (VEGF).

[0036] A dosage unit for use of the agent of the present invention, may be a single compound or mixtures thereof with other compounds. The compound may be mixed together, form ionic or even covalent bonds. The agent of the present invention may be administered in oral, intravenous (bolus or infusion), intraperitoneal, subcutaneous, or intramuscular form, all using dosage forms well known to those of ordinary skill in the pharmaceutical arts. Depending on the particular location or method of delivery, different dosage forms, e.g., tablets, capsules, pills, powders, granules, elixirs, tinctures, suspensions, syrups, and emulsions may be used to provide the agent of the present invention to a patient in need of therapy that includes the agent. The agent may also be administered as any one of known salt forms.

[0037] The agent is typically administered in admixture with suitable pharmaceutical salts, buffers, diluents, extenders, excipients and / or carriers (collectively referred to herein as a pharmaceutically acceptable carrier or carrier materials) selected based on the intended form of administration and as consistent with conventional pharmaceutical practices. Depending on the best location for administration, the agent may be formulated to provide, e.g., maximum and / or consistent dosing for the particular form for oral, rectal, topical, intravenous injection or parenteral administration. While the agent may be administered alone, it will generally be provided in a stable salt form mixed with a pharmaceutically acceptable carrier. The carrier may be solid or liquid, depending on the type and / or location of administration selected.

[0038] As used herein, the term phrase “pharmaceutically acceptable carrier” is art recognized and includes a pharmaceutically acceptable material, composition or vehicle, suitable for administering compounds of the present invention to mammals. The carriers include liquid or solid filler, diluent, excipient, solvent or encapsulating material, involved in carrying or transporting the subject agent from one organ, or portion of the body, to another organ, or portion of the body. Each carrier must be “acceptable” in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient. Some examples of materials which can serve as pharmaceutically acceptable carriers include: sugars, such as lactose, glucose and sucrose; starches, such as corn starch and potato starch; cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients, such as cocoa butter and suppository waxes; oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, com oil and soybean oil; glycols, such as propylene glycol; polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; esters, such as ethyl oleate and ethyl laurate; agar; buffering agents, such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen- free water; isotonic saline; Ringer’s solution; ethyl alcohol; phosphate buffer solutions; and other non-toxic compatible substances employed in pharmaceutical formulations, particularly phosphate buffered saline solutions which are preferred for intraocular delivery. Wetting agents, emulsifiers and lubricants, such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, release agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and agents can also be present in the compositions.

[0039] Formulations of the present invention include those suitable for oral, nasal, topical, transdermal, buccal, sublingual, intramuscular, intraperotineal, intraocular, intravitreal, posterior juxtascleral, anterior juxtascleral, retrobulbar, subretinal, and / or other routes of parenteral administration. The specific route of administration will depend, inter alia, on the specific cell to be targeted. The formulations may conveniently be presented in unit dosage form and may be prepared by any methods well known in the art of pharmacy. The amount of active ingredient that can be combined with a carrier material to produce a single dosage form will generally be that amount of the compound that produces a therapeutic effect.

[0040] As used herein, “plurality” is understood to mean more than one. For example, a plurality refers to at least two, three, four, five, or more.

[0041] As used herein, the term a “nucleic acid” or “nucleotides” is understood as two or more independently selected natural or non-natural nucleic acids joined by a covalent bond. A polynucleotide can include 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 75, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000 or more natural or non-natural nucleotidesjoined by covalent bonds. Polynucleotides as described herein include full-length non-coding and coding sequences (e.g., fully processed mRNA or its precursors) as well as shorter polynucleotide sequences (e.g., fragments of naturally occurring nucleotides or synthetic polynucleotide fragments).

[0042] As used herein, the term “primary RNA Transcript” refers to an RNA sequence that is directly transcribed from a genetic DNA without any RNA processing or modification, which may be selected from the group consisting of hnRNA, pre-mRNA, rRNA, tRNA, snoRNA, snRNA, pri-microRNA (pri-miRNA), viral RNA and their RNA precursors as well as derivatives. After transcription, uracil (U) is substituted for thymine (T).

[0043] As used herein, the term “precursor messenger RNA (pre-mRNA): refers to a primary messenger RNA transcripts of a protein-coding gene, which are produced by eukaryotic type-II RNA polymerase (Pol-II) machineries in eukaryotes through an intracellular mechanism termed transcription. A pre-mRNA sequence contains a 5 '-untranslated region (UTR), a 3'-UTR, exons and introns.

[0044] As used herein, the term “messenger RNA (mRNA)” refers to an assembly of pre-mRNA exons, which is formed after intron removal by intracellular RNA splicing machineries (spliceosomes) and served as a protein-coding RNA for peptide / protein synthesis. The peptides / proteins encoded by mRNAs include, but not limited, enzymes, growth factors, insulin, antibodies and their analogs / homologs as well as derivatives.

[0045] As used herein, the term “complementary DNA (cDNA)” refers to a single-stranded or double-stranded DNA that contains a sequence complementary to an mRNA sequence and does not contain any intronic sequence.

[0046] As used herein, the term “microRNA (miRNA)” refers to a single-stranded RNA capable of binding to targeted gene transcripts (mRNAs) that have partial complementarity to the sequence of microRNA. Mature microRNA is usually sized about 17-27 oligonucleotides in length and is able to either directly degrade its intracellular mRNA target(s) or suppress the protein translation of its targeted mRNA(s), depending on the complementarity between the microRNA and its target mRNA(s). Native microRNAs are found in almost all eukaryotes, functioning as a defense against viral infections and allowing regulation of specific gene expression during development of plants and animals. In principle, one microRNA often target multiple target mRNAs to fulfill its full functionality while on the other hand multiple miRNAs may target the same gene transcripts to enhance the effect of gene silencing.

[0047] As used herein, the term “microRNA Precursor (pri- / pre-miRNA)” refers to a hairpin-like single-stranded RNA containing stem-arm and stem-loop regions for interacting with RNase III Dicer endoribonucleases to produce one or multiple mature microRNAs (miRNAs) capable of silencing a targeted gene or a specific group of targeted genes that contain full or partial complementarity to the mature microRNA sequence(s). The stem-arm of a pri- / pre-miRNA can form either a perfectly (100%) or a partially (mis-matched) hybrid duplexes, while the stem-loop connects one end of the stem-arm duplex to form a circle or hairpin-loop conformation required for being assembled into an RNA-induced silencing complex (RISC) with some argonaute proteins (AGO).

[0048] As used herein, the term “small interfering RNA (siRNA)” refers to a short doublestranded RNA sized about 18-27 perfectly base-paired ribonucleotide duplexes and capable of degrading target gene transcripts with almost perfect complementarity.

[0049] As used herein, the term “small or short hairpin RNA (shRNA)” refers to a single-stranded RNA that contains a pair of partially or completely matched stem-arm nucleotide sequences divided by an unmatched loop oligonucleotide to form a hairpin-like structure. Many natural miRNAs are derived from hairpin-like RNA precursors, namely precursor microRNA (pre- miRNA).

[0050] As used herein, the term “vector” refers to a recombinant nucleic acid composition such as recombinant DNA (rDNA) capable of movement and residence in different genetic environments. Generally, another nucleic acid is operatively linked therein. The vector can be capable of autonomous replication in a cell in which case the vector and the attached segment is replicated. One type of preferred vector is an episome, i.e., a nucleic acid molecule capable of extrachromosomal replication. Preferred vectors are those capable of autonomous replication and expression of nucleic acids. Vectors capable of directing the expression of genes encoding for one or more polypeptides and / or non-coding RNAs are referred to herein as “expression vectors” or “expression-competent vectors”. Particularly important vectors allow cloning of cDNA from mRNAs produced using a reverse transcriptase. A vector may contain components consisting of a viral or a type-II RNA polymerase (Pol-II or pol-2) promoter, or both, a Kozak consensus translation initiation site, polyadenylation signals, a plurality of restriction / cloning sites, a pUC origin of replication, a SV40 early promoter for expressing at least an antibiotic resistance gene in replication-competent prokaryotic cells, an optional SV40 origin for replication in mammalian cells, and / or a tetracycline responsive element. The structure of a vector can be a linear or circular form of single- or double-stranded DNA selected form the group consisting of plasmid, viral vector, transposon, retrotransposon, DNA transgene, jumping gene, and a combination thereof.

[0051] As used herein, the term “promoter” refers to a nucleic acid to which a polymerase molecule recognizes, or perhaps binds to, and initiates RNA transcription. For the purposes of the instant invention, a promoter can be a known polymerase or its cofactor binding site, an enhancer and the like, any sequence that can initiate synthesis of RNA transcripts by a desired polymerase.

[0052] As used herein, “prevention” is understood as to limit, reduce the rate or degree of onset, or inhibit the development of at least one sign or symptom of a disease or condition particularly in a subject prone to developing the disease or disorder. The age of onset of one or more symptoms of the disease can sometimes be determined by the specific conditions or a mutation. Prevention can include the delay of onset of one or more signs or symptoms of ROP and / or DR and need not be prevention of appearance of at least one sign or symptom of the disease throughout the lifetime of the subject. Prevention can require the administration of more than one does of an agent or therapeutic.

[0053] As used herein, the term “small molecule” refers to a compound, typically an organic compound, having a molecular weight of no more than about 1500 Da, 1000 Da, 750 Da, or 500 Da. In an embodiment, a small molecule does not include a polypeptide or nucleic acid including only natural amino acids and / or nucleotides.

[0054] As used herein, the term “subject” refers to living organisms, in particular, humans. In certain embodiments, the living organism is an animal, in certain preferred embodiments, the subject is a mammal, in certain embodiments, the subject is a domesticated mammal or a primate including a non-human primate. Examples of subject include humans, monkeys, dogs, cats, mice, rates, cows, horses, goats, and sheep. A human subject may also be referred to as a subject or patient.

