Non-human primate model for geographic atrophy

Abstract

The present disclosure provides a non-human primate model for geographic atrophy related to macular degeneration such as age-related macular degeneration, methods of generating the model, and methods of using the model to screen therapeutic compounds useful in treating macular degeneration, for example, by slowing progression of geographic atrophy.

Description

NON-HUMAN PRIMATE MODEL FOR GEOGRAPHIC ATROPHYCROSS-REFERENCE TO RELATED APPLICATION

[0001] This application claims priority from U.S. Patent Application No. 63 / 753,214, filed February 3, 2025, the disclosure of which is incorporated herein by reference in its entirety.BACKGROUND

[0002] Age-related macular degeneration (AMD) is a leading cause of vision loss in adults over 60 years of age, with prevalence expected to grow to 288 million worldwide in 2030. As AMD progresses, the macula deteriorates and leads to advanced forms of disease -either wet AMD or dry AMD. Wet AMD, also called neovascular AMD, is characterized by neovascularization in the retina and leakage of blood or other fluids into the macula. Dry AMD, on the other hand, does not involve vascular leakage. Geographic atrophy (GA) is a progressive, irreversible, advanced form of AMD (such as dry AMD) characterized by the death of photoreceptor and retinal pigment epithelial (RPE) cells.

[0003] GA often starts in the parafovea and perifovea areas of the macula and then spreads to the fovea, eventually leading to irreversible central blindness in fewer than ten years. As the eye ages, oxidative damage accumulates, and in some individuals, results in the formation of drusen, which is yellow deposits of lipids between the RPE and Bruch’s membrane. Excessive drusen accumulation triggers chronic inflammation and destruction of photoreceptor, RPE, and choriocapillaris cells, causing the appearance of sharply defined atrophic lesions. These lesions resemble geographic areas on a map due to the appearance of choroidal vessels in retinal regions with missing RPE layer. See, e.g., Bakri et al., JManag Care Spec Pharm. 2023;29(5):Sl-S10.

[0004] While wet AMD can be treated with therapies targeting vascular endothelial growth factor (VEGF) to stop neovascularization and vascular leakage, treatment options for dry AMD, including GA, are very limited. There also is a lack of non-human primate (NHP) models that recapitulate GA lesion growth. Since primates are the only animal species with a macula, rodent models cannot accurately recapitulate human disease features. Thus, there is a need to generate an NHP model that can accurately recapitulate key features of GA and serve as a reliable system for studying GA and developing GA-targeting therapeutics.SUMMARY

[0005] The present disclosure provides a non-human primate model for geographic atrophy (GA) and a method for generating the model. In this method, laser with a power of 30-50 milliwatts (mW) is delivered for 30-60 seconds to one or more spots in a macula of a non-human primate (e.g., cynomolgus macaque), wherein the spot(s) are outside the fovea of the macula and each have a diameter of about 1 mm. The laser treatment induces macular lesions in the treated spot(s). The lesions will then develop into geographic atrophy.

[0006] In a related aspect, the present disclosure provides the use of a laser to model geographic atrophy lesions in a non-human primate, wherein the laser is to be delivered with a power of 30-50 milliwatts (mW) for 30-60 seconds to one or more spots in a macula of the non-human primate, wherein each spot is outside the fovea of the macula and has a diameter of about 1 mm.

[0007] In some embodiments, laser is delivered to two or more (e.g., four, five, six, seven, or eight) spots in the macula and the spots are spaced about 0.5 mm or more apart (e.g., about 0.5 mm to about 0.1 mm apart).

[0008] In some embodiments, the laser delivered is at a wavelength of about 480 nm to about 550 nm, optionally about 488 nm to 540 nm. In further embodiments, the laser has a wavelength of about 532 nm.

[0009] In some embodiments, the laser is delivered with a power of 40 mW for about 30 seconds. In further embodiments, the laser is laser (e.g., an argon laser) with a wavelength of about 532 nm and delivered to four to six spots in the macula with a power of 40 mM for 30 seconds.

[0010] In some embodiments, the laser is delivered through a scleral port.

[0011] In another aspect, the present disclosure provides laser for use in generating the present NHP model.

