Methods of inhibiting telomerase activity to treat choroidal neovascularization

Abstract

Methods of treating choroidal neovascularization in a patient in need thereof are described herein. In some aspects, the method comprises inhibiting telomerase activity within a biological compartment of the patient. In some instances, inhibiting telomerase activity comprises disposing a telomerase inhibitor and / or disposing an antisense oligonucleotide within the biological compartment of the patient. In some embodiments, the biological compartment of the patient is the eye. Further, in some instances, the patient has neovascular age-related macular degeneration, diabetic retinopathy, retinopathy of prematurity (ROP), corneal neovascularization, and / or neovascular glaucoma.

Description

METHODS OF INHIBITING TELOMERASE ACTIVITY TO TREAT CHOROIDAL NEOVASCULARIZATION CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Application No.63 / 565,804, filed on March 15, 2024, the entire contents of which are hereby incorporated by reference. STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

[0002] This invention was made with government support under R21 EY030651 awarded by the National Institutes of Health. The government has certain rights in the invention. SEQUENCE LISTING

[0003] An electronic sequence listing (069596-00077.xml; size 8.6 KB; date of creation February 28, 2025) submitted herewith is incorporated by reference in its entirety. FIELD

[0004] The present invention relates to the field of treating choroidal neovascularization, specifically through the inhibition of telomerase activity. BACKGROUND

[0005] In neovascular age-related macular degeneration (nvAMD), abnormal blood vessel growth beneath the macula leads to retinal damage and severe vision loss. Often, nvAMD is treated with VEGF-targeted ocular therapeutics. However, several challenges persist with VEGF-targeted therapies. VEGF plays a role in the maintenance of retinal pigment epithelium (RPE), photoreceptors, and other retinal cells. As a result, several adverse effects on multiple retinal cells have been reported in animal models and in patients receiving prolonged anti-VEGF therapy. Furthermore, loss in therapeutic efficacy, coupled with increased rates of macular atrophy and subretinal fibrosis, are also significant risks associated with the long-term use anti- VEGF therapy. In many cases of neovascular eye disease, patients fail to respond to anti-VEGF therapy altogether. Therefore, the development of improved therapeutic strategies for thetreatment of choroidal neovascularization (CNV) with minimal effect on homeostatic cellular functions are needed. SUMMARY

[0006] In one aspect, described herein are methods of treating choroidal neovascularization in a patient in need thereof. In some embodiments, such a method comprises inhibiting telomerase activity within a biological compartment of the patient.

[0007] In one aspect of the present disclosure, there is provided a method of treating choroidal neovascularization in a patient in need thereof. The method includes inhibiting telomerase activity within a biological compartment of the patient.

[0008] In some embodiments, the inhibiting of telomerase activity includes disposing a telomerase inhibitor within the biological compartment of the patient. The telomerase inhibitor is BIBR 1532 in some cases. In some instances, BIBR 1532 is disposed within an eye of the patient at a therapeutic dosage. In some embodiments, the therapeutic dosage is about 660-1326 picogram per eye. In some embodiments, the therapeutic dosage is sufficient to achieve an intravitreal concentration of about 100 nM to about 1 μM.

[0009] In some cases, the telomerase inhibitor comprises BIBR 1532 and anti-VEGF antibodies. In some instances, each of BIBR 1532 and anti-VEGF antibodies are disposed within an eye of the patient at a sub-therapeutic dosage. In some embodiments, the sub-therapeutic dosage may be about 166-660 picograms per eye of BIBR 1532 and about 0.1-5 ng per eye anti- VEGF antibodies. In some embodiments, the sub-therapeutic dosage is about 5 mg to about 20 mg of anti-VEGF antibody and the sub-therapeutic dosage is sufficient to achieve a BIBR 1532 concentration of about 50 nM to about 300 nM in a vitreous cavity of the patient.

[0010] In some embodiments, the inhibiting of telomerase activity includes disposing an antisense oligonucleotide within the biological compartment of the patient. In some cases, the antisense oligonucleotide targets Tert, Terc, or a combination thereof. In some cases, the antisense oligonucleotide reduces Tert and / or Terc gene expression in the patient.

[0011] In some embodiments, the inhibiting of telomerase activity includes disposing a recombinant adeno-associated virus (rAAV) that expresses small interfering RNA (siRNA) within the biological compartment of the patient. In some cases, the siRNA target Tert, Terc, or acombination thereof. In some cases, the siRNA reduce Tert and / or Terc gene expression in the patient.

[0012] In some instances, the biological compartment of the patient is the eye. In some instances, the inhibiting of telomerase activity includes intravitreally administering a telomerase inhibitor or an antisense oligonucleotide to the patient. In some instances, the patient has neovascular age-related macular degeneration, diabetic retinopathy, retinopathy of prematurity (ROP), corneal neovascularization, or neovascular glaucoma.

[0013] In another aspect of the present disclosure, there is provided a composition for treating choroidal neovascularization in a patient in need thereof. The composition includes BIBR 1532 in a microparticle or nano-formulation. In some embodiments, the microparticle or nano-formulation includes at least one liposome, nanoparticle, nano emulsion, dendrimer, or combination thereof.

[0014] In yet another aspect of the present disclosure, there is provided a composition for inhibiting telomerase. The composition includes a sub-therapeutic dosage of BIBR 1532 and a sub-therapeutic dosage of anti-VEGF antibody. In some embodiments, the sub-therapeutic dosage of BIBR 1532 is about 166-660 pg / μL and the sub-therapeutic dosage of anti-VEGF antibody is about 0.1-5 ng / μL. In some embodiments, the sub-therapeutic dosage is about 5 mg to about 20 mg of anti-VEGF antibody and the sub-therapeutic dosage is sufficient to achieve a BIBR 1532 concentration of about 50 nM to about 300 nM in a vitreous cavity of the patient. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] FIG.1A depicts graphical representations of telomerase activity that promotes laser induced choroidal neovascularization (CNV). Tert mRNA levels in the retinal pigment epithelium (RPE) and choroid tissue from control or laser-induced CNV mice 3 days post laser photocoagulation (n = 7 samples) are presented. Data are presented means ± SEM. P-values are obtained by Mann-Whitney. A p-value of <0.05 was considered statistically significant. (*P<0.05).

[0016] FIG.1B depicts graphical representations of telomerase activity that promotes laser induced CNV. Quantification of the telomerase activity in RPE / choroid tissue from control or laser-induced CNV mice 7 days post laser photocoagulation (n = 3 samples) is presented. Dataare presented means ± SEM. P-values are obtained by unpaired t-test. A p-value of <0.05 was considered statistically significant. (*P<0.05).

[0017] FIG.1C depicts fluorescence image representations of telomerase activity that promotes laser induced CNV. Representative images of FITC-isolectin B4 stained CNV lesions in RPE / choroid / scleral flat mounts from wild-type (WT) mice and G1 Tert knock-out mice (Tert– / –) mice after day 7 of laser injury are presented. Scale bar, 100µm.

[0018] FIG.1D depicts graphical representations of telomerase activity that promotes laser induced CNV. The confocal images of the CNV lesions from WT and Tert– / –mice, as exemplified in FIG.1C, were quantified using Nikon analysis software. Relative volumes of the CNV lesions are presented (n = 23 CNV lesions for WT, and 21 CNV lesions for Tert– / –). Data are presented means ± SEM. P-values are obtained by Mann-Whitney. A p-value of <0.05 was considered statistically significant. (*P<0.05).

[0019] FIG.1E depicts fluorescence image representations of telomerase activity that promotes laser induced CNV. Representative images of the FITC-isolectin B4 stained CNV lesions in WT mice and G1 and G3 Terc– / –mice 7 days post-laser photocoagulation. Scale bar, 100µm.