[0055] As used herein, a subject “suffering from or suspected of suffering from” a specific disease, condition, or syndrome has a sufficient number of risk factors or presents with a sufficient number or combination of signs or symptoms of the disease, condition, or syndrome such that a competent individual would diagnose or suspect that the subject was suffering from the disease, condition or syndrome, such as ROP and / or DR. Methods for identification of subjects suffering from or suspected of suffering from conditions such as ROP and / or DR is within the ability of those in the art. Subjects suffering from, and suspected of suffering from, a specific disease, condition, or syndrome are not necessarily two distinct groups.

[0056] As used herein, the phrase “therapeutically effective amount” refers to an amount of an agent that is effective, upon single or multiple does administration to the cell or subject, in prolonging the survivability of the patient with such a disorder, reducing one or more signs orsymptoms of the disorder, preventing or delaying and the like beyond that expected in the absence of such treatment, e.g., ROP and / or DR.

[0057] An agent or other therapeutic intervention can be administered to a subject, either alone or in combination with one or more additional therapeutic agents or interventions, as a pharmaceutical composition in mixture with conventional excipient, e.g., pharmaceutically acceptable carrier, or therapeutic treatments.

[0058] The pharmaceutical agents may be conveniently administered in unit dosage form and may be prepared by any of the methods well known in the pharmaceutical arts, e.g., as described in Remington’s Pharmaceutical Sciences (Mack Pub. Co., Easton, PA, 1985). Formulations for parenteral administration may contain as common excipients such as sterile water or saline, polyalkylene glycols such as polyethylene glycol, oils of vegetable origin, hydrogenated naphthalenes and the like. In particular, biocompatible, biodegradable lactide polymer, lactide / glycolide copolymer, or polyoxyethylene-polyoxypropylene copolymers may be useful excipients to control the release of certain agents.

[0059] The present invention is directed to the use of an implant with agent to treat ROP and / or DR. In one embodiment, the present invention includes a method for the treatment of age-related macular degeneration in a human that comprises administering to the human therapeutically effective amount of agent. In some embodiments, the agent is provided in or with a pharmaceutically acceptable carrier. In other embodiments, the agent implant is administered in or about the eye, e.g., intraocularly, subretinally, intravitreally, posterior juxtascleral, anterior juxtascleral, retrobulbar, intramuscularly, or topically. The implants of the present invention can be injected into one or more non-limiting locations, such as pre-determined locations, that can include, e.g., intravitreal, intrastromal, intracameral, subtenon, retinal, subretinal, retrobulbar, peribulbar, suprachoroidal, subchoroidal, conjunctival, subconjunctival, episcleral, posterior juxtascleral, anterior juxtascleral, circumcorneal, topical, and tear duct.

[0060] Administration of a therapeutic agent such as an agent through the use of one or more intraocular implants may improve the treatment of ocular diseases or conditions involving oxidative stress. Implants comprise a pharmaceutically acceptable polymeric composition and are formulated to release one or more pharmaceutically active agents over an extended period of time. In certain embodiments involving the delivery of one agent, the dosage regimen may be formulated to provide one drug to the anterior or posterior segment of the eye. For example, for ocular drug delivery to the posterior region of the eye, the implant would likely be introduced as an intravitreal implant, inserted into the vitreous cavity.

[0061] In certain embodiments involving the delivery of more than one agent, the dosage regiment may be formulated to provide two or more drugs to the posterior segment of the eye under different dosage regimens. For example, the dosage of the drug in an implant may be made to be discontinuous over the treatment period while a non-discontinuous dosage of an auxiliary agent is administered in an implant over the same overall time period. The implant containing the agent and the implant containing the auxiliary agent may be different implants or the same implant comprising means of differentially administering the agent and auxiliary agent, such means including different coatings or shells which may contain, neither, one or both drugs, or covalent linkage of one or both drugs to a biodegradable polymer of the implant by way of a biodegradable linkage, thus permitting regulation of the delivery of one or more drug over the time of the treatment. The implants are effective to provide a therapeutically effective dosage of the agent or agents directly to a region of the eye to treat one or more ocular diseases or conditions involving oxidative stress. Thus, with a single administration, therapeutic agents will be made available at the site where they are needed and may be maintained for an extended period of time, rather than subjecting the patient to repeated injections or, in the case of self-administered drops, ineffective treatment with only limited bursts of exposure to the active agent or agents.

[0062] As used herein, the term “auxiliary agents” refers to agents such as lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, colorings, flavorings and / or aromatic substances, and the like, which do not deleteriously interact with the agents of the formulation.

[0063] One such intraocular implant in accordance with the disclosure herein comprises a therapeutic component and a drug release sustaining component associated with the therapeutic component. In accordance with the present invention, the therapeutic component comprises, consists essentially of, or consists of, the agent. The drug release sustaining component is associated with the therapeutic component to sustain release of a therapeutically effective amount of the agent into an eye in which the implant is placed. The therapeutic amount of the agent is released into the eye for a period of time greater than about two months after the implant is placed in the eye.

[0064] For the purposes of this description, we use the following terms as defined in this section, unless the context of the word indicates a different meaning.

[0065] As used herein, an “intraocular implant” refers to a device or element or drug product that is structured, sized, or otherwise configured to be placed “in an eye”, including the subconjunctival space. Intraocular implants are generally biocompatible with physiological conditions of an eyeand do not cause adverse side effects. Intraocular implants may be placed in an eye without disrupting vision of the eye.

[0066] As used herein, a “therapeutic component” refers to a portion of an intraocular implant comprising one or more therapeutic agents or substances used to treat a medical condition of the eye. The therapeutic component may be a discrete region of an intraocular implant, or it may be homogenously distributed throughout the implant. The therapeutic agents of the therapeutic component are typically ophthalmically acceptable, and are provided in a form that does not cause adverse reactions when the implant is placed in an eye.

[0067] As used herein, a “drug release sustaining component” refers to a portion of the intraocular implant that is effective to provide a sustained release or extended-release of the therapeutic agents of the implant. A drug release sustaining component may be a biodegradable polymer matrix, or it may be a coating covering a core region of the implant that comprises a therapeutic component.

[0068] As used herein, “associated with” means mixed with, dispersed within, coupled to, covering, or surrounding. With respect to intraocular implants which comprise a therapeutic component associated with a biodegradable polymer matrix, “associated with” specifically excludes biodegradable polymeric coatings that may be provided on or around the matrix.

[0069] As used herein, an “ocular region” or “ocular site” refers generally to any area of the eyeball, including the anterior and posterior segment of the eye, and which generally includes, but is not limited to, any functional (e.g., for vision) or structural tissues found in the eyeball, or tissues or cellular layers that partly or completely line the interior or exterior of the eyeball. Specific examples of areas of the eye in an ocular region include the anterior chamber, the posterior chamber, the vitreous cavity, the choroid, the suprachoroidal space, the conjunctiva, the subconjunctival space, the episcleral space, the intracorneal space, the epicorneal space, the sclera, the pars plana, surgically-induced avascular regions, the macula, and the retina.

[0070] As used herein, an “ophthalmic or ocular disease” or “ophthalmic or ocular condition” is a disease, ailment or condition which affects or involves the eye or one of the parts or regions of the eye. Broadly speaking the eye includes the eyeball, or globe, the tissues and fluids which constitute the eye, the periocular muscles (such as the oblique and rectus muscles) and the portion of the optic nerve which is within or adjacent to the eye.

[0071] An anterior ocular condition is a disease, ailment or condition which affects or which involves an anterior (i.e., front of the eye) ocular region or site, such as a periocular muscle, an eye lid or an eye ball tissue or fluid which is located anterior to the posterior wall of the lens capsule or ciliary muscles. Thus, an anterior ocular condition primarily affects or involves theconjunctiva, the cornea, the anterior chamber, the iris, the posterior chamber (behind the retina but in front of the posterior wall of the lens capsule), the lens or the lens capsule and blood vessels and nerve which vascularize or innervate an anterior ocular region or site.

[0072] As used herein, the term “biodegradable” refers to a material that will breakdown to soluble species or that will degrade under physiologic conditions to smaller units or chemical species that are, themselves, non-toxic (biocompatible) to the subject and capable of being metabolized, eliminated, or excreted by the subject. The terms biodegradable, bioabsorbable, and bioerodible as used herein are equivalent and are used interchangeably herein.

[0073] As used herein, the term “nonbiodegradable” refers to a material that will not breakdown to soluble species or that will not degrade under physiologic conditions to smaller units or chemical species that are, themselves, non-toxic (biocompatible) to the subject and capable of being metabolized, eliminated, or excreted by the subject. The terms nonbiodegradable, nonbioerodible and nonbioabsorbable as used herein are equivalent and are used interchangeably herein.

[0074] As used herein, the term “biodegradable polymer” refers to a polymer or polymers which degrade in vivo, and wherein erosion and / or breakdown of the polymer or polymers over time occurs concurrently with or subsequent to release of the therapeutic agent. Specifically, hydrogels such as methylcellulose which act to release drug through polymer swelling are specifically excluded from the term “biodegradable polymer”. A biodegradable polymer may be a homopolymer, a copolymer, or a polymer comprising more than two different polymeric units.

[0075] As used herein, the term “treat”, “treating”, or “treatment” refers to reduction or resolution or prevention of an ocular condition, ocular injury or damage, or to promote healing of injured or damaged ocular tissue.

[0076] As used herein, the term “therapeutically effective amount” refers to the level or amount of agent needed to treat an ocular condition, or reduce or prevent ocular injury or damage without causing significant negative or adverse side effects to the eye or a region of the eye.