[0012] In another aspect, the present disclosure provides a method of determining whether a compound is capable of treating geographic atrophy in humans, comprising: administering the compound to a laser-treated eye of the non-human primate, and monitoring lesion growth and / or retinal thinning in the eye, wherein reduced lesion growth and / or retinal thinning as compared to a control eye not treated with the compound is indicative of the compound’s capability of treating geographic atrophy in humans. In some embodiments, the administering step is performed before, concurrent with, and / or after the delivery of laser (e.g., performed once or repeatedly). In some embodiments, the administering step is performed within about six weeks after the laser treatment. The geographic atrophy inhumans may be associated with macular degeneration, e.g., secondary to age-related macular degeneration (AMD) such as dry AMD. In a related aspect, the present disclosure provides a method of evaluating the efficacy of a compound in treating geographic atrophy in humans, comprising obtaining a macula image of a laser-treated eye of the non-human primate described herein after the laser-treated eye has been treated with the compound, analyzing the image for lesion growth and / or retinal thinning, wherein reduced lesion growth and / or retinal thinning as compared to a control eye not treated with the compound is indicative of the compound’s efficacy in treating geographic atrophy in humans.

[0013] The present methods for generating the NHP GA model and for therapy screening may further comprise monitoring lesion growth and / or retinal thinning at the lesion sites over a period of about six weeks to about ten weeks after laser treatment. In some embodiments, the methods also comprise evaluating the existence of choroidal neovascularization (CNV) formation, e.g., by fluorescein angiography imaging, in the laser-treated eye. In some embodiments, the methods comprise measuring complement activation levels in the laser-treated eye. In some embodiments, the methods comprise measuring the levels of vascular endothelial growth factor (VEGF) in the laser-treated eye.

[0014] In another aspect, the present disclosure provides non-human primates with geographic atrophy for use in the compound screening method described herein.

[0015] Other features, objectives, and advantages of the invention are apparent in the detailed description that follows. It should be understood, however, that the detailed description, while indicating embodiments and aspects of the invention, is given by way of illustration only, not limitation. Various changes and modifications within the scope of the invention will become apparent to those skilled in the art from the detailed description.BRIEF DESCRIPTION OF THE FIGURES

[0016] FIG. 1 is a schematic diagram of the study design showing the laser treatment of cynomolgus macaques which were then monitored for either 4 weeks or 8 weeks. Laser was applied in the macula of the non-human primate (NHP) eye, both superior and inferior to the fovea, in patterns of either 4 laser spots or 3 laser spots.

[0017] FIG. 2A shows representative fluorescein angiography (FA) images of Cohort 1 at 4 weeks post-laser treatment. The images represent animal ID 1001 at 4 weeks post-laser treatment. The left eye (OS) was treated with a 100 mW laser for 30 seconds with a 0.5 mm spot. The right eye (OD) was untreated.

[0018] FIG. 2B shows optical coherence tomography (OCT) images of the retinal layers in the macula for animal ID 1001. The upper image shows the untreated OD and the lower image shows the OS treated with a 100 mW laser for 30 seconds with a 0.5 mm spot.

[0019] FIG. 2C shows FA images of animal ID 2501 at 4 weeks post-laser treatment. The OD was treated with an 80 mW laser for 30 seconds, targeting a 0.5 mm spot. The OS was treated with an 80 mW laser for 60 seconds, targeting a 0.5 mm spot.

[0020] FIG. 2D shows OCT images of the retinal layers in the macula for animal ID 2501. The upper image shows the OD treated with an 80 mW laser for 30 seconds with a 0.5 mm spot. The lower image shows the OS treated with an 80 mW laser for 60 seconds with a 0.5 mm spot.

[0021] FIG. 2E shows FA images for animal ID 2001 at 4 weeks post-laser treatment. The OD was treated with a 50 mW laser for 30 seconds, targeting a 0.5 mm spot. The OS was treated with a 50 mW laser for 60 seconds, targeting a 0.5 mm spot.

[0022] FIG. 2F shows OCT images of the retinal layers in the macula for animal ID 2001. The upper image shows the OD treated with a 50 mW laser for 30 seconds with a 0.5 mm spot. The lower image shows the OS treated with a 50 mW laser for 60 seconds with a 0.5 mm spot.