[0020] FIG.1F depicts graphical representations of telomerase activity that promotes laser induced CNV. The confocal images of the CNV lesions from WT and Terc– / –mice, as exemplified in FIG.1E, were quantified using ImageJ software. Relative volumes of the CNV lesions are presented (n = 32 CNV lesions for WT, and 28 CNV lesions for Terc– / –). Data are presented means ± SEM. P-values are obtained by Mann-Whitney. A p-value of <0.05 was considered statistically significant. (*P<0.05).

[0021] FIG.2A is a schematic representation of Tert- and Terc-specific antisense oligonucleotides (ASOs) suppressing CNV growth. Schematic representation of the ASO treatment in a laser-induced CNV mouse model with ASO dosing at three days before and three days after laser photocoagulation.

[0022] FIG.2B is a fluorescence image representation of Tert- and Terc-specific antisense oligonucleotides (ASOs) suppressing CNV growth. Representative confocal images of FITC- isolectin B4 stained RPE / choroid / scleral flatmount showing CNV lesion (7 day lost laser injury) in mice administered with control (Ctr), Tert and Terc ASOs. Scale bar, 100µm.

[0023] FIG.2C is a graphical representation of Tert- and Terc-specific antisense oligonucleotides (ASOs) suppressing CNV growth. Quantification of CNV volume using ImageJ is presented (n = 40 CNV lesions for Ctr, 25 CNV lesions for Tert ASO, 27 CNV lesions for Terc ASO). Data are mean ± SEM. Statistical analysis was performed using one-way analysis of variance (ANOVA). A P-value of less than 0.05 indicates statistical significance (*P < 0.05, ns = not significant).

[0024] FIG.3A is a fluorescence image representation of how telomerase activity in non- bone marrow derived cells contributes to the CNV growth. Recipient WT and Tert– / –mice were transplanted with bone marrow cells derived from either from WT and Tert– / –mice. The laser- induced CNV was produced in bone marrow chimera mice 45 days post bone marrow transplantation. Representative confocal image of isolectin B4 stained CNV lesions are presented. Scale bar, 100 µm.

[0025] FIG.3B is a graphical representation of how telomerase activity in non-bone marrow derived cells contributes to the CNV growth. Quantification of CNV lesion volume using Nikon analysis software (n = 38 CNV lesions for WT->WT (R), 28 CNV lesions for Tert- / -->WT (R), 37 CNV lesions for Tert- / -->Tert- / -(R), 26 CNV lesions for WT-> Tert- / -(R)) is presented. (R) represents the recipient mouse. Data are represented as the mean ± SEM. P-values were obtained using Kruskal Wallis test with Dunn’s correction. (*P<0.05, ns=not significant).

[0026] FIG.4A is a schematic representation of the effect of telomerase inhibitor BIBR 1532 on laser induced CNV in mice. Schematic representation of the BIBR 1532 treatment timeline.

[0027] FIG.4B is a graphical representation of the effect of telomerase inhibitor BIBR 1532 on laser induced CNV in mice. Telomerase activity in RPE / choroid tissue as determined by TRAP assay at day 7 after laser injury with or without BIBR 1532 (1326pg / eye) is presented (n = 4 samples each group). Data are mean ± SEM. Statistical analysis was performed using unpaired t-test. A P-value of less than 0.05 indicates statistical significance (*P<0.05, ns = not significant).

[0028] FIG.4C is a fluorescence image representation of the effect of telomerase inhibitor BIBR 1532 on laser induced CNV in mice. Representative images of the isolecton B4 stained CNV lesions in RPE / choroid / scleral flat mounts (at 7 days post laser injury) after intravitreal vehicle or BIBR 1532 (1326pg / eye) administration. Scale bar, 100 µm. (n = 24 CNV lesions for Control, 19 CNV lesions for BIBR 1532).

[0029] FIG.4D is a graphical representation of the effect of telomerase inhibitor BIBR 1532 on laser induced CNV in mice. Confocal images of the CNV lesions from mice intravitreally administered with vehicle or BIBR 1532 were quantified using ImageJ. Relative CNV lesion volumes are presented. Data are mean ± SEM. Statistical analysis was performed using unpaired t-test. A P-value of less than 0.05 indicates statistical significance (*P<0.05, ns = not significant).

[0030] FIG.4E is a graphical representation of the effect of telomerase inhibitor BIBR 1532 on laser induced CNV in mice. Dose-dependent effect of intravitreal BIBR 1532 administration was assessed by quantifying the confocal images of the CNV lesions via ImageJ analysis. Relative CNV lesion volumes are presented (n = 39 lesions for vehicle control, 35 lesions for 166 pg BIBR 1532, 37 lesions for 331 pg BIBR 1532, 43 lesions for 663 pg BIBR 1532, and 33 lesions for 1326 pg BIBR 1532). Data are mean ± SEM. Statistical analysis was performed using Kruskal-Wallis with Dunn’s correction. A P-value of less than 0.05 indicates statistical significance (*P<0.05, ns = not significant).

[0031] FIG.4F is a fluorescence image representation of the effect of telomerase inhibitor BIBR 1532 on laser induced CNV in mice. Representative confocal images of laser-induced CNV lesions are presented from Tert- / -mice intravitreally administered with BIBR 1532 (1326pg / eye) or vehicle. The images were captured at day 7 post laser. Scale bar, 100µm.

[0032] FIG.4G is a graphical representation of the effect of telomerase inhibitor BIBR 1532 on laser induced CNV in mice. Quantification of laser-induced CNV lesions from Tert- / -mice intravitreally administered with BIBR 1532 (1326 pg / eye) or vehicle is presented. The images were analyzed using Nikon Elements analysis software. Relative CNV volumes are presented (n = 16 CNV lesions for vehicle, 19 CNV lesions for 1326 pg BIBR). Data are mean ± SEM. Statistical analysis was performed using unpaired t-test. A P-value of less than 0.05 indicates statistical significance (*P<0.05, ns = not significant).

[0033] FIG.5A is a schematic of how BIBR 1532 action is similar to anti-VEGF therapy in inhibiting CNV growth. Schematic representation of the BIBR 1532 and anti-VEGF treatment timeline.

[0034] FIG.5B is a graphical representation of how BIBR 1532 action is similar to anti- VEGF therapy in inhibiting CNV growth. Quantification of the laser induced- CNV lesions from mice following intravitreal administration of a 1320 pg dose of BIBR 1532 and 10 ng dose of anti-VEGF antibodies alone or in combination is presented. CNV lesion images were analyzedby Nikon Elements software. Relative CNV volumes are presented. Data are represented as the mean ± SEM. Statistical analysis was performed using one-way analysis of variance (ANOVA). A P-value of less than 0.05 indicates statistical significance (*P<0.05, ns = not significant).

[0035] FIG.5C is a graphical representation of how BIBR 1532 action is similar to anti- VEGF therapy in inhibiting CNV growth. Quantification of the laser induced- CNV lesions from mice following intravitreal administration of a 660 pg dose of of BIBR 1532 and a 5 ng dose of anti-VEGF antibodies alone or in combination is presented. CNV lesion images were analyzed by Nikon Elements software. Relative CNV volumes are presented. Data are represented as the mean ± SEM. Statistical analysis was performed using one-way analysis of variance (ANOVA). A P-value of less than 0.05 indicates statistical significance (*P<0.05, ns = not significant).

[0036] FIG.5D is a graphical representation of how BIBR 1532 action is similar to anti- VEGF therapy in inhibiting CNV growth. Quantification of the laser induced- CNV lesions from mice following intravitreal administration of a 330 pg dose of BIBR 1532 and a 2 ng dose of anti-VEGF antibodies alone or in combination is presented. CNV lesion images were analyzed by Nikon Elements software. Relative CNV volumes are presented. Data are represented as the mean ± SEM. Statistical analysis was performed using one-way analysis of variance (ANOVA). A P-value of less than 0.05 indicates statistical significance (*P<0.05, ns = not significant).

[0037] FIG.6A is a graphical representation of Tert and Terc ASO efficiency as determined in cell culture. RAW 264.7 cells were transfected with the ASOs and transcript levels Tert were assessed by qPCR. Relative transcripts abundance is presented (n = 4 cell culture replicates each group). Data are mean ± SEM. P-values were obtained using unpaired t-test. A P-value of less than 0.05 indicates statistical significance (*P<0.05).