[0077] The present invention provides new drug delivery formulations or preparations, and methods of using such formulations and preparations, for extended or sustained drug release into an eye, for example, to achieve sustained desired therapeutic effects. The drug delivery formulations or preparations are in the form of implants or implant elements that may be placed in an eye.

[0078] Intraocular implants in accordance with the disclosure herein comprise an agent drug. The agent drug may be present in or on the same implant or different implants. The agent drug may reduce oxidative stress in the eye in an acceptable range.

[0079] Such intraocular implants may comprise a therapeutic component and a drug release sustaining component associated with the therapeutic component. In accordance with the present invention, the therapeutic component comprises, consists essentially of, or consists of, an agent drug. The drug release sustaining component is associated with the therapeutic component to sustain release of a therapeutically effective amount of the steroid into an eye in which the implant is placed. The therapeutically effective amount of the agent is preferably released into the eye for a period of time greater than about one or two or more months after the implant is placed in the eye.

[0080] In one embodiment, the intraocular implants comprise an agent drug and a biodegradable polymer matrix. The agent drug is associated with a biodegradable polymer matrix that releases drug, such as by dissolving, breaking apart or degrading, at a rate effective to sustain release of a therapeutically effective amount of the agent drug from the implant for a time greater or longer than about one or two or more months from a time the implant is placed in an ocular site or region of an eye. The intraocular implant is biodegradable or bioerodible and provides a sustained release of the agent drug in an eye for extended periods of time, such as for more than a few days, weeks, two months, or for about three months or more and up to about six months or more.

[0081] The biodegradable polymer component of the implant may be a mixture of biodegradable polymers, wherein at least one of the biodegradable polymers is a polylactide or poly(lactide-co- glycolide) polymer having a molecular weight less than 40 kiloDaltons (kD). Additionally or alternatively, the implants may comprise a first biodegradable polymer having terminal free acid groups, and a different second biodegradable polymer having terminal free acid groups. Additionally or alternatively, the implants may comprise a first biodegradable polymer having a terminal ester, and a different second biodegradable polymer having terminal ester groups Furthermore, the foregoing implants may comprise a mixture of different biodegradable polymers, each biodegradable polymer having an inherent viscosity in a range of about 0.16 deciliters / gram (dL / g) to about 0.24 dL / g. Examples of suitable biodegradable polymers include polymers synthesized from lactide monomer, glycolide monomer and mixtures thereof. Other examples of suitable biodegradable polymers include polymers synthesized from lactic acid monomer, glycolic acid monomer and mixtures thereof.

[0082] In another embodiment, intraocular implants comprise a therapeutic component that comprises an agent drug, and a polymeric outer layer covering the therapeutic component. Thepolymeric outer layer may include one or more orifices or openings or holes that are effective to allow a liquid to pass into the implant, and to allow the steroid to pass out of the implant. The therapeutic component is provided in a core or interior portion of the implant, and the polymeric outer layer covers or coats the core. The polymeric outer layer may include one or more biodegradable portions. The implant can provide an extended release of the steroid for more or longer than about two months, and for more than about one year, and even for more than about five or about ten years.

[0083] In one embodiment, the polymeric outer layer of the implant may comprise two or more layers or coats of biodegradable material, with each such layer having a different composition or rate of degradation than the layer immediately adjoining it. For example, the polymeric outer layer of the implant may comprise concentric rings or nested coatings comprising a first layer, wherein the first layer may comprise, for example, a biodegradable polymer and the absence of agent drug, a biodegradable polymer comprising a therapeutically effective amount of an agent drug, a biodegradable polymer comprising an amount of an auxiliary agent effective to reduce at least one side effect of agent drug, and a biodegradable polymer comprising a therapeutically effective amount of agent drug and an amount of an auxiliary agent effective to reduce at least one side effect of a steroid, and a biodegradable polymer without any added drug.

[0084] A second layer may also comprise, for example, a biodegradable polymer and the absence of agent drug, a biodegradable polymer comprising a therapeutically effective amount of an agent drug, a biodegradable polymer comprising an amount of an auxiliary agent able to reduce at least one side effect of an agent drug, and a biodegradable polymer comprising a therapeutically effective amount of a steroid and an amount of an auxiliary agent able to reduce at least one side effect of a steroid, and a biodegradable polymer without any added drug, with the additional provisos that the first and second layers are located adjoining one another in the biodegradable implant, that the first and second layers are not identical, and that the first layer is designed to erode substantially before the second layer. Additional layers may be present; preferably, each such layer will not be identical to the layers immediately surrounding it.

[0085] Example 1. Blocking retinal capillary regression to prevent retinopathy.

[0086] It was found that vascular regression (defined here as the apoptotic eradication of blood vessels) in the neonatal and juvenile mouse eye (defined a “recipe” for ocular capillary regression) involved two observations: slow blood flow and inhibition of ETS transcription factors. The pathological retinal capillaries (i.e., neovascular tufts) that form in the eyes of mice exposed to an oxygen-induced retinopathy (OIR) challenge model (FIG 1). However, it has now been found that regression of these pathological tufts is not effective at rescuing visual function in mice exposedto the OIR model, based on optokinetic tracking and scotopic electroretinograms. By way of explanation, the inventors suspected that the hyperoxia phase of the OIR model (postnatal days P7-P12) — a period in which regression of normal retinal capillaries occurs — results in ischemic damage to photoreceptors once mice are returned to room air at P12. Furthermore, this ischemia causes the growth of the pathological neovascular tufts that are characteristic of retinopathies — eye diseases that include retinopathy of prematurity and diabetic retinopathy.

[0087] FIG. 1 is a schematic of the oxygen-induced retinopathy (OIR) protocol in mice, with respective phases of oxygen exposure (in blue) and retinal vascular effects (in the boxes below the oxygen levels). Note that the formation of neovascular tufts peaks at P17, and the tufts undergo a spontaneous regression process (i.e., resolution) by P25.

[0088] It was found that that blocking hyperoxia-induced retinal capillary regression is effective at preventing subsequent growth of neovascular tufts in mouse eyes. First, the inventors defined two ETS transcription factors that need to be downregulated / inhibited to drive regression of neovascular tufts: ERG and FLU. Tamoxifen-inducible genetic deletion of these genes in endothelial cells Flil / ErECkomice) causes tufts to regress in the OIR model (FIG. 2). Having determined that these two ETS factors (which are very similar to each other) need to be inhibited to drive regression, the inventors sought to determine whether restoration of one of the factors would be sufficient to prevent regression during the hyperoxia stage of the OIR model. In support of this idea, a genetic mouse line was used in which tamoxifen drives overexpression of ERG in endothelial cells (ErECoeand the inventors reduced hyperoxia-induced regression (FIG. 3). Next, it was possible to reduce regression resulting in fewer neovascular tufts. Indeed, it was found that tufts were significantly reduced in ERG-overexpression mice compared to littermate controls (FIG. 4). This is important evidence that blocking regression can prevent retinopathy in a mouse model that particularly mimics human retinopathy of prematurity.

[0089] FIG. 2: Neovascular tufts undergo regression in Flil / ErECkomice exposed to OIR. Littermate control and FlH / ErECkopups were subjected to OIR and induced with tamoxifen at P16-P17. Retinas were immunostained at P18 for CD31+ vasculature and were quantified with the Imaged plugin SWIFT NV. Numbers of NV tufts were comparable in controls and mutants (not shown), but mutant tufts were smaller, resulting in decreased NV area (n=6-10). Stats: unpaired two-tailed t-test.

[0090] Fig. 3: Endothelial Erg overexpression reduces hyperoxia-induced retinal capillary regression. Littermate control and ErECoepups were induced with tamoxifen at P5-P7 and subjected to OIR. Retinas were immunostained at P8 for CD31+ vasculature, which was analyzed with the Imaged plugin SWIFT NV to quantify avascular area (n=2-5).

[0091] Fig. 4: Reduced hyperoxia-induced retinal capillary regression in ErgiECoemice correlates with reduced formation of neovascular tufts. Littermate control and ErECoepups were induced with tamoxifen at P5-P7 and subjected to OIR. Retinas were immunostained at P17 for CD31+ vasculature, which was analyzed with the ImageJ plugin SWIFT NV to quantify avascular area (n=4-8). Stats: unpaired two-tailed t-test.

[0092] Another approach was used to block hyperoxia-induced regression in young mice is related to the slow flow ingredient in the regression recipe. By way of explanation, but not a limitation of the present invention, the flow-stimulated transcription factors KLF2 and KLF4 promote survival of endothelial cells under normal flow conditions, while ERG and FLU promote survival under slow flow conditions. This could explain why slow-flow vessels like neovascular tufts are susceptible to regression after ETS factor inhibition. One way that flow drives expression of KLF2 / 4 is via a cell surface mechanosensing channel called PIEZO1. Yodal is a chemical agonist for PIEZO 1 that can stimulate KLF2 / 4 expression in endothelial cells, even in the absence of flow. When Yodal was administered to mouse pups, the inventors successfully reduced hyperoxia- induced retinal capillary regression (FIG. 5). Together with the genetic experiments shown in FIG. 4, these data demonstrate that hyperoxia-induced capillary regression can be blocked by either: (1) expressing the ETS factor ERG; or (2) stimulating the perception of flow with Yodal.

[0093] FIG. 5: The PIEZO 1 agonist Yodal reduces hyperoxia-induced retinal capillary regression. Littermate C57B1 / 6J pups were injected (s.c. to back) with vehicle (PBS) or Yodal (600nmol / kg / day) from P5-P7. Pups were exposed to hyperoxia (75% O2) for 24 hr starting at P7. Retinas were subsequently immunostained at P8 for CD31+vasculature, which was analyzed with the ImageJ plugin SWIFT NV to quantify avascular area (n=5-6). Stats: unpaired two-tailed t- test.