[0023] FIG. 3A shows FA images of animal ID 4001 at 4 weeks post-laser treatment. Right eye (OD) shows three laser spots treated with 30 mW for 30 seconds (about 1 mm, superior) and three spots treated with 30 mW for 60 seconds (about 1 mm, inferior). Left eye (OS) shows three spots treated with 30 mW for 60 seconds (about 1 mm, inferior) and three spots treated with 30 mW for 30 seconds (about 1 mm, superior).

[0024] FIG. 3B shows OCT retinal images of animal ID 4001. The upper image corresponds to a lesion generated with laser setting 30 mW for 60 seconds (about 1 mm). The lower image corresponds to a lesion generate with laser setting 30 mW for 30 seconds (about 1 mm).

[0025] FIG. 3C shows FA images of animal ID 4501 at 4 weeks post-laser treatment. OD shows three spots treated with 40 mW for 30 seconds (about 1 mm, superior) and three spots treated with 50 mW for 30 seconds (about 1 mm, inferior). OS shows three spots treated with 40 mW for 30 seconds (about 1 mm, superior) and three spots treated with 50 mW for 30 seconds (about 1 mm, inferior).

[0026] FIG. 3D shows OCT images of animal ID 4501. The upper image corresponds to lesions generated with 50 mW for 30 seconds (about 1 mm). The lower image corresponds to lesions generated with 40 mW for 30 seconds (about 1 mm).

[0027] FIG. 4A shows FA images of animal ID 6001. At 4 weeks post-laser treatment, the left eye (OS) shows three laser spots treated with 30 mW for 60 seconds (about 1 mm, superior and inferior), and the right eye (OD) shows three laser spots treated with 40 mW for 30 seconds (about 1 mm, superior and inferior).

[0028] FIG. 4B shows FA images of animal ID 6001 at 8 weeks post-laser treatment. The treatment details for OS and OD are the same as at 4 weeks.

[0029] FIG. 4C shows FA images of animal ID 6501 at 4 weeks post-laser treatment. The left eye (OS) shows three spots treated with 30 mW for 60 seconds (about 1 mm, superior and inferior), and the right eye (OD) shows three spots treated with 40 mW for 30 seconds (about 1 mm, superior and inferior).

[0030] FIG. 4D shows FA images of animal ID 6501 at 8 weeks post-laser treatment. The treatment details for OS and OD are the same as at 4 weeks.

[0031] FIGs. 5A-5E show OCT images of the left eye (OS) of animal ID 6001. This eye was treated with three laser spots using 30 mW for 60 seconds (about 1 mm, superior and inferior). The images show the lesion area at different time points. FIG. 5A: immediately post-treatment (post-dose); FIG. 5B: one week post-treatment; FIG. 5C: two weeks posttreatment; FIG. 5D: four weeks post-treatment; FIG. 5E: eight weeks post-treatment.

[0032] FIGs. 5F-5J show OCT images of the right eye (OD) of animal ID 6501. This eye was treated with three laser spots using 40 mW for 30 seconds (about 1 mm, superior and inferior). The images show the lesion area at different time points. FIG. 5F: immediately post-treatment (post-dose); FIG. 5G: one week post-treatment; FIG. 5H: two weeks posttreatment; FIG. 51: four weeks post-treatment; FIG. 5 J: eight weeks post-treatment.

[0033] FIG. 6A shows a confocal scanning laser ophthalmoscopy (cSLO) image illustrating the lesion markings used to measure surface area.

[0034] FIG. 6B shows the average lesion surface area for lesions created with two different laser settings: 30 mW for 60 seconds and 40 mW for 30 seconds. Statistics were collected using two-way ANOVA, Tukey’s multiple comparison was used for analysis, with significance levels indicated as *p < 0.05, **p < 0.01, and ***p < 0.001.

[0035] FIG. 6C shows the progression of lesion surface area over an 8-week period.

[0036] FIG. 7A shows the average retinal thickness measurements taken within the lesion area and outside the lesion area to assess changes over time. Statistical analysis was performed using two-way ANOVA with Tukey's multiple comparisons. *p < 0.05, **p < 0.01, and ***p < 0.001.