[0038] FIG.6B is a graphical representation of Tert and Terc ASO efficiency as determined in cell culture. RAW 264.7 cells were transfected with the ASOs and transcript levels Terc were assessed by qPCR. Relative transcripts abundance is presented (n = 4 cell culture replicates each group). Data are mean ± SEM. P-values were obtained using unpaired t-test. A P-value of less than 0.05 indicates statistical significance (*P<0.05).

[0039] FIG.6C is a heat map representation of how BIBR 1532 mediates the repression of angiogenic factors that are induced in CNV. Heat map demonstrating the relative abundance of angiogenic factors in RPE and choroid tissue of laser-induced CNV mice with or without intravitreal BIBR 1532 administration. Protein lysates at 7 day post laser injury were analyzed byLuminex multiplex assay. Numerical value within each cell of the heat map represents change in the cytokine level are presented (n = 4 tissue lysates sample each group).

[0040] FIG.6D is a graphical representation of how BIBR 1532 mediates the repression of angiogenic factors that are induced in CNV. VEGF promoter activity was measured using dual- Luciferase® Reporter Assay System in HEK 293T cells transfected with indicated plasmids (+). Ratio of Firefly / Renilla luciferase activity are presented in the bar graph. Data are represented as mean ± SEM. Statistical analysis was performed using one-way analysis of variance (ANOVA). A P-value of less than 0.05 indicates statistical significance (*P<0.05, ns = not significant).

[0041] FIG.7A is an imaging representation of the effect of BIBR 1532 on retinal health. Representative fundus examination of mice injected with BIBR 1532 (1326 pg) or vehicle after 7 days post laser treatment (n = 6 eyes each group).

[0042] FIG.7B is an imaging representation of the effect of BIBR 1532 on retinal health. Representative spectral-domain optical coherence tomography (SD-OCT) images of the BIBR 1532 (1326 pg) or vehicle injected eyes 7 days post laser injury (n = 6 eyes each group).

[0043] FIG.7C is a graphical representation of the effect of BIBR 1532 on retinal health. The retinal layer thickness was calculated using the InVivoVue Diver software (RPE, ONL and INL) (n = 4 eyes for each group). Values are mean ± SEM. Statistical analysis was performed using 2-way ANOVA. A P-value of less than 0.05 indicates statistical significance (ns = not- significant).

[0044] FIG.7D is an imaging representation of the effect of BIBR 1532 on retinal health. Histological analysis of retinal morphology using H&E staining on paraffin embedded sections of control and BIBR 1532 (1326 pg) -treated eyes at 7 days post laser injury. Scale bar, 50µm.

[0045] FIG.7E is a graphical representation of the effect of BIBR 1532 on retinal health. H&E stained images were analyzed using Nikon Elements software to determine thickness of retinal layers (GCL, INL and ONL) (n = 3 eyes each group). Values are mean ± SEM. Statistical analysis was performed using 2-way ANOVA. A P-value of less than 0.05 indicates statistical significance (ns = not-significant).

[0046] Further implementations, features, and aspects of the disclosed technology, and the advantages offered thereby, are described in greater detail hereinafter, and can be understood with reference to the following detailed description, accompanying drawings, and claims.DETAILED DESCRIPTION

[0047] Embodiments described herein can be understood more readily by reference to the following detailed description, examples, claims, and examples. Elements, apparatus and methods described herein, however, are not limited to the specific embodiments presented in the detailed description, examples, claims, and examples. In particular, these embodiments are merely illustrative of the principles of the present invention. Accordingly, this disclosure is not intended to embrace all such alternatives, modifications and variations that fall within the spirit and broad scope of the specification and in view of the claims.

[0048] All publications, patents and patent applications mentioned in this specification are incorporated herein in their entirety by reference, to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated herein by reference. In addition, citation or identification of any reference in this application shall not be construed as an admission that such reference is available as prior art to the present invention. To the extent that section headings are used, they should not be construed as necessarily limiting.

[0049] In addition, all ranges disclosed herein are to be understood to encompass any and all subranges subsumed therein. For example, a stated range of “l.0 to 10.0” should be considered to include any and all subranges beginning with a minimum value of 1.0 or more and ending with a maximum value of l0.0 or less, e.g., 1.0 to 5.3, or 4.7 to 10.0, or 3.6 to 7.9. All ranges disclosed herein are also to be considered to include the end points of the range, unless expressly stated otherwise. For example, a range of “between 5 and 10,” “from 5 to 10,” or “5-l0” should generally be considered to include the end points 5 and 10.

[0050] Further, when the phrase “up to” is used in connection with an amount or quantity, it is to be understood that the amount is at least a detectable amount or quantity. For example, a material present in an amount “up to” a specified amount can be present from a detectable amount and up to and including the specified amount.

[0051] Additionally, in any disclosed embodiment, the terms “substantially,” “approximately,” and “about” may be substituted with “within [a percentage] of” what is specified, where the percentage includes 0.1, 1, 5, and 10 percent.

[0052] It is also to be understood that the article “a” or “an” refers to “at least one,” unless the context of a particular use requires otherwise.

[0053] Terms such as “administering” or “administration” include acts such as prescribing, dispensing, giving, or taking a substance such that what is prescribed, dispensed, given, or taken is actually contacts the patient’s body externally or internally (or both). It is specifically contemplated that instructions or a prescription by a medical professional to a subject or patient to take or otherwise self-administer a substance is an act of administration.

[0054] The term “therapeutic dosage” refers to a commonly used dose in clinical practice for the treatment of a disease or condition. For BIBR 1532, a therapeutic dosage may range from about 100 nM to about 1 µM (concentration in vitreous cavity) for treatment of patients having choroidal neovascularization. For anti-VEGF antibody, a therapeutic dosage may range from about 50 µg to about 500 µg (concentration in vitreous cavity) for treatment of patients having choroidal neovascularization.

[0055] The term “sub-therapeutic dosage” refers to a dosage that is lower than the optimal or commonly used dosage for a therapeutic agent when used as a single agent, but when used in the combinations described herein, provides a therapeutic result. Sub-therapeutic dosages may provide an improved safety profile relative to a therapeutic dosage of the same therapeutic agent used as a single agent. For BIBR 1532, a sub-therapeutic dosage may range from about 50 nM to about 300 nM (concentration in vitreous cavity) for treatment of patients having choroidal neovascularization when used in combination with one or more other therapeutic agents. For anti-VEGF antibody, a sub-therapeutic dosage may range from about 5 μg to about 20 μg for treatment of patients having choroidal neovascularization when used in combination with one or more other therapeutic agents. METHODS OF TREATMENT AND PREVENTION

[0056] The teachings of the present disclosure provide for the treatment and / or prevention of choroidal neovascularization (CNV) or neovascular diseases of the eye in a subject in need of such treatment. Neovascular diseases of the eye include neovascular age-related macular degeneration, diabetic retinopathy, retinopathy of prematurity (ROP), corneal neovascularization, or neovascular glaucoma.

[0057] The method of treatment and / or prevention comprises inhibiting telomerase activity within a biological compartment of the patient. In some instances, inhibiting telomerase activity comprises disposing a telomerase inhibitor within a biological compartment of the patient. Insome cases, inhibiting telomerase activity comprises disposing an antisense oligonucleotide within a biological compartment of the patient. In some instances, inhibiting telomerase activity comprises disposing a recombinant adeno-associated virus (rAAV) that expresses small interfering RNA (siRNA) within the biological compartment of the patient. In some embodiments, the biological compartment of the patient is the eye.

[0058] Inhibiting telomerase activity is accomplished by the genetic and pharmacological targeting of telomerase components telomerase reverse transcriptase (TERT) and / or telomerase RNA component (TERC). It has been shown that telomerase activity is induced in CNV, and that the genetic and pharmacological targeting of telomerase components TERT and / or TERC suppresses CNV. Herein, compounds or agents capable of inhibiting telomerase activity are provided to patients in need of neovascular eye disease treatment or management.