[0094] In terms of preventing capillary regression in the OIR model (a model for retinopathy of prematurity (ROP) in humans), three additional aspects were explored. First, using Erg- overexpression mice (ErECoe) — which have reduced hyperoxia-induced regression and neovascular tuft formation (see FIGS. 3 and 4 in summary section) — to determine if visual acuity is also rescued. The inventors predicted that these mice will have less ischemic damage to their photoreceptors because of the reduced capillary regression, and this will preserve their visual function as measured through optokinetic tracking and scotopic electroretinograms. Second, it is shown herein that Yodal can reduce hyperoxia-induced regression (see FIG. 5) by showing that it reduced tuft formation.

[0095] In the context of diabetic retinopathy (DR), like ROP, DR is characterized by an initial period of retinal capillary regression that is followed by pathological angiogenesis (i.e., tuftformation). Unfortunately, no bona fide rodent model of DR exists, possibly because rodents do not live long enough to develop retinopathy in response to diabetic stressors (1). However, retinal capillary regression has been detected in murine STZ and high fat diet challenge models of diabetes (2, 3). The inventors studied STZ-induced retinal vascular regression and to contrast its mechanisms to those seen in the hyperoxia phase of the OIR model. Consistent with a previous study (4), the inventors observed retinal capillary regression in adult C57B1 / 6J mice at 10 weeks after STZ challenge (FIG. 6).

[0096] Three different approaches to block STZ-induced capillary regression can be used. First, ErgiECoe mice with successfully reduced hyperoxia-induced regression in the OIR model are used to determine if they likewise reduce STZ-induced regression, as predicted herein. Second, Klf4ECoe mice are used to determine if STZ-induced regression is reduced in these animals, as predicted herein. Third, the inventors recognized that blood flow reduction occurs in STZ- challenged mice based on the inability of a systemically administered lectin (concanavalin A = conA) to perfuse into some retinal capillaries (FIG. 7A). These vessels are also CD31- / ColIV+, indicating that they are regressed capillaries (5). These data demonstrate that flow ceases after capillary regression, but they do not address whether flow slows down prior to regression, as we predict. However, conA also stains immune cells (6), and the inventors found conA+ cells at the junctions of collagen sleeves (FIG. 7B). Given the location of these conA+ cells near regressed vessels, and knowing that capillary ECs in diabetic eyes express adhesion molecules like ICAM- 1 that attract vessel-occluding leukocytes that reduce perfusion (i.e., leukostasis) (7), the inventors predict that reduced flow is an important ingredient in the DR capillary regression recipe. Therefore, the inventors can take advantage of eye drops containing lifitegrast, which serves as an antagonist for ICAM-l / LFA-1 interactions (8) and thus block leukostasis in the eye (as it has in STZ-challenged rats [9]). By way of explanation, but not a limitation of the present invention, the inventors hypothesize that leukostasis contributes to capillary slow flow and regression (see Fig. 7B), and it is predicted that lifitegrast drops will reduce STZ-induced capillary regression. These observations result from the findings in the OIR model that blocking retinal capillary regression prevents retinopathy and will block regression in a DR model.

[0097] FIG. 6: Capillary regression is elevated in STZ-challenged mice. 8-week-old male C57B1 / 6J mice were injected with STZ (50 mg / kg) for 5 consecutive days. Hyperglycemia (>250 mg / dl) was confirmed 2 weeks after the last STZ dose. At 6, 10, and 14 weeks after STZ treatment, retinas were harvested and immunostained for CD31 and the basement membrane component ColIV. Empty collagen sleeves (CD31- / ColIV+; arrows) — which are indicative of capillary regression! — were manually counted in 20X confocal fields (3 per eye) in the deep retinal layerand were compared against eyes from age-matched, vehicle-treated controls (“No STZ”). Stats: one-way ANOVA compared to controls; each dot = one mouse.

[0098] FIGS. 7A and 7B show evidence of low flow in regressed retinal vessels after STZ challenge. Mice were treated with STZ (as in FIG. 6), and 8 weeks later were perfused with FITC- conjugated conA for visualization of patent vessels. Retinas were also immunostained for CD31 and collagen IV (ColIV). (FIG. 7A) Orange arrows indicate unperfused conA- / CD31- / ColIV+ collagen sleeves (i.e., regressed capillaries). (FIG. 7B) conA+ cells (blue arrows) can be found at the junction of some regressed capillaries (orange arrow), where they potentially interfered with blood flow prior to regression.

[0099] Example 2.

[0100] Retinopathy of prematurity (ROP) and diabetic retinopathy (DR) are ocular disorders in which a loss of retinal vasculature leads to ischemia followed by a compensatory neovascularization response. In mice, this is modeled using oxygen-induced retinopathy (OIR), whereby neonatal animals are transiently housed under hyperoxic conditions that result in central retina vessel regression and subsequent neovascularization. Using endothelial cell (EC)-specific gene deletion, the inventors found that loss of two ETS-family transcription factors, ERG and FLU, led to regression of OIR-induced neovascular vessels but failed to improve visual function, suggesting that relevant retinal damage occurs prior to and independently of neovascularization. In the initial stage of OIR, it was found that hyperoxia repressed ERG expression in retinal ECs of wild type mice, raising the possibility that oxygen-induced ERG downregulation promotes vessel regression during the initiation of OIR-induced pathology.

[0101] A murine model of EC-specific ERG overexpression was developed, and overexpression was sufficient to prevent hyperoxia-induced vascular regression, neuronal cell death, and neovascularization in the OIR model. Importantly, ERG overexpression also improved visual function in OIR-challenged mice. Moreover, it is shown herein that both ERG and FLU are downregulated in the retinal vessels of human patients with early stages of DR, showing that neovascular disorders of the eye may share common mechanisms underlying pathological retinal capillary regression. These results demonstrate that the regulation of vascular regression by EC- expressed ETS transcription factors can be adapted by novel therapeutic approaches for the prevention and / or alleviation of ocular neovascular disorders.

[0102] Deletion of endothelial ERG and FLU promotes neovascular tuft regression.

[0103] Oxygen-induced retinopathy (OIR) is a murine model of ROP in which neonatal mice are housed under hyperoxia (75% O2) from postnatal day (P) 7 to Pl 2, leading to oxygen-inducedregression of retinal blood vessels (FIG. 8A). As in humans, the resulting vascular insufficiency leads to a compensatory phase of retinal neovascular tuft formation that peaks around P17 (S). As shown above, neovascular tufts can be selectively regressed by treatment with a pharmacological inhibitor of ETS family transcription factors.

[0104] Because ERG and FLU are EC-expressed ETS family transcription factors that play a critical role in maintaining vascular homeostasis (- / , 5), it was hypothesized that inhibition of one or both of these factors would lead to the regression of OIR-induced neovascular tufts. Though ERG and FLU are highly expressed by ECs under normal circumstances, numerous studies have documented the downregulation of either factor during inflammatory diseases such as bacterial / viral infection, pulmonary arteriolar hypertension, autoimmune disease, liver disease, and aging (6, 16, 24-26). The inventors generated Er^lox / llox;Cdh5(PAC)-CreERT2(ErgE'kand Flilflox / flox;Cdh5(PAC)-CreERT2(FlHiECko) mice to enable tamoxifen-inducible and EC-specific deletion of Erg and Flil, respectively. ErgECkoand FliPECkomice were exposed to 75% O2 from P7 to P12, and tamoxifen was administered at P16 and P17 after neovascular tufts had already formed (FIG. 1 A). At P18, retinal flat mount preparations were analyzed by immunofluorescence for the EC marker CD31, and the Imaged plugin SWIFT NV (S) was used to quantify total neovascular area, neovascular tuft number, and avascular area (FIGS. IB, 1C, 1G, 1H, II).

[0105] Though there was a trend towards less neovascularization for both Er '2 k'' and FliPECkomice, neither genotype reached significance (FIGS. IB, 1C). ERG and FLU have highly homologous DNA binding domains (9) and play synergistic roles in maintaining endothelial homeostasis (6), suggesting that the presence of either protein may functionally compensate for the loss of the other. The inventors generated Er^lox / floxFlilflox / fl°x;Cdh5(PAC)-CreERT2(Erg / FHrECko) mice to achieve EC-specific deletion of both Erg and Flil genes and subjected them to the OIR model, as described above (FIGS. 1 J, IK). In contrast to the individual knockouts, simultaneous deletion of Erg and Flil had an additive effect, resulting in a significant reduction (-40%) in neovascular area compared to littermate controls (FIGS. ID, IE). Moreover, average tuft sizes were significantly reduced in Erg / FUrECkomice (FIGS. ID, IF), and this was likely due to tuft regression rather than compromised tuft growth since there were no significant differences in overall tuft numbers between control and mutant genotypes (FIG. II). Meanwhile, for mice raised in room air, there were a comparable number of empty basement membrane sleeves (CD3 T ColIV+) between Erg / Fli liECkomice and littermate controls (FIGS. IL, IM). Since empty basement membrane sleeves are indicative of capillary regression 10), these data show that regression occurs specifically in tufts of Erg / FliliECkomice but not in the normal retinal vasculature.

[0106] Visual function is not improved following / / ' / ’7 / 7' / / ;''-induced neovascular tuft regression.

[0107] The success in regressing neovascular tufts in Erg / FUrECkomice prompted the inventors to ask if this led to an improvement in OIR-induced visual defects (77, 72). Erg / FUrECkomice were exposed to 75% O2 from P7 to Pl 2, induced gene deletion as before at P16 / P17, and measured retinal function and visual acuity at P21 using electroretinogram and optokinetic tracking (OKT), respectively (FIG. 2A). Using both scotopic and photopic electroretinograms, similar decreases in a- and b-wave amplitudes for OIR Erg / FUrECkoand OIR control mice were observed compared to mice of both genotypes that were raised in room air (FIGS. 2B-2F). Similarly, both genotypes had comparable reductions in their threshold for spatial frequency detection as determined by OKT (FIG. 2G). Therefore, despite the regression of neovascular tufts achieved by deleting both Erg and Flil from ECs (FIGS. 1D-F), no signs of improved visual function in OIR-challenged Erg / FUrECkomice were observed.