[0037] FIG. 7B shows the change in retinal thickness within the lesion area for the two different laser settings.

[0038] FIGs. 8A-E show the analysis of aqueous humor by Olink® proteomics of the OS and OD eyes of animals 3001, 4001, and 4501 analyzed using Olink® proteomics. Samples were collected at three time points: pre-laser treatment (0), 2 weeks post-laser (2), and 4 weeks post-laser (4). Significant differences between time points for each analyte are indicated by horizontal lines above the corresponding groups.

[0039] FIGs. 9A-C show the VEGF A (FIG. 9A), B (FIG. 9B), and D (FIG. 9C) levels in aqueous humor of OS and OD eyes of animals 3001, 4001, and 4501 pre-laser (0), 2 weeks post-laser (2) and 4 weeks post laser (4) by Olink® proteomics. The p values indicate no significant change from baseline.DETAILED DESCRIPTION

[0040] The present disclosure provides a non-human primate (NHP) model for geographic atrophy (GA), in which macular lesions are induced by applying laser photocoagulation technology at specific settings to non-human primates such as cynomolgus macaque. The GA model demonstrates the key characteristic of progressive outer retinal atrophy (i.e., increase in lesion size) mimicking the human disease.

[0041] Development of GA may be associated with macular degeneration, other retinal diseases that cause macular atrophy, or eye injury. GA often represents an advanced stage of AMD, especially dry AMD. RPE and photoreceptor atrophy and lesion growth in the macula have served as key readouts for AMD disease progression and therapeutic efficacy in patients with AMD. The present NHP model provides a useful tool for understanding early changes in lesion growth and molecular pathways involved in GA progression. Given that GA can be observed in the NHP model within eight weeks after laser treatment (as opposed to the typical development of GA over months in humans), the present disclosure provides an accelerated animal model to facilitate the study of GA. The present model also is useful for testing potential therapeutics for their ability to halt lesion growth and preserve retinal structure in GA related to dry AMD and other forms of macular degeneration.I. Generation of a NHP Model for GA

[0042] Primates are the only species with a macula, the region affected in GA. Thus, prior rodent models (e.g., Ibbett et al., Scientific Reports (2019) 9:7475) cannot appropriately recapitulate human disease features. In the two prior NHP models for retinal atrophy (Liu etal., Invest Ophthalmol Vis Sci. (2021) 62(13):8; Rajagopalan et al., Experimental Eye Research (2021) 209:108678), lesion growth and / or the molecular pathways involved in lesion growth were not characterized; nor were signs associated with potential wet AMD fully examined (e.g., both VEGF levels and development of choroidal neovascularization (CNV)). It is thus unclear how accurately these models recapitulate human GA (typically associated with dry AMD) and whether they can serve as a reliable animal model for human GA or for drug screening. By contrast, the present GA model has been validated by observations of clinically validated targets - complement activation, atrophy of photoreceptor and RPE, lesion growth, and the absence of CNV. Inflammation caused by complement activation has been shown to play a critical role in GA development. Further, the present method applies laser photocoagulation to a high number (e.g., four to six) of nonconfluent macular spots outside the fovea, where the clearly demarcated lesions induced in those spots can be individually monitored and measured, thereby increasing the statistical power of studies done with each animal and reducing the number of animals needed for each study.

[0043] In the present invention, macular lesions are generated in macular regions outside the fovea (in parafovea and perifovea regions) by laser photocoagulation using specific laser settings. The laser treatment reproducibly produces lesions with localized atrophy of the retinal pigment epithelium (RPE) and photoreceptor layers. The present laser treatment can achieve selective thinning of the outer retinal layers, including the RPE and outer nuclear layer, a key feature in dry AMD and GA secondary to dry AMD. The treatment does not lead to VEGF upregulation, vascular leakage, or choroidal neovascularization (CNV), which are key features in wet AMD, a different advanced form of AMD. Wet AMD patients are typically treated with anti-VEGF therapeutics. Complement activation, on the other hand, is a clinically validated target for GA and is successfully recapitulated in the present NHP GA model. In the present NHP model, the atrophic lesion surface area increases over an eight-week period, concordant with loss in retinal thickness seen in geographic atrophy; no significant changes in full field electroretinogram recordings are present, consistent with GA patients not having significant full field ERG deficits.