[0059] In some instances, the inhibiting of telomerase activity is accomplished by disposing a telomerase inhibitor within a biological compartment of the patient. The telomerase inhibitor may be BIBR 1532 with or without anti-VEGF antibody. In some cases, BIBR 1532 is disposed within a biological compartment of the patient at a therapeutic dose. In some cases, each of BIBR 1532 and anti-VEGF antibodies are disposed with a biological compartment of a patient, either as a solution containing both BIBR 1532 and anti-VEGF antibodies or as separate solutions applied sequentially or concurrently to the patient. In such cases, each of BIBR 1532 and anti-VEGF antibodies are disposed within a biological component of the patient at doses that are considered sub-therapeutic doses when used for a single agent.

[0060] In some instances, the inhibiting of telomerase activity is accomplished by disposing an antisense oligonucleotide (ASO) within a biological compartment of the patient. The ASO targets Tert, Terc, or a combination thereof. In some cases, the ASO reduces Tert and / or Terc gene expression in the patient. Exemplary ASO for inhibiting telomerase activity in accordance with the present disclosure are found in Table 1 as SEQ ID NO: 2 and / or SEQ ID NO: 3. The ASO are delivered at a therapeutic dosage, which may be 50 µg to 1 mg.

[0061] In some instances, the inhibiting of telomerase activity is accomplished by disposing a recombinant adeno-associated virus (rAAV) that expresses small interfering RNA (siRNA) within a biological compartment of the patient. In some cases, the siRNA target Tert, Terc, or a combination thereof. In some cases, the siRNA reduce Tert and / or Terc gene expression in the patient. The siRNA are delivered at a therapeutic dosage, which may be 50 µg to 1 mg.COMPOSITIONS AND MEDICAMENTS

[0062] Useful compositions of the present disclosure may comprise one or more active agents, which may include telomerase inhibitors, ASO, and rAAV that express siRNA, as described above. In one embodiment, such compounds are in the form of compositions, such as but not limited to, pharmaceutical compositions and medicaments. The compositions disclosed may comprise one or more of such compounds, in combination with a pharmaceutically acceptable carrier.

[0063] The pharmaceutical compositions of the disclosure may be used in the treatment and prevention methods of the present disclosure. Such compositions are administered to a subject in amounts sufficient to deliver a therapeutically effective amount of the compound(s) so as to be effective in the treatment and prevention methods disclosed herein. The therapeutically effective amount may vary according to a variety of factors such as, but not limited to, the subject’s condition, weight, sex and age. Other factors include the mode and site of administration. The pharmaceutical compositions may be provided to the subject in any method known in the art. Exemplary routes of administration include, but are not limited to, intravitreal, subcutaneous, intravenous, topical, epicutaneous, oral, intraosseous, intramuscular, intranasal and pulmonary. In preferred embodiments, the route of administration is intravitreal.

[0064] The compositions of the present disclosure may be administered only one time to the subject or more than one time to the subject. Furthermore, when the compositions are administered to the subject more than once, a variety of regimens may be used, such as, but not limited to, one per hour, one per day, once per week, once per month or once per year. The compositions may also be administered to the subject more than one time per day. The therapeutically effective amount of the compositions and appropriate dosing regimens may be identified by routine testing in order to obtain optimal activity, while minimizing any potential side effects. In addition, co-administration or sequential administration of other agents may be desirable.

[0065] The compositions of the present disclosure may further comprise agents which improve the solubility, half-life, absorption, etc. of the compound(s). Furthermore, the compositions of the present disclosure may further comprise agents that attenuate undesirable side effects and / or or decrease the toxicity of the compounds(s).

[0066] The compound(s) may be administered in a physiologically acceptable diluent, such as a sterile liquid or mixture of liquids, including water, saline, aqueous dextrose and related sugar solutions, an alcohol, such as ethanol, isopropanol, or hexadecyl alcohol, glycols, such as propylene glycol or polyethylene glycol such as poly(ethyleneglycol) 400, glycerol ketals, such as 2,2-dimethyl-1,3-dioxolane-4-methanol, ethers, an oil, a fatty acid, a fatty acid ester or glyceride, or an acetylated fatty acid glyceride with or without the addition of a pharmaceutically acceptable surfactant, such as, but not limited to, a soap, an oil or a detergent, suspending agent, such as, but not limited to, pectin, carbomers, methylcellulose, hydroxypropylmethylcellulose, or carboxymethylcellulose, or emulsifying agents and other pharmaceutical adjuvants.

[0067] The examples below describe the foregoing and other embodiments in further detail. EXAMPLES EXAMPLE 1

[0068] The following disclosure and non-limiting specific example describe the inhibition of telomerase as a treatment for choroidal neovasculiarizaton (CNV). Telomerase is a ribonucleoprotein complex with a telomerase reverse transcriptase (TERT) component and a telomerase RNA component (TERC), a non-coding RNA that serves as a template for TERT enzymatic activity. Telomerase levels in somatic cells are either very low or undetectable.

[0069] In this non-limiting example, a proangiogenic role for telomerase in a mouse model of nvAMD is identified. It is shown that telomerase activity is induced in CNV, and it was found that the genetic and pharmacological targeting of telomerase components TERT and TERC suppress CNV. These studies identify telomerase as a therapeutic target for the treatment of neovascular diseases of the eye. These studies also provide a foundation for telomerase inhibition as a novel and effective therapeutic strategy for managing nvAMD and other ocular diseases driven by pathological neovascularization, such as diabetic retinopathy (DR), retinopathy of prematurity (ROP), corneal neovascularization, and neovascular glaucoma. Telomerase expression and activity are upregulated in the laser-induced CNV mouse model

[0070] To examine the involvement of telomerase in CNV, Tert gene expression was analyzed in vivo. Elevated levels of Tert mRNA were observed in the retinal pigment epithelium (RPE) and choroid of laser-induced CNV mice compared to control mice without laser injury(FIG.1A). To further investigate the activity level of telomerase in CNV, RPE and choroid lysates were analyzed by a Telomeric Repeat Amplification Protocol (TRAP) assay. The RPE and choroid from eyes with CNV displayed elevated levels of telomerase activity compared to these tissues from control mice (FIG.1B). These findings collectively indicate that telomerase gene expression and activity are significantly induced in angioproliferative tissue in CNV. Telomerase deficiency attenuates CNV growth after laser injury

[0071] The functional role of telomerase in CNV development was investigated by using Tert– / –and Terc– / –mice. The Tert– / –and Terc– / –mice used in this study are on the 129 / C57BL / 6J mixed genetic background, which have unusually long telomeres. As a result, no phenotype is observed in these mice until the telomeres become critically short in late generation homozygous knockout mice. CNV growth was compared between WT and first generation (G1) Tert– / –mice generated through intercrossing of Tert+ / –animals. CNV lesions were labeled with isolectin B4, and the volume of the neovascular lesion was quantified using confocal microscopy. It was found that Tert– / –mice had significantly reduced CNV growth (FIG.1C-D). Next, CNV in Terc+ / –generation 1 (G1) and 3 (G3) Terc– / –mice were examined. Similar to Tert– / –mice, CNV volume was significantly reduced in both G1 and G3 Terc– / –mice (FIG.1E-F). Collectively, these findings show the role of telomerase in the process of CNV growth. Since the telomere shortening is not overserved in G1 Tert– / –and Terc– / –mice, not intending to be bound by theory, it is likely that the reduced CNV in Tert– / –and Terc– / –mice stems from mechanisms independent of telomere length. Antisense oligonucleotide (ASO) therapies targeting Tert and Terc suppress laser-induced CNV in mice