[0108] In wild type mice subjected to OIR, revascularization of the central retina occurs spontaneously following the resolution of neovascular tufts by P25 (73). The inventors reasoned that compromised revascularization might contribute to the visual defects detected in Erg / Eli liECkomice at P21. However, persistent visual defects were observed in OIR-treated wild type mice even after the retina had been fully revascularized at P30 (FIGS. 2H-2N), showing that visual defects associated with the OIR model are due to neuronal damage that occurred prior to the neovascular phase rather than to failed revascularization of the retina after tuft resolution.

[0109] Endothelial ERG expression is downregulated by hyperoxia exposure during OIR.

[0110] Like ROP, OIR-induced neovascularization occurs because of an initial hyperoxic challenge that leads to vascular regression and the impairment of nutrient delivery to the metabolically consumptive retina. The inventors show that hyperoxia-induced regression of central retinal vessels was rapid in the OIR model, with a loss of nearly one third of all retinal vessels by 24hr of hyperoxia exposure from P7 to P8 in wild type mice (FIGS. 3 A, 3B). Following this, vascular regression spread outward from the central retina during the subsequent 4 days of hyperoxia exposure from P8 to P12. Loss of endothelial ERG during this phase of hyperoxia exposure has previously been reported (74), and the discovery that neovascular tuft regression is accelerated in Erg / FliliECkomice led the inventors to ask if the downregulation of either transcription factor might also contribute to hyperoxia-induced vessel regression. Therefore, endothelial ERG and FLU expression was assessed in the initial 24hr window of hyperoxia exposure to determine if they were downregulated during this period in which the most substantial amount of retinal vascular regression occurred.

[0111] Immunofluorescence imaging for ERG at 24hr exposure to 75% O2 revealed robust nuclear expression in most retinal ECs (FIG. 3C). However, nuclear ERG was notably absent from a subset of ECs (FIG. 3C, yellow arrows) located along the border of the retinal avascular region where active vessel regression was occurring. Furthermore, immunoblot analysis of whole retina tissue lysates revealed a reduction in ERG expression in wild type mice exposed to hyperoxia for 12hr compared to littermates exposed to room air (FIGS. 3D, 3E). Importantly, ERG expression was normalized to the capillary marker endomucin (EMCN; FIGS. 3D, 3E), indicating that the observed reduction in ERG expression occurred in viable ECs and was not due to the overall loss of retinal vessels. By contrast, a similar reduction in retinal FLU expression was not observed after mice were exposed to hyperoxia for 12hr (FIGS. 3H, 31).

[0112] To determine if oxygen may have a direct effect on ERG expression in ECs, the inventors cultured primary human retinal endothelial cells (HRECs) under 21% and 75% O2 and assessed ERG expression by immunoblot. By 8 and 24h of 75% O2, a moderate reduction of ERG expression was observed (FIGS. 3F, 3G). Like the in vivo findings, oxygen had little effect on FLU expression in vitro (FIGS. 3J, 3K). The inventors and others have previously reported that ERG downregulation can occur after its ubiquitination and subsequent proteasomal degradation, and TRIM25 is a ubiquitin ligase that putatively marks ERG for proteolysis in certain contexts (75, 1 ). Intriguingly, the inventors found that oxygen exposure upregulates TRIM25 expression in HRECs, suggesting a potential mechanism for Ch-dependent ERG downregulation (FIG. 3L). In agreement with this, siRNA-mediated deletion of TRIM25 in ECs led to a significant upregulation of ERG protein without affecting ERG mRNA transcript expression (FIGS. 3M-3O). Collectively, the in vivo and in vitro data show that the expression of ERG is more robustly regulated by oxygen exposure in retinal ECs than is FLI1 expression, with higher oxygen levels resulting in more downregulation of ERG.

[0113] Overexpression of endothelial ERG reduces hyperoxia-induced vascular regression and neovascularization.

[0114] Since it was found that endothelial ERG downregulation correlated with hyperoxia- induced vascular regression, the inventors hypothesized that the overexpression of endothelial ERG would counteract this process. Therefore, the inventors developed a mouse in which murine Erg coding sequence was inserted into the ROSA26 locus downstream of a floxed STOP cassette. These mice were crossed with the EC-specific Cdh5(PAC)-CreERT2line (77). Administration of tamoxifen to ROSAErs / Ers;Cdh5(PAC)-CreERT2(Ergl2 oe) mice from P5 to P7 led to a ~7-fold upregulation of ERG protein in whole retinas that could be detected by immunoblot andimmunofluorescence at P8 compared to littermate controls (ROSAErg / Erg) subjected to the same tamoxifen administration scheme (FIGS. 4A-D).

[0115] After tamoxifen induction from P5 to P7 and exposure to hyperoxia for 24hr (FIG. 4A), immunofluorescence imaging of CD31 -stained flatmounted retinas revealed that vascular regression was significantly attenuated in ErgECoemice compared to control littermates (FIG. 4D, 4E). We then asked if the observed reduction in hyperoxia-induced vascular regression led to a reduced neovascular response in ErgE!'oemice. At P17, the peak time of neovascularization in the OIR model, a reduction in both neovascular area and avascular area were observed in ErgE!'oemice compared to littermate controls (Fig. 4F-H). This shows that the prevention of early-stage vascular regression in the OIR model by ERG overexpression mitigates a compensatory neovascular response.

[0116] Overexpression of endothelial ERG preserves retinal function following OIR.

[0117] To determine if the preservation of retinal vessels in OIR-treated ErgiECoemice had a positive impact on visual function, the inventors induced endothelial ERG overexpression from P5 to P7, subjected mice to hyperoxia from P7 to Pl 2, and then performed a TUNEL stain of retinal cross sections at Pl 3 to assess neuronal cell death (FIG. 4 A). A significant reduction in OIR-induced apoptosis of neuronal retinal cells in ErgECoemice was observed compared to littermate controls, particularly in the inner nuclear layer of the central retina (FIGS. 5A, 5B).

[0118] The prevention of retinal neovascularization and the reduction of neuronal cell death in Erll°emice prompted us to ask if these animals also had diminished OIR-induced visual defects. Therefore, ErgiECoemice were administered tamoxifen from P5 to P7 (via eyedrop), exposed to 75% O2 (or room air) from P7 to P12, and then analyzed by electroretinogram at P21 (FIG. 4A). Note that both oral and eyedrop routes of tamoxifen administration resulted in comparably elevated ERG expression in ErgiECoeretinas (FIGS. 41 and 4 J), although ErgE!'oemice survived longer and appeared healthier at P21 when tamoxifen was administered via eyedrops. OIR- challenged ErgE!'oemice showed a significant improvement in a- and b-wave amplitudes for scotopic electroretinogram and b-wave amplitudes for photopic electroretinograms compared to control littermates (FIGS. 5C-5G). OIR-challenged ErECoemice also showed a significant improvement in spatial frequency detection by OKT (FIG. 5H). Therefore, prevention of hyperoxia-induced retinal vascular regression correlates with improved visual function in the OIR model.

[0119] ERG and FLU expression are downregulated in retinal capillaries of patients with early stages of DR.

[0120] Finally, to confirm the clinical significance of these findings, the inventors assessed ERG and FLU expression in human eye sections from postmortem donors with non-proliferative DR (NPDR) — the initial stage of DR during which most retinal capillary regression occurs. After immunostaining the eye sections, it was found that both ERG and FLU were significantly downregulated in retinal capillaries of patients with NPDR compared to age-matched patients without DR (FIG. 6A, yellow arrows). Notably, choroidal vessels, which are also visualized in retinal sections, do not regress in the early stage of diabetes and therefore can be used as an internal control (7S). Importantly, the inventors found that neither ERG nor FLU were downregulated in choroidal vessels of NPDR patients (FIG. 6A, purple arrows), indicating that downregulation of ERG and FLU correlates specifically with ocular vessels undergoing regression at this stage of DR. Together with the OIR mouse studies, these results demonstrate that ERG and / or FLU downregulation is a conserved feature of early-stage retinopathies and a therapeutic target for mitigating disease progression by inducing the expression of ERG and FLU mitigates ocular vessels undergoing regression at this stage of DR.

[0121] Neovascular disorders of the eye are a substantial healthcare burden worldwide. There are -50,000 annual ROP diagnoses, and incidence rates continue to rise due to improvements in survival rates for preterm infants. Though there are substantial differences in the etiologies and affected populations of ROP and DR, both diseases are characterized by an initial loss of retinal vasculature that in turn leads to the formation of neovascular tufts. All currently available treatments target the stabilization of these neovascular tufts, and though they have efficacy in preventing severe consequences such as retinal detachment, no treatment yet addresses the underlying trigger of vascular insufficiency. This is due in part to a lack of understanding regarding the basic mechanisms of vascular regression that occur in ECs during the initiation of ROP and DR.

[0122] This example follows the inventors showing that a broad pharmacological inhibitor of ETS-family transcription factors promoted regression of neovascular tufts in the OIR challenge model (3). Here, genetic models of EC-specific gene deletion were used to confirm that the loss of two specific ETS family members, Erg and Flil, is sufficient to regress OIR-induced neovascular tufts. ERG and, to a lesser extent, FLU are predominantly expressed by ECs (79). Because of the high homology of these proteins and previous observations of partially compensatory functions for ERG and FLU (5, 6), it is not surprising that it was necessary to delete both Erg and Flil to drive tuft regression.