[0044] In some embodiments, the laser has a wavelength of about 480 nm to about 550 nm (e.g., about 488 nm to about 540 nm). In further embodiments, the laser has a wavelength of 532 nm. Laser used herein may be generated with a gaseous medium or a solid-state medium. In some embodiments, the laser is an argon laser.

[0045] In some embodiments, the laser treatment is conducted with laser power between about 30 mW and about 50 mW, e.g., between about 35 mW and 45 mW, for a duration ofabout 30 to about 60 seconds. In particular embodiments, the laser treatment is conducted with laser energy of about 40 mW for a duration of about 30 seconds.

[0046] In some embodiments, the laser spots on the retina may have a diameter of about 0.5 mm to about 1.5 mm, e.g., about 0.75 mm to about 1.25 mm, such as about 1 mm. If more than one spot in the macula is treated with laser, the spots are nonoverlapping (nonconfluent) so as to allow characterization of individual lesions occurring at the laser-treated spots. In some embodiments, the multiple spots (e.g., two, three, four, five, six, seven, or eight spots) may be spaced about 0.5 mm or more (e.g., 0.5, 0.6, 0.7, 0.8, 0.9, 1.0 or more mm) apart. The laser spot(s) are in the macula outside the fovea and may be superior and / or inferior to the fovea.

[0047] In some embodiments, the laser treatment is conducted with a laser having a power of about 40 mM and a wavelength of about 488-540 nm (e.g., about 532 nm) for a duration about 30 seconds, with the size of the laser spot being about 1 mm. In further embodiments, more than one laser spot may be applied to each retina, e.g., in groups of 3 or 4 nonconfluent spots (e.g., about 0.5 mm or more apart).

[0048] In some embodiments, an endolaser probe is used to deliver the laser energy into the eye. Typically, before doing so, the animal is administered anesthesia in the eye and scleral ports are created for probe insertion. For example, the target eye may be anesthetized using topical proparacaine and the conjunctival fomices may be flushed with betadine-containing saline; scleral entry points may be marked posterior to the limbus using a caliper; and then scleral ports may be created using a microvitreoretinal blade and a 25-gauge or 23-gauge valved cannula. An endoilluminator probe may be inserted through a scleral port to facilitate visualization of the posterior segment during laser photocoagulation. Once laser treatment is completed, scleral ports may be removed and any lateral canthotomy may be sutured.

[0049] After laser treatment, the treated eye is monitored in-life for lesion growth and retinal atrophy over time using imaging techniques. In some embodiments, the imaging is performed through fluorescein angiography (FA) and / or optical coherence tomography (OCT) imaging. FA may also be performed at baseline (before laser treatment) and at similar timepoints to OCT measurements, so as to monitor potential CNV. In some embodiments, the imaging is performed with blue laser confocal scanning laser ophthalmoscopy (cSLO) for autofluorescence assessment of the lesioned area. Retinal thickness and lesion surface areas may be measured. The lesions may be monitored over four to eight weeks post-treatment.For example, baseline OCT imaging is performed at baseline, immediately post-laser, and subsequently weekly or biweekly to assess retinal thickness and atrophy.II. Use of the NHP Model for GA

[0050] Pathology of GA may be observed within about eight weeks after laser treatment. During and after this time, the model can be used for its intended purposes, such as studying lesion growth characteristics and testing the efficacy of potential therapeutics for AMD (e.g., dry AMD) and GA associated with AMD.

[0051] In some embodiments, aqueous humor is collected in the GA model and analyzed to determine the levels of certain proteins, such as inflammation-related proteins (e.g., complements and complement components involved in the classical pathway and the non-classical pathway, such as Cla and Bb) and angiogenesis-related proteins (e.g., VEGF). For example, RNA sequencing, ELISA, and proteomics analysis may be performed to identify molecular changes associated with lesion growth. Identification of the molecule changes may facilitate the discovery of druggable therapeutic targets for AMD (e.g., dry AMD) and AMD-related GA.

[0052] In some embodiments, a potential therapeutic compound may be administered to the laser-treated eye of the NHP and the lesion