[0072] The studies in Tert– / –and Terc– / –mice suggested a proangiogenic role of telomerase in laser-induced CNV development (FIG.1C-F). To further confirm the role of telomerase in CNV development and to assess whether telomerase can be therapeutically targeted, the effect of Tert- and Terc-targeted synthetic ASOs on the development of laser-induced CNV was tested. The ASOs effectively reduced the Tert and Terc gene expression (FIG.6A-B). The CNV volume was notably decreased in WT mice administered with intravitreous Tert- and Terc-targeting ASOs,compared to mice administered scrambled ASO (FIG.2A-C). These results provide further evidence supporting the role of telomerase in the development of CNV pathology. Telomerase activity in non-bone marrow-derived cells contributes to CNV development

[0073] To determine the cellular origin of the proangiogenic activity of telomerase, experiments in bone marrow chimeric mice were performed. Laser-induced CNV volumes were not significantly different in WT mice receiving Tert– / –bone marrow (Tert– / –→WT) compared to the control group, WT mice receiving WT bone marrow (WT →WT) (FIG.3A-B). Likewise, there was no difference in CNV volumes between Tert– / –mice receiving WT bone marrow (WT →Tert– / –) and Tert– / –mice receiving Tert– / –bone marrow (Tert– / –→Tert– / –) (FIG.3A-B). However, Tert– / –→Tert– / –mice had a significantly low level of angiogenesis compared to WT →WT mice (FIG.3A-B). Taken together, not intending to be bound by theory, these observations suggest that telomerase activity in non-bone marrow-derived cells is important for the development of CNV. Pharmacological targeting of telomerase suppresses laser-induced CNV

[0074] To assess the therapeutic potential of telomerase inhibition as an angiosuppressive strategy, BIBR 1532, a selective telomerase inhibitor, was utilized. BIBR 1532 has excellent selectivity for human TERT (hTERT (IC50) = 93 nM). The structure of BIBR 1532 is provided below.

[0075] Based on the observation that laser-induced CNV has elevated telomerase activity (FIG.1B), the telomerase inhibitory activity of BIBR 1532 was first confirmed in mouse CNV. BIBR 1532 was administered via intravitreous injection in wild type mouse eyes with laser- induced CNV (FIG.4A). Protein lysates from RPE and choroid tissues were analyzed by TRAP assay to assess telomerase activity. As expected, intravitreous administration of BIBR 1532 in wild type mice significantly reduced telomerase activity in CNV (FIG.4A-B). Next, the impactof BIBR 1532 on neovascular growth in vivo was evaluated. Intravitreous administration of BIBR 1532 significantly reduced laser-induced CNV (FIG.4A, and FIG.4C-D). Furthermore, the inhibitory effect of BIBR on CNV volume using a range of BIBR 1532 doses (166-1326 pg / eye) was investigated. While there was a progressive reduction in CNV volume with increasing BIBR 1532 dose, the inhibitor effects were statistically significant at 1326 pg / eye (FIG.4E). To determine the specificity of BIBR 1532 in reducing CNV size through a telomerase-dependent mechanism, BIBR 1532 was administered intravitreouslly to Tert- / -mice and determined its effect on growth of CNV. No changes in CNV growth in Tert- / -mice treated with BIBR 1532 were observed, indicating that BIBR 1532 induces CNV suppression specifically through the inhibition of telomerase (FIG.4F-G). Telomerase inhibition suppresses induction of angiogenic factors

[0076] Considering the effective suppression of angiogenesis by BIBR 1532 in laser-induced CNV, the underlying molecular processes were further investigated. The impact of BIBR 1532 was investigated on various angiogenic and growth factors using Luminex-based quantification. The results indicated that several of the potent angiogenic mediators, such as angiopoietin-2, MIP-1a, Endoglin, FGF-2, HGF, SDF-1, and KC, induced by laser injury were suppressed with the administration of BIBR 1532 (FIG.6C). The anti-angiogenic effect of BIBR 1532 was compared with anti-VEGF neutralizing antibodies. Both treatments demonstrated comparable efficacy in significantly reducing CNV growth (FIG.5A-B). Notably, a combination therapy utilizing sub-therapeutic doses of BIBR 1532 and anti-VEGFA antibodies yielded a statistically significant suppression of CNV, exceeding the effect of either drug alone (FIG.5D). However, this enhanced effect was not evident when using therapeutic doses of either BIBR 1532 or anti- VEGF antibodies, indicating a potential “floor effect” phenomenon where the efficacy of the treatment plateaus at higher doses (FIG.5B-C). The effect of TERC and TERT on VEGF promoter activity was examined by co-transfecting a VEGF promoter firefly luciferase construct with TERT and TERC expression plasmids in HEK293T cells. These studies revealed that both TERC and TERT induce VEGF promoter activity, and their co-expression has a cumulative effect on this activity (FIG.6D). These findings collectively suggest that the telomerase inhibitor BIBR 1532 suppresses angiogenesis potentially through VEGF-dependent and independentmechanisms, such as by suppressing other cytokines, which are involved in the proangiogenic activity of telomerase in CNV. BIBR 1532 does not induce morphological and histological changes in the healthy mouse eye

[0077] Considering the potent antiangiogenic efficacy of BIBR in laser-induced CNV model, it was examined if the inhibitor has any adverse effect on retinal structure. BIBR 1532 (1326 pg / eye) was administered via intravitreal injection in WT mice, and the retinal structure was assessed by fundus photography, spectral domain-optical coherence tomography (SD-OCT), and histology. There were no observable morphological changes in the fundus (FIG.7A). Similarly, SD-OCT imaging showed no visible abnormalities or differences in the thickness of retinal layers between the BIBR 1532- and PBS-treated groups (FIG.7B-C). To further investigate any potential histological alterations, hematoxylin and eosin (H&E) staining was performed on paraffin-embedded eye sections. Consistent with the fundus and SD-OCT findings, these histological analyses revealed no evidence of tissue abnormalities or changes in retinal layer thickness (FIG.7D-E). Discussion

[0078] The role of telomerase in ocular neovascularization and its potential contribution to pathologies such as nvAMD was previously unknown. To address this, the potential involvement of telomerase in laser-induced CNV, a mouse model of nvAMD, was explored.

[0079] This non-limiting Example presents novel evidence of the involvement of telomerase in the development of CNV in a mouse model. Telomerase has a catalytic subunit called TERT and an essential RNA component called TERC, which serves as a template for telomeric DNA elongation. These results demonstrated here show that telomerase expression and activity are upregulated in CNV tissues compared to control tissues without laser injury. The upregulation of telomerase expression and activity in CNV tissue supports the notion that telomerase plays a role in the development of CNV. Additionally, the functional role of telomerase in CNV was investigated using G1 Tert– / –and Terc– / –mice, which revealed a significant reduction in CNV growth in these knockout mice compared to wild-type mice. Furthermore, the therapeutic potential of Tert- and Terc-targeted ASOs and TERT inhibitor BIBR 1532 was explored, andeach effectively suppressed the growth of laser-induced CNV. The pharmacological targeting of telomerase using BIBR 1532 demonstrates promising therapeutic potential. The specificity of BIBR 1532 in inhibiting telomerase-dependent CNV growth is supported by the lack of its effect in Tert– / –mice, further validating its mechanism of action.

[0080] Previous research suggests that because of their inherently long telomeres, Tert– / –and Terc– / –mice exhibit normal phenotypes until telomere shortening reaches critical levels in later generations (G4-G6). Therefore, the interventions targeting telomerase (i.e., G1 Tert– / –and Terc– / –mice, BIBR1532, and ASOs targeting Tert and Terc) are unlikely to induce critical telomere shortening in the timeframe of these experiments. Hence, the observed reduction in CNV likely arises from mechanisms independent of telomere length. Telomerase exhibits several extra- telomeric activities, with potential avenues for its proangiogenic effects. For example, hTERT has been reported to induce VEGF promoter activity via interaction with Sp1 transcription factor. In this context, tumor implanted in G1 Tert– / –mice displayed retarded growth and VEGF expression. Consistent with these studies, here, it is reported that both TERT and TERC augment VEGF promoter activity in a luciferase reporter system (FIG.6D).