[0123] These data show that neovascular disorders of the eye offer two windows in which the regulation of vascular regression by ETS factors can be therapeutically useful. First, ERG andFLU inhibition can be used to promote selective regression of pathological neovascular tufts. This approach can complement current interventions that focus on inhibiting VEGF to reduce both retinal neovascularization and vascular leakage that causes scarring and retinal detachment (7). Secondly, stabilizing ERG expression can be used as a preventive measure to attenuate the hyperoxia-induced regression that initially drives tuft formation. The present invention demonstrates the beneficial impact of preventing hyperoxic vessel regression on visual function. It is shown herein that photoreceptor damage is associated with early stages of the OIR challenge model, providing an explanation for OIR-induced visual defects that persist well after the spontaneous resolution of retinal neovessels (72). Thus, preventing hyperoxic vessel regression has the additive benefits of alleviating both neuronal tissue ischemia and subsequent retinal neovascularization.

[0124] When the inventors previously reported that pharmacological ETS factor inhibition could drive ocular vascular regression, it was acknowledged that this process independently requires slow blood flow in capillaries (3). Capillaries within neovascular tufts are thin and tortuous with slow flow as opposed to normal retinal vessels that maintain physiological flow, which explains why the broad ETS factor inhibitor that the inventors administered to eyes of OIR-challenged mice at P17 could selectively regress neovascular tufts by P19. Hyperoxia drives retinal vasoconstriction in mice and humans (20, 21 indicating that both slow flow and ERG downregulation (FIGS. 3 A to 3G) occur during the hyperoxia stage of the OIR model. In contrast, DR is largely restricted to adult populations and occurs as a sequela of hyperglycemia rather than hyperoxia (2). DR is associated with more complicated retinal blood flow dynamics, although retinal capillaries are reported to have reduced diameters in early stages of this disease in humans and mice (22, 23). It is shown herein that prevention of just one of these triggers — ETS factor downregulation — is sufficient to block the regression process.

[0125] Study design. This study aimed to manipulate retinal vascular regression to prevent pathologies associated with ROP. Using the murine OIR challenge model that mimics ROP progression, the inventors first attempted to promote regression of pathological neovascular tufts with genetic deletion of the ETS transcription factors Erg and Flil in ECs. When this failed to rescue visual function as measured by electroretinograms and optokinetic tracking in OIR- challenged mice, the inventors then shifted focus to an earlier stage of the model in which pathological regression of normal retinal vasculature is triggered through exposure to hyperoxia. At this stage, the inventors attempted to counteract hyperoxia-induced regression by genetically overexpressing endothelial Erg since the inventors found ERG downregulation occurred in mouse retinal ECs and in cultured primary human retinal ECs after exposure to high oxygen. Theinventors also assessed ERG and FLU expression in the postmortem eyes of patients with nonproliferative DR to determine if these ETS factors are likewise downregulated in the early stage of this retinopathy, which is associated with pathological regression of retinal capillaries similarly to ROP.

[0126] For the mouse studies, each experimental group consisted of a minimum of six mice to ensure a robust demonstration of differences in regression phenotypes and the expression of relevant genes. The minimum animal numbers and sample sizes required to achieve statistical significance were determined by power analyses based on pilot experiments and published literature. Littermate mice were used as controls for all the animal studies. The investigators were blinded to the sample analyses and quantification. Sample sizes and specific statistical tests used for each experiment are outlined in the figure legends.

[0127] Study approvals. Histological sections from deidentified donor eyes from patients with NPDR and from nondiabetic patients were obtained from Lions Gift of Sight Eye Bank (Saint Paul, MN). All methods were performed in accordance with federal and institutional guidelines. Protocols involving mouse breeding, tamoxifen induction, the OIR model, and visual function testing followed the guidelines of the Association for Research in Vision and Ophthalmology (ARVO) Statement for the Use of Animals in Ophthalmic and Visual Research. All studies were conducted following protocols approved by the Oklahoma Medical Research Foundation Institutional Animal Care and Use Committee (#23-12 and #24-27).

[0128] Animals. Mice used in this study included C57BL / 6J (The Jackson Laboratory; #000664), Er^ox(gift of Joshua Wythe, Baylor College of Medicine; available through The Jackson Laboratory; #030988) (#3), Flilflox(gift of Maria Trojanowska, Boston University) (37), and Cdh5(PAC)-CreERT2(gift of Ralf Adams, Max Planck Institute for Molecular Biomedicine; available through Taconic; #13073) (26). ROSAErg / Erg(generated by Graeme Birdsey and Anna Randi, Imperial College London), were constructed by genOway (Lyon, France). Briefly, a knock- in vector was designed, containing murine Erg cDNA inserted upstream of an IRES-tdTomato- Reporter cassette. A loxP flanked Stop cassette was inserted between a CAG promoter and the Erg cDNA to allow expression to be dependent upon induction by Cre recombinase. The inducible targeting vector was inserted into the permissive Rosa26 locus of C57BL / 6 mice. Mice were housed at the Oklahoma Medical Research Foundation animal facility in a specific pathogen-free (SPF) environment at 25°C under a 12 / 12 hr light / dark cycle. Both male and female were included in all studies. Genotyping of ROSAErg / Ergwas performed using the primers 5'- GTTTTGGAGGCAGGAAGCACTTGC-3' (SEQ ID NO:1), 5'-GCAGTGAGAAGAGTACCACCATGAGTCC-3’ (SEQ ID NO:2), and 5'-CGAGGCGGATCACAAGCAATA-3' (SEQ ID N0:3). Genotyping of Er^oxwas performed using the primers 5 '-GAGATGGCGC AACGC AATTAATG-3 ' (SEQ ID NO:4), 5'- AGAGTCTCTGCACACAGAACTTCC-3' (SEQ ID NO: 5), and 5'- AATGCTCTGGTAAGGCACACAAGG-3' (SEQ ID NO:6), as previously described (77). Genotyping of Flilfloxj s performed using the primers 5'-GACTCAAACCAGGGAAAGTTGC- 3' (SEQ ID NO:7), and 5'-TTGGGAAGGTGGAATCTAGCAG-3' (SEQ ID NO:8). Genotyping of Cdh5(PAC)-CreERT2mice was performed using the primers 5'- TCCTGATGGTGCCTATCCTC-3' (SEQ ID NO: 9) and 5'-CGAACCTGGTCGAAATCAGT-3' (SEQ ID NO: 10). Tamoxifen-induced Cdh5(PAC)-CreERT2activation was achieved by oral gavage of 20 mg / ml tamoxifen (Cat#T5648, Sigma) dissolved in peanut oil, administered to pups at P5- P7 (3 pl / day) or P16-P17 (5 pl / day). An eyedrop route of tamoxifen administration (20 mg / ml, 3 pl / day from P5 to P7) was used for ErECoeand littermate control mice that were subsequently used for electroretinogram measurements at P21.

[0129] Oxygen-induced Retinopathy (OIR) model. The mouse OIR model was performed as described previously (7). Neonatal mouse pups were housed in room air (RA; 21% oxygen) until P7. Then, mouse pups were housed in hyperoxia chambers (75% oxygen; Biospherix) for 5 days from P7 to P12, during which time hyperoxia-induced retinal vascular regression occurred. Mice were next removed from the hyperoxia chambers and were housed in RA until terminal experiments were performed. Note that the peak time of neovascular tuft formation occurred at Pl 7. Visual function tests (OKT and electroretinograms) were performed at P21. Littermate Cre^^-negative mice were used as controls in the OIR model and received tamoxifen treatment at the same time as their experimental counterparts. Age-matched mice were used as RA controls for animals that were subjected to the OIR model.

[0130] Immunofluorescent staining and imaging. Human eyes were collected within 32 hours postmortem and were overnight fixed in Davidson's fixative. Antigen epitopes were retrieved with heat induction using EnVision FLEX Target Retrieval Solution (Citrate buffer pH 6.1, Agilent Technologies, Inc., Santa Clara, CA). Tissue autofluorescence was quenched with 0.1% Sudan Black. Mouse whole-mount retinal staining was performed as described previously (28). Briefly, mouse eyeballs were enucleated and fixed in 4% paraformaldehyde (PF A) in phosphate-buffered saline (PBS, pH 7.4) for 1 hr. Retinas were isolated from fixed eyeballs under a dissection microscope. The retinas were incubated in a blocking buffer [3% bovine serum albumin (BSA), 10% donkey serum, and 0.5% Triton X-100 in PBS] overnight at 4°C. The retinas were incubated in primary antibodies overnight at 4°C followed by secondary antibodies overnight at 4°C and were then flatmounted. 4’,6-diamidino-2-phenylindole (DAPI) was used for counterstaining, andProLong Diamond Antifade Mountant (Cat P36930, Invitrogen) was used as a mounting buffer. Images were collected using a Nikon AXR confocal microscope and analyzed using Nikon Elements software. SWIFT NV plugin in ImageJ software was used to quantify neovascular and avascular areas in the retinal flatmounts (77). Details of primary and secondary antibodies used in this study are listed in Table 1.

[0131] Table 1. List of antibodies used in this study.

[0132] Terminal Deoxynucleotidyl Transferase-dUTP Nick End Labeling (TUNEL) staining.Ocular cryosections were prepared as described previously (28). Sagittal sections (5 pm) were generated through the peripheral and central regions of the retina and through the optic nerve. TUNEL staining was performed using In Situ Cell Death Detection Kit (Cat 11684795910, Roche) according to the manufacturer’s protocol (29). Sections were incubated in TUNEL reactionmixture for 1 hr at 37°C. DAPI was used for counterstaining. TUNEL+cells were quantified using ImageJ software.

[0133] Immunoblot analysis. Immunoblot analysis was performed as described previously 30). Briefly, retinas were dissected and snap-frozen in liquid nitrogen and stored at -80°C until use. Tissues were lysed in RIPA buffer (Sigma) supplemented with 1% proteinase inhibitor cocktail (Sigma). After sonication, the samples were centrifuged at 12,000 x g for 10 min at 4°C. Protein concentrations within the supernatant were determined by BCA assay (ThermoFisher). Samples with equal amounts of proteins were resolved in SDS-PAGE gels (Invitrogen) and transferred to nitrocellulose membranes (Invitrogen). After blocking with 10% nonfat milk in Tris-buffered saline with 0.1% Tween 20 (TBST), the membranes were incubated with primary antibodies overnight at 4°C, followed by incubation with secondary antibodies for 2 hr. All antibodies were diluted in TBST containing 5% BSA. Details of primary and secondary antibodies used in this study are listed in Table 1.

[0134] Quantitative reverse transcription polymerase chain reaction (qPCR). Cultured endothelial cells were lysed in Trizol reagent (Invitrogen). RNA was extracted using an RNeasy Mini Prep Kit (QIAGEN). cDNA was generated using an iScript cDNA Synthesis Kit (Bio-Rad). Target gene mRNA levels were measured by qPCR using SsoAdvanced Universal SYBR Green Sypermix (BioRad) and analysis on a CFX96 Real-Time PCR thermocycler (Bio-Rad). The following primers were used for measuring human ERG mRNA: 5'- AACGAGCGCAGAGTTATCGTGC-3' (SEQ ID NO:11) and 5'- GTGAGCCTCTGGAAGTCGTCC-3' (SEQ ID NO: 12). Human 18s and ACTB were used as housekeeping gene transcripts for normalization and were measured using the primers 5'- CCCGAAGCGTTTACTTTGAAA-3' (SEQ ID NO: 13) and 5'- CGCGGTCCTATTCCATTATTC-3' (SEQ ID NO: 14) (for 18s) and the primers 5'- CTCTTCCAGCCTTCCTTCCT-3' (SEQ ID NO: 15) and 5'- AGC ACTGTGTTGGCGT AC AG-3 ' (for ACTB) (SEQ ID NO: 16).

[0135] Electroretinograms. Scotopic and photopic responses to electroretinograms were measured using a Celeris system (Diagnosys), as described previously (31, 32). Briefly, mice were dark- adapted overnight (> 18 hr). Under dim red light, mice were anesthetized by an intraperitoneal injection of 120 pg / g ketamine and 9 pg / g xylazine diluted in saline. Pupils were sequentially dilated with 0.5% proparacaine hydrochloride ophthalmic solution (Sandoz), 1% tropicamide hydrochloride ophthalmic solution (Sandoz), and 10% phenylephrine hydrochloride ophthalmic solution (Sandoz). Body temperature was maintained at 37°C on the Celeris platform. Systane lubricant eye gel (Alcon) was applied to conduct the electrical signal between the cornea andelectrode. The contralateral unstimulated eye was used as the reference. Scotopic electroretinograms were performed using light flashes of intensities ranging from -2.0 to 1 log cd.s / m2. For each intensity, at sufficient intervals (60 to 300s), mice were allowed to readapt to darkness, as to recover from any photobleaching effects. Photopic electroretinogram recordings were performed after 5-minute intervals of white light adaptation at 10 cd.s / m2to desensitize rods. For photopic electroretinograms, the cone response was measured at 3 different light intensities (1, 3, and 10 cd.s / m2) in the presence of white light (10 cd.s / m2).

[0136] Optokinetic Tracking (OKT). Visual acuity thresholds were measured using an Optometry apparatus and software (Cerebral Mechanics) as described previously 33). Briefly, the spatial frequency and contrast sensitivity were assessed in awake, freely moving mice at P21. Mice were placed on a pedestal inside a chamber with a virtual cylinder consisting of vertical lines projected on 4 computer screens. The vertical lines rotated at varying frequencies, and tracking behavior was assessed in a stepwise manner. Spatial frequency was represented as the highest frequency (cycle / degree) at which mice tracked the rotating cylinder. The contrast threshold was measured at a spatial frequency of 0.064 cycles / degree, based on a previous publication of the OIR model (34). Contrast sensitivity was identified as the highest value that still elicited a response in the mouse. Both spatial frequency and contrast sensitivity were measured from 3 different litters at P21.

[0137] Cell culture. Primary human retinal microvascular endothelial cells (HREC; ACBRI 181, Cell Systems) or human umbilical vein endothelial cells (HUVECs; PCS- 100-010, ATCC) were cultured in endothelial complete media (4Z0-500, Cell Systems) or EGM2 (Lonza), respectively. Only cells that were passed from 2 to 8 times were used for experiments. Culture under variable oxygen levels was accomplished using a ProOx model C21 (BioSpherix, NY). For siRNA treatments, 1.0-1.5X105HUVECs were plated in 6-well dishes and incubated overnight at 37°C. The following morning, media was replaced with OptiMEM (Thermo) containing either Silencer Select Negative Control No. l siRNA or ATA / A 25-targeting siRNA (Thermo; si 5206) diluted in Lipofectamine RNAiMAX transfection agent (Thermo) following the manufacturer’s guidelines.

[0138] Statistics. At least six mice per group were used for all animal experiments, and mice were derived from at least two separate litters. At least three technical replicates were performed for in vitro experiments. Results are presented as mean ± SD. Tests for normality (D’Agostino-Pearson and Shapiro-Wilk) and equal variance (Brown-Forsythe) were conducted to determine the appropriate parametric or nonparametric statistical models. Statistical analyses were conducted using a two-tailed Student’s t test for comparison of two groups, or using a one-way ANOVA followed by the Student-Newman-Keuls multiple comparison test when more than two groupswere compared. P values of <0.05 were considered statistically significant. Prism 10.0 software (GraphPad) was used for all statistical assessments.

[0139] It is contemplated that any embodiment discussed in this specification can be implemented with respect to any method, kit, reagent, or composition of the invention, and vice versa. Furthermore, compositions of the invention can be used to achieve methods of the invention.

[0140] It will be understood that particular embodiments described herein are shown by way of illustration and not as limitations of the invention. The principal features of this invention can be employed in various embodiments without departing from the scope of the invention. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, numerous equivalents to the specific procedures described herein. Such equivalents are considered to be within the scope of this invention and are covered by the claims.

[0141] All publications and patent applications mentioned in the specification are indicative of the level of skill of those skilled in the art to which this invention pertains. All publications and patent applications are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

[0142] The use of the word “a” or “an” when used in conjunction with the term “comprising” in the claims and / or the specification may mean “one,” but it is also consistent with the meaning of “one or more,” “at least one,” and “one or more than one.” The use of the term “or” in the claims is used to mean “and / or” unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive, although the disclosure supports a definition that refers to only alternatives and “and / or.” Throughout this application, the term “about” is used to indicate that a value includes the inherent variation of error for the device, the method being employed to determine the value, or the variation that exists among the study subjects.

[0143] As used in this specification and claim(s), the words “comprising” (and any form of comprising, such as “comprise” and “comprises”), “having” (and any form of having, such as “have” and “has”), “including” (and any form of including, such as “includes” and “include”) or “containing” (and any form of containing, such as “contains” and “contain”) are inclusive or open- ended and do not exclude additional, unrecited elements or method steps. In embodiments of any of the compositions and methods provided herein, “comprising” may be replaced with “consisting essentially of’ or “consisting of’. As used herein, the phrase “consisting essentially of’ requires the specified integer(s) or steps as well as those that do not materially affect the character or function of the claimed invention. As used herein, the term “consisting” is used to indicate thepresence of the recited integer (e.g., a feature, an element, a characteristic, a property, a method / process step or a limitation) or group of integers (e.g., feature(s), element(s), characteristic(s), propertie(s), method / process steps or limitation(s)) only.

[0144] The term “or combinations thereof’ as used herein refers to all permutations and combinations of the listed items preceding the term. For example, “A, B, C, or combinations thereof’ is intended to include at least one of: A, B, C, AB, AC, BC, or ABC, and if order is important in a particular context, also BA, CA, CB, CBA, BCA, ACB, BAC, or CAB. Continuing with this example, expressly included are combinations that contain repeats of one or more item or term, such as BB, AAA, AB, BBC, AAABCCCC, CBBAAA, CABABB, and so forth. The skilled artisan will understand that typically there is no limit on the number of items or terms in any combination, unless otherwise apparent from the context.

[0145] As used herein, words of approximation such as, without limitation, “about”, "substantial" or "substantially" refers to a condition that when so modified is understood to not necessarily be absolute or perfect but would be considered close enough to those of ordinary skill in the art to warrant designating the condition as being present. The extent to which the description may vary will depend on how great a change can be instituted and still have one of ordinary skilled in the art recognize the modified feature as still having the required characteristics and capabilities of the unmodified feature. In general, but subject to the preceding discussion, a numerical value herein that is modified by a word of approximation such as “about” may vary from the stated value by at least ±1, 2, 3, 4, 5, 6, 7, 10, 12 or 15%.

[0146] Additionally, the section headings herein are provided for consistency with the suggestions under 37 CFR 1.77 or otherwise to provide organizational cues. These headings shall not limit or characterize the invention(s) set out in any claims that may issue from this disclosure. Specifically and by way of example, although the headings refer to a “Field of Invention,” such claims should not be limited by the language under this heading to describe the so-called technical field. Further, a description of technology in the “Background of the Invention” section is not to be construed as an admission that technology is prior art to any invention(s) in this disclosure. Neither is the “Summary” to be considered a characterization of the invention(s) set forth in issued claims. Furthermore, any reference in this disclosure to “invention” in the singular should not be used to argue that there is only a single point of novelty in this disclosure. Multiple inventions may be set forth according to the limitations of the multiple claims issuing from this disclosure, and such claims accordingly define the invention(s), and their equivalents, that are protected thereby. In all instances, the scope of such claims shall be considered on their own merits in light of this disclosure, but should not be constrained by the headings set forth herein.