[0081] Additionally, telomerase has been reported to promote NF-κB-driven gene expression. Thus, not intending to be bound by theory, this suggests that telomerase inhibition during CNV might suppress inflammation, a component of angiogenesis. Another potential mechanism involving TERT is as a modulator of the Wnt–β-catenin signaling pathway. TERT, in complex with the Wnt transcription factor BRG1 / SMARCA4, physically binds to Wnt-target genes, thereby promoting their expression. Notably, several proangiogenic genes, including VEGFA, FGF, Norrin, MMP7, c-Myc, SDF-1, and endothelin-1, are direct targets of Wnt signaling. Consistent with this, FGF, endothelin-1, VEGF protein levels were reduced in BIBR 1532-adminisered CNV tissue (FIG.6C). Additionally, TERT has been reported to impact DNA methylation and chromatin remodeling. Hence, not intending to be bound by theory, it can be speculated that elevated TERT activity might induce epigenetic changes leading to altered transcriptional program in the context of angioproliferative conditions. Interestingly, sub- therapeutic doses of BIBR 1532 and anti-VEGFA antibodies suppressed CNV in statistically significant manner, exceeding the effect of either drug alone (FIG.5D). However, not intending to be bound by theory, this cooperative effect was not evident when using therapeutic doses ofeither BIBR 1532 or anti-VEGF antibodies, indicating a potential “floor effect” phenomenon where the efficacy of the treatment plateaus at higher doses (FIGS.5B-C).

[0082] The bone marrow chimera studies indicate that the proangiogenic activity of telomerase in CNV likely arises from endothelial or other stromal cells (e.g., microglia and pericytes) in the local environment. In this non-limiting Example, these findings emphasize the role of telomerase activity in tissue-resident cells. Not intending to be bound by theory, it is possible that telomerase activity in tissue-resident EPCs plays a role in endothelial cell proliferation in response to angiogenic stimuli.

[0083] The data showed that BIBR 1532 suppresses laser-induced CNV with comparable efficacy to anti-VEGF neutralizing antibodies. These findings provide insights into the role of telomerase in CNV pathology and as a therapeutic target for nvAMD. With the challenges of anti-VEGF therapeutics, telomerase presents an attractive alternative for treating neovascular diseases of the eye. One advantage of targeting telomerase for nvAMD is its minimal expression in non-proliferating somatic cells, which would minimize off-target effects of the inhibitor.

[0084] In conclusion, this non-limiting Example provides shows that telomerase plays a role in the development of experimental choroidal neovascularization and that telomerase inhibition is a therapeutic target for nvAMD. Materials and Methods Animals

[0085] Male and female C57BL / 6J, Tert– / –and Terc– / –mice aged between 8 to 12 weeks were used in this study. C57BL / 6J, Tert– / –and Terc– / –mice were obtained from the Jackson laboratory. Tert+ / –(Strain #:004132, JAX) and Terc+ / –(Strain #:005423, JAX) mice were bred as heterozygotes, and resulting generation 1 (G1) homozygous knockout and wildtype litter mates were used for experiments. All experimental procedures were approved by the Institutional Animal Care and Use Committee of The Ohio State University and / or University of Virginia. Studies were conducted in accordance with the principles outlined in the Association for Research in Vision and Ophthalmology (ARVO) Statement for Use of Animals in Ophthalmic and Vision Research. The mice were housed in a standard sterile facility with ventilated cage systems. The environment maintained a controlled temperature and humidity, with a 12-hour light and 12-hour dark cycle. The animals had access to a sterilized, irradiated rodent diet (astandard laboratory mouse diet) ad libitum and received sterile water through automatic water systems. For all the procedures, the mice were anesthetized by intraperitoneal injection of avertin (500 µg / g of body weight). Antisense oligonucleotides

[0086] In vivo ready ASOs, Antisense LNA GapmeR targeting mouse Tert, and Terc genes or scramble were purchased from Qiagen (Cat# 339523). The ASO sequence information is provided in Table 1. Table 1qPCR m18s rRNA Forward: SEQ ID IDT NA TTC GTA TTG CGC CGC TAG A NO: 6

[0087] RAW 264.7 cells (ATCC, #TIB-71) were cultured in Dulbecco’s modified Eagle’s medium (DMEM; ThermoFisher, #12430062) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin–streptomycin. The cells were maintained at 37oC with 5% CO2 in a suitable incubator. Prior to transfection, the cells were seeded in a 12-well plate and allowed to reach approximately 70% confluency. The cells were transfected with 0.5 µg of Tert / Terc (ASOs) along with Scrambled ASO (synthesized by Qiagen) using Lipofectamine 3000 (Thermofisher, #L3000001) for 48 hours as described previously (Banerjee, D., Langberg, K., Abbas, S., Odermatt, E., Yerramothu, P., Volaric, M., Reidenbach, M.A., Krentz, K.J., Rubinstein, C.D., Brautigan, D.L., et al. (2021). A non-canonical, interferon-independent signaling activity of cGAMP triggers DNA damage response signaling. Nat Commun 12, 6207.10.1038 / s41467-021- 26240-9.). Luciferase promoter activity assay

[0088] HEK293T cells cultured in 12-well plates were used to assess VEGF promoter activity using luciferase reporter system. VEGF promoter Firefly luciferase construct (300 ng) (Addgene, Plasmid #66128) was co-transfected with an empty vector, TERC (300 ng) (Abmgood Cat# 46496061), or TERT (300) (Addgene Plasmid #51631) expression plasmids alone or in combination. Renilla reporter plasmid (50ng) under HSV-thymidine kinase promoter (Promega Cat# E2241) was also transfected and was used for normalization. Cells were transfected at 50- 60% confluence using Lipofectamine 3000 reagent (Invitrogen, cat# L3000001). Experimental design ensured each well received equal amount of total plasmid DNA.40 hours post transfection,cells were lysed and Firefly and Renilla luciferase activity were measured sequentially using Dual- Luciferase® Reporter Assay System (Promega, Cat# E1910) following manufacture’s protocol. Laser-induced choroidal neovascularization mouse model

[0089] Laser photocoagulation was performed using either a slit lamp delivery system or the Phoenix MICRON® Image-Guided Laser System. Using slit lamp delivery system laser photocoagulation (532 nm, 200 mW, 100 ms, 75 μm; Iridex, Mountain View, CA) was performed as previously described (Kleinman, M.E., Yamada, K., Takeda, A., Chandrasekaran, V., Nozaki, M., Baffi, J.Z., Albuquerque, R.J., Yamasaki, S., Itaya, M., Pan, Y., et al. (2008). Sequence- and target-independent angiogenesis suppression by siRNA via TLR3. Nature 452, 591-597. 10.1038 / nature06765).

[0090] The laser photocoagulation using the Micron IV Phoenix image-guided laser system was performed following a previously established procedure (Gong, Y., Li, J., Sun, Y., Fu, Z., Liu, C.H., Evans, L., Tian, K., Saba, N., Fredrick, T., Morss, P., et al. (2015). Optimization of an Image- Guided Laser-Induced Choroidal Neovascularization Model in Mice. PLoS One 10, e0132643. 10.1371 / journal.pone.0132643). This system utilizes Meridian Merilas 532 green laser photocoagulator. Laser photocoagulations lesions were induced with spot diameter of 50 µm, delivering a 70 ms pulse of 220 mW power. Prior to each experiment, the laser system was calibrated to ensure accurate power output. To facilitate the procedure, the eyes of the mice were dilated using tropicamide and phenylephrine drops, and a general eye lubricant was applied to the cornea. Once the pupils were dilated, the mice were positioned to visualize the fundus on a computer screen connected to the micron system, and the laser beam was directed onto the fundus. Four laser burns were created around the optic disc at the 3, 6, 9, and 12 o’clock positions, approximately 2–3 optic disc diameter away from the optic nerve. Confirmation of the Bruch’s membrane rupture was observed by the formation of a bubble at the center of the laser spot. CNV lesions with hemorrhagic complications were included in the study. Flat mount staining and CNV volume measurement