[0147] All of the compositions and / or methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the compositions and / or methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the invention. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.

[0148] To aid the Patent Office, and any readers of any patent issued on this application in interpreting the claims appended hereto, applicants wish to note that they do not intend any of the appended claims to invoke paragraph 6 of 35 U.S.C. § 112, U.S.C. § 112 paragraph (f), or equivalent, as it exists on the date of filing hereof unless the words “means for” or “step for” are explicitly used in the particular claim.

[0149] For each of the claims, each dependent claim can depend both from the independent claim and from each of the prior dependent claims for each and every claim so long as the prior claim provides a proper antecedent basis for a claim term or element.REFERENCES - EXAMPLE 1

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Claims

What is claimed is:

1. A method of preventing ocular vascular regression, preserving visual function, or both, comprising: identifying a subject in need of treatment to prevent ocular vascular regression; and providing the subject with an effective amount of an agent that is: an agonist that increases expression of at least one gene selected from: erythroblast transformation-specific (ETS)-Related Gene (ERG), Fli-1 Proto-Oncogene, ETS Transcription Factor (FLU); Kriippel-like factor (KLF) transcription factor 2 (KLF2); KLF transcription factor 4 (KLF4); or a Piezo-Type Mechanosensitive Ion Channel Component 1 (PIEZO1) agonist, or both; wherein the agent is provided in an amount sufficient to prevent the ocular vascular regression, preserving visual function, or both.

2. The method of claim 1, wherein the subject is in need of treatment for retinopathy of prematurity (ROP), diabetic retinopathy (DR), capillary regression in heart or kidney.

3. The method of claim 1, wherein the agent that increases the expression of at least one of ERG, FLU, KLF2, or KLF4, is a nucleic acid-based therapeutic.

4. The method of claim 3, wherein the nucleic acid-based therapeutic is selected from a DNA, an RNA, an mRNA, a siRNA, RNAi, promoter RNA (pRNAs), enhancer RNA (eRNAs), or superenhancer RNA (seRNAs).

5. The method of claim 1, wherein the agent is a small molecule agonist that increases the expression of ERG, FLU, KLF2, or KLF4 mRNA, stabilized an ERG, FLU, KLF2, or KLF4 mRNA, or both.

6. The method of claim 1, wherein the agent comprises one or more vectors that expresses ERG, FLU, or both.

7. The method of claim 1, wherein the ocular vascular regression is retinal capillary regression.

8. The method of claim 1, wherein the PIEZO1 agonist is Yoda-1.

9. The method of claim 1, wherein the agent is administered topically, subconjunctivally, intracamerally, subtenonally, subretinally, subchoroidally, suprachoroidally, supraorbitally, retrobulbarlly, as an ocular implant, or intravitreally, or wherein the agent is formulated into an eye drop, gel, ointment, spray, a reservoir, or mist.

10. The method of claim 1, wherein the agent prevents least one of: decrease retinal neovessels by at least 40% or a retinal avascular area by at least 60% compared to a vehicle- injected contralateral eye.

11. The method of claim 1, wherein the agent does not inhibit vascular endothelial growth factor (VEGF).

12. A method for preventing or reducing a retinopathy of prematurity (ROP) or diabetic retinopathy (DR)in a patient comprising the steps of: identifying the patient in need of prevention or treatment for ocular vascular regression; and providing the patient with an effective amount of an agent that: increases expression of at least one gene selected from at least one of: erythroblast transformation-specific (ETS)-Related Gene (ERG), Fli-1 Proto-Oncogene, ETS Transcription Factor (FLU); Kriippel-like factor (KLF) transcription factor 2 (KLF2); KLF transcription factor 4 (KLF4); or is a Piezo-Type Mechanosensitive Ion Channel Component 1 (PIEZO1) agonist, or both; wherein the agent is provided in an amount effective to prevent or reduce the ocular vascular regression, preserving visual function, or both.

13. The method of claim 12, wherein the agent that increases the expression of at least one of ERG, FLU, KLF2, or KLF4, is a nucleic acid-based therapeutic.

14. The method of claim 13, wherein the nucleic acid-based therapeutic is selected from a DNA, an RNA, an mRNA, a siRNA, RNAi, promoter RNA (pRNAs), enhancer RNA (eRNAs), or superenhancer RNA (seRNAs).

15. The method of claim 12, wherein the agent is a small molecule agonist that increases the expression of ERG, FLU, KLF2, or KLF4 mRNA, stabilized an ERG, FLU, KLF2, or KLF4 mRNA, or both.

16. The method of claim 12, wherein the agent comprises one or more vectors that expresses ERG, FLU, or both.

17. The method of claim 12, wherein the ocular vascular regression is retinal capillary regression.

18. The method of claim 12, wherein the PIEZO1 agonist is Yoda-1.

19. The method of claim 12, wherein the agent is administered topically, subconjunctivally, intracamerally, subtenonally, subretinally, subchoroidally, suprachoroidally, supraorbitally, retrobulbarlly, as an ocular implant, or intravitreally, or wherein the agent is formulated into an eye drop, gel, ointment, spray, a reservoir, or mist.

20. The method of claim 12, wherein the agent prevents least one of: decrease retinal neovessels by at least 40% or a retinal avascular area by at least 60% compared to a vehicle- injected contralateral eye.

21. The method of claim 12, wherein the agent does not inhibit vascular endothelial growth factor (VEGF).

22. A method of preventing or treating retinopathy of prematurity (ROP), diabetic retinopathy (DR), or both, in a subject exposed to high levels of oxygen comprising providing the subject with an effective amount of an agent that: increases expression of at least one of: erythroblast transformation-specific (ETS)- Related Gene (ERG), Fli-1 Proto-Oncogene, ETS Transcription Factor (FLU); Kriippel-like factor (KLF) transcription factor 2 (KLF2); KLF transcription factor 4 (KLF4); or is a Piezo-Type Mechanosensitive Ion Channel Component 1 (PIEZO1) agonist, or both; wherein the agent is provided in an amount sufficient to prevent or treat the ROP, DR, or both.

23. The method of claim 22, wherein the agent that increases the expression of at least one of ERG, FLU, KLF2, or KLF4, is a nucleic acid-based therapeutic.

24. The method of claim 23, wherein the nucleic acid-based therapeutic is selected from a DNA, an RNA, an mRNA, a siRNA, RNAi, promoter RNA (pRNAs), enhancer RNA (eRNAs), or superenhancer RNA (seRNAs).

25. The method of claim 22, wherein the agent comprises one or more vectors that expresses ERG, FLU, or both.

26. The method of claim 22, wherein the PIEZO1 agonist is Yoda-1.

27. The method of claim 22, wherein the agent is administered topically, subconjunctivally, intracamerally, subtenonally, subretinally, subchoroidally, suprachoroidally, supraorbitally, retrobulbarlly, as an ocular implant, or intravitreally, and wherein the agent s formulated into an eye drop, gel, ointment, spray, a reservoir, or mist.

28. A method of preventing or treating retinal disease in a subject with diabetes comprising:identifying a subject with diabetes in need of treatment to prevent or treat ocular vascular regression; and providing the subject with diabetes with an effective amount of an agent that is: an agonist that increases expression of at least one gene selected from: erythroblast transformation-specific (ETS)-Related Gene (ERG), Fli-1 Proto-Oncogene, ETS Transcription Factor (FLU); Kriippel-like factor (KLF) transcription factor 2 (KLF2); KLF transcription factor 4 (KLF4); or a Piezo-Type Mechanosensitive Ion Channel Component 1 (PIEZO1) agonist, or both; wherein the agent is provided in an amount sufficient to prevent the ocular vascular regression, preserving visual function, or both in the patient with diabetes.

29. The method of claim 28, wherein the subject is in need of treatment for capillary regression in heart or kidney.

30. The method of claim 28, wherein the agent that increases the expression of at least one of ERG, FLU, KLF2, or KLF4, is a nucleic acid-based therapeutic.

31. The method of claim 30, wherein the nucleic acid-based therapeutic is selected from a DNA, an RNA, an mRNA, a siRNA, RNAi, promoter RNA (pRNAs), enhancer RNA (eRNAs), or superenhancer RNA (seRNAs).

32. The method of claim 28, wherein the agent is a small molecule agonist that increases the expression of ERG, FLU, KLF2, or KLF4 mRNA, stabilized an ERG, FLU, KLF2, or KLF4 mRNA, or both.

33. The method of claim 28, wherein the agent comprises one or more vectors that expresses ERG, FLU, or both.

34. The method of claim 28, wherein the ocular vascular regression is retinal capillary regression.

35. The method of claim 28, wherein the PIEZO1 agonist is Yoda-1.

36. The method of claim 28, wherein the agent is administered topically, subconjunctivally, intracamerally, subtenonally, subretinally, subchoroidally, suprachoroidally, supraorbitally, retrobulbarlly, as an ocular implant, or intravitreally, or wherein the agent is formulated into an eye drop, gel, ointment, spray, a reservoir, or mist.

37. The method of claim 28, wherein the agent prevents least one of: decrease retinal neovessels by at least 40% or a retinal avascular area by at least 60% compared to a vehicle- injected contralateral eye.

38. The method of claim 28, wherein the agent does not inhibit vascular endothelial growth factor (VEGF).

39. The method of claim 28, wherein the agent unblocks blood flow in diabetic capillaries clogged with adherent leukocytes by blocking the binding of lymphocyte function-associated antigen 1 (LFA-1) and intercellular adhesion molecule 1 (ICAM-1).

40. The method of claim 39, wherein the agent that unblocks blood flow is lifitegrast.

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