[0091] On day 7 after laser photocoagulation, all mice were sacrificed, and the eyes were carefully removed using curved forceps and immediately fixed in 4% paraformaldehyde (PFA) for 1 hour at room temperature. Under a dissecting microscope, the anterior part of the eye, includingthe cornea and lens, was dissected away using spring scissors. The whole neural retina was then gently removed, leaving behind the posterior cup consisting of the RPE, choroid and sclera. The posterior eye cups were subsequently washed with PBS and dehydrated followed by rehydration by immersing the tissue in increasing and decreasing concentrations of methanol solution (75%, 50% and 25%). Afterwards, posterior eye cups were washed twice with phosphate-buffered saline (PBS) and then blocked using a solution containing 1% goat serum and 0.5% Triton X-100 for 1 hour at room temperature. To stain the CNV lesions, the eye cups were incubated overnight at 4oC with FITC-labeled Griffonia Simplicifolia Lectin I (GSL1) Isolectin B4 (0.7%, Vector Laboratory, #FL-1201). Afterward, the eye cups were washed with 0.1% Triton X-100 in PBS. To prepare flat mounts, the posterior eye cups were carefully flattened onto glass slides by making 4 radial cuts, ensuring that the sclera faced downwards. The flat mounts were then mounted with an antifade mounting medium (Vector Laboratory, #H-1400). The Isolectin B4-stained CNV lesion were imaged using a Nikon A1R or AXR confocal microscope, and fluorescence images of the CNV lesions were captured using consistent laser power and gain settings to minimize instrumental variability across lesions. The 3D volume of the CNV lesions was calculated using Nikon NIS- Elements software or as described previously using ImageJ. Intravitreal injections

[0092] For intravitreal injections, the ASOs and antibodies were dissolved in PBS. A BIBR 1532 stock solutions was prepared in dimethyl sulfoxide (DMSO) and diluted in PBS before injection. 1 µl (1 µg) ASO in PBS was administrated twice; the first injection occurred 3 days before the laser photocoagulation (days -3), and the second injection took place 3 days after the laser treatment (day 3). Similarly, 1 µl of BIBR 1532 was administered to deliver the compound at 166-1326 picogram / eyes. BIBR 1532 was injected twice into the vitreous of the eye. The first dose was administered on day 1 before (day -1) the laser treatment, and the second dose was given after 3 days (day 3) of laser photocoagulation. Equivalent amounts of DMSO diluted in PBS was used as the vehicle for these injections. Furthermore, 1 µl of anti-VEGF (2, 5, or 10 ng; R&D Systems, #AF-493) or IgG (2, 5, or 10 ng; ThermoFisher, #02-6202) was delivered once immediately after the laser treatment. All intravitreal injections were performed on anesthetized mice under a surgical microscope using a 33-gauge needle fitted in a Hamilton syringe.Real-Time Polymerase Chain Reaction

[0093] To examine the gene expression in the RPE and choroid, tissues were harvested three day-post-laser injury. Tissues from both eyes of a mouse were combined to generate one pooled sample per mouse. The total RNA was extracted using RNeasy Mini Kit (Qiagen #74104) following the manufacturer’s instructions. Subsequently, cDNA synthesis was performed using the QuantiTect Rev. Transcription Kit (Qiagen, #205311) according to the manufacturer’s protocol. The resulting cDNA samples were stored at −20°C until further analysis. Real-time polymerase chain reaction (RT-PCR) was conducted using SYBR green master mix on an Applied Biosystems QuantStudioTM3 machine. The reference gene for normalization was 18s rRNA. The relative gene expression of Tert was determined using the ΔΔCt method. Primer sequences utilized for gene expression analysis can be found in Table 1. Telomerase Activity

[0094] Telomerase activity in the RPE and choroid tissue was assessed using TeloTAGGG Telomerase PCR ELISAPLUSkit, following the manufacturer’s instructions (Sigma Aldrich, #12013789001). The RPE / choroid tissues collected at 7 days post laser injury were lysed in 200 µl of lysis buffer and incubated on ice for 2 hours. The lysates were then centrifuged, and the supernatant was carefully transferred to new 1.5 ml tubes. The protein content in the samples was estimated using a bicinchoninic acid (BCA) assay following the kit protocol (Fisher Scientific, #P123225). Negative controls were prepared by treating the tissue extract with RNase. The TRAP reaction was performed using 3 µg of total protein in a thermocycler, employing the P1-TS1 primer and anchor primer P2 to generate telomerase-specific products with 6 nucleotide increments. An internal standard product of 216 bp was included. After PCR amplification, the products were split into 2 aliquots. Each aliquot was denatured and hybridized separately to digoxigenin-labeled detection probes specific for telomeric repeats and the Internal Standard (IS), incubating at 37oC for 2 hours. The resulting products were immobilized to a streptavidin-coated microplate via the biotin-labeled primer. Finally, they were detected using an antibody against digoxigenin conjugated to horseradish peroxidase. Color development was achieved by adding the TMB substrate, and the absorbance was measured at 450 nm (with a reference wavelength of approximately 690 nm) using a microtiter plate (ELISA) reader within 30 minutes after adding the stop reagent. The absorbance values were reported as A450 nm – A690 nm. To calculate therelative telomerase activity, the following formula was used: RTA = [(absorbance of sample – absorbance of negative control) / (absorbance of internal control (IS) of sample)] / [(absorbance of control template – absorbance of lysis buffer) / absorbance of IS of control template] X 100. Histology

[0095] The eyes were fixed in 4% paraformaldehyde (PFA) and processed for paraffin embedding.10 µm-thick serial sections were stained with H&E and imaged using a slide scanning microscope (Olympus VS200). The thickness of the retinal layers was measured using Nikon NIS elements software. Spectral-domain Optical coherence tomography (SD-OCT)

[0096] OCT was conducted on mice 7 days after the administration of BIBR (1326 pg / eye) or vehicle injections. The mice were anesthetized, and their pupils were dilated using tropicamide and phenylephrine drops. SD-OCT imaging was performed using the Envisu R2200 VHR system and InVivoVue software. Linear scan, radial scan, and rectangular scan were obtained for each eye. The acquired scans were averaged suing the InVivoVue reader and subsequently analyzed in InVivoVue Diver. Quantification of cytokines

[0097] Multiple cytokines, chemokines, and growth factors in mouse RPE / choroid samples were simultaneously analyzed using the Luminex™ 200 system (Luminex, Austin, TX, USA). Eve Technologies’ Mouse Angiogenesis & Growth Factor 16-Plex Discovery Assay® service was utilized to perform these analyses. The assay included markers such as Amphiregulin, Angiopoietin-2, EGF, Endoglin, Endothelin-1, Fas Ligand, FGF-2, Follistatin, G-CSF, HGF, IL- 1β, IL-6, IL-17, KC, Leptin, MCP-1, MIP-1α, PLGF-2, Prolactin, sALK-1, SDF-1, TNFα, VEGF- A, VEGF-C, and VEGF-D. The sensitivities of these markers in the assay ranged from 0.2 to 50.3 pg / mL. Bone marrow transplantation

[0098] 8-12 week old recipient mice were identified and offered antibiotic-containing drinking water (Neomycin sulfate at 2 mg / ml) 3 days prior to the planned irradiation day. Recipient micewere prepared by exposing them to a total dose of 13 Gy X ray irradiation (RS-2000 Biological System, Rad Source Technologies inc.) delivered in two equal fractions (6.5 Gy) with a 3-hour interval. Donor bone marrow cells were isolated from the femoral and tibial bones and resuspended DMEM medium at a concentration of 2.5 × 107cells / ml. Donor bone marrow cells (5 × 106in 200 µl) were injected in recipient mice (24 hr post irradiation) via the tail vein. The transplanted bone marrow cells were allowed to reconstitute in the recipient mice for 45-60 days. Engraftment of the donor cells in the recipient mice was assessed by PCR-based genotyping of DNA purified from peripheral blood. Statistical analysis

[0099] Statistical analyses were conducted using GraphPad Prism software (version 9.0, GraphPad Software Inc., CA, USA). The normality of the data distribution was assessed using the D’Agostino and Pearson omnibus normality tests. For comparing differences between the two groups, unpaired t-tests or Mann-Whitney tests were utilized depending on the normality of the data. One-way or two-way ANOVA or Kruskal Wallis tests were employed for comparing difference among more than two groups. Statistical significance was defined as a P-value less than 0.05. Illustrations in FIG.2A, FIG.4A, and FIG.5A were created with BioRender. EXAMPLE 2

[0100] Genetic material to be used as a gene therapy may be incorporated into a virus-based vector system, such as an adeno-associated virus (AAV), which is produced by expression of the viral vector components in immortalized living cells maintained in tissue culture. AAVs can be modified to express small interfering RNA (siRNA) that target genes related to ocular diseases, such as telomerase components TERT and TERC. Thus, the AAV-delivered siRNA can inhibit telomerase as a therapeutic strategy for managing ocular disease. For example, AAV vectors may be used as a platform for siRNA delivery for the treatment of nvAMD and other ocular diseases driven by pathological neovascularization, such as DR, ROP, corneal neovascularization, and neovascular glaucoma.

[0101] AAV is a small (approximately 25 nm), non-enveloped virus of the Parvoviridae family, including twelve (12) different AAV serotypes that infects humans and some other primate species. They are replication-deficient and in nature have linear single-stranded DNA (ssDNA)genomes. The AAV genome contains replication and packaging, capsid, and accessory protein genes, and the AAV lifecycle requires co-infection with a helper virus. Generally, a helper virus is at least one of adenovirus E2A, E4 and VA RNA, which can impart helper function to support propagation of AAV.

[0102] A recombinant AAV (rAAV) vector is derived from the wild type genome of AAV by removing all or a portion the wild-type genome from the AAV genome, for example the rep / cap genes, and replacing it with a non-native nucleic acid sequence, often referred to as a transgene. Typically, one or both inverted terminal repeat (ITR) sequences of the AAV genome are retained and flank the cloned non-native sequence in the AAV vector, referred to as an AAV transfer plasmid.

[0103] rAAV production without a helper virus is desirable because the use of adenovirus for helper functions can risk production of a replication-competent adenovirus. Cell lines such as HEK293 provide the required helper functions in a helper plasmid, which contains all helper genes necessary for production of rAAV except the E1 gene, which is provided by the cell line. Transfection of a packaging cell line with the helper plasmid, a plasmid with the replication and capsid genes of AAV, and a plasmid with a transgene flanked by ITRs, results in production of rAAV that contains the transgene.

[0104] For gene therapy applications in treating ocular disease, an siRNA-expressing plasmid may be co-transfected with a helper plasmid and a rep / cap plasmid in HEK293 cells to produce rAAV for AAV-mediated siRNA delivery. In some instances, the rep / cap and helper functions are provided in one plasmid, which is co-transfected with the siRNA-expressing plasmid. Where the siRNA target Tert and Terc, rAAV delivery of the siRNA may result in the inhibition of telomerase in subjects with ocular diseases driven by pathological neovascularization.

[0105] Delivery may include administration of the rAAV that expresses the tert and / or terc targeting siRNA via intravitreous injection. For intravitreal injections, the rAAV may be dissolved in PBS and injected for delivery to the subject’s eye or eyes.References Altamura, G., Degli Uberti, B., Galiero, G., De Luca, G., Power, K., Licenziato, L., Maiolino, P., and Borzacchiello, G. (2020). The Small Molecule BIBR1532 Exerts Potential Anti-cancer Activities in Preclinical Models of Feline Oral Squamous Cell Carcinoma Through Inhibition of Telomerase Activity and Down-Regulation of TERT. Front Vet Sci 7, 620776. 10.3389 / fvets.2020.620776. Andriessen, E.M.M.A., Binet, F., Fournier, F., Hata, M., Dejda, A., Mawambo, G., Crespo-Garcia, S., Pilon, F., Buscarlet, M., Beauchemin, K., et al. (2021). Myeloid-resident neuropilin-1 promotes choroidal neovascularization while mitigating inflammation. EMBO Molecular Medicine 13, e11754. https: / / doi.org / 10.15252 / emmm.201911754. 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Claims

CLAIMS1. A method of treating choroidal neovascularization in a patient in need thereof, themethod comprising: inhibiting telomerase activity within a biological compartment of the patient.

2. The method of claim 1, wherein inhibiting telomerase activity comprises disposing atelomerase inhibitor within the biological compartment of the patient.

3. The method of claim 2, wherein the telomerase inhibitor is BIBR 1532.

4. The method of claim 3, wherein BIBR 1532 is disposed within an eye of the patient at atherapeutic dosage.

5. The method of claim 4, wherein the therapeutic dosage is sufficient to achieve anintravitreal concentration of about 100 nM to about 1 μM.

6. The method of claim 4, wherein the therapeutic dosage is about 660-1326 picogram pereye.

7. The method of claim 2, wherein the telomerase inhibitor comprises BIBR 1532 and anti-VEGF antibodies.

8. The method of claim 7, wherein each of BIBR 1532 and anti-VEGF antibodies aredisposed within an eye of the patient at a sub-therapeutic dosage.

9. The method of claim 8, wherein the sub-therapeutic dosage is about 5 mg to about 20 mgof anti-VEGF antibody and the sub-therapeutic dosage is sufficient to achieve a BIBR 1532 concentration of about 50 nM to about 300 nM in a vitreous cavity of the patient.

10. The method of claim 8, wherein the sub-therapeutic dosage is about 166-660 picogramsper eye of BIBR 1532 and about 0.1-5 ng per eye anti-VEGF antibodies.

11. The method of claim 1, wherein inhibiting telomerase activity comprises disposing anantisense oligonucleotide within the biological compartment of the patient.

12. The method of claim 11, wherein the antisense oligonucleotide targets Tert, Terc, or acombination thereof.

13. The method of claim 11, wherein the antisense oligonucleotide reduces Tert and / or Tercgene expression in the patient.

14. The method of claim 1, wherein inhibiting telomerase activity comprises disposing arecombinant adeno-associated virus (rAAV) that expresses small interfering RNA (siRNA) within the biological compartment of the patient.

15. The method of claim 14, wherein the siRNA target Tert, Terc, or a combination thereof.

16. The method of claim 14, wherein the siRNA reduce Tert and / or Terc gene expression inthe patient.

17. The method of claim 1, wherein the biological compartment of the patient is the eye.

18. The method of claim 1, wherein inhibiting telomerase activity comprises intravitreallyadministering a telomerase inhibitor or an antisense oligonucleotide to the patient.

19. The method of claim 1, wherein the patient has neovascular age-related maculardegeneration, diabetic retinopathy, retinopathy of prematurity (ROP), corneal neovascularization, or neovascular glaucoma.

20. A composition for treating choroidal neovascularization in a patient in need thereof, thecomposition comprising BIBR 1532 in a microparticle or nano-formulation.

21. The composition of claim 20, wherein the microparticle or nano-formulation comprises atleast one liposome, nanoparticle, nano-emulsion, dendrimer, or combination thereof.

22. A composition for inhibiting telomerase comprising:a sub-therapeutic dosage of BIBR 1532; and a sub-therapeutic dosage of anti-VEGF antibody.

23. The composition of claim 22, wherein the sub-therapeutic dosage of BIBR 1532 is about166-660 pg / μL and the sub-therapeutic dosage of anti-VEGF antibody is about 0.1-5 ng / μL.

24. The composition of claim 22, wherein the sub-therapeutic dosage is about 5 mg to about20 mg of anti-VEGF antibody and the sub-therapeutic dosage is sufficient to achieve a BIBR 1532 concentration of about 50 nM to about 300 nM in a vitreous cavity of a patient.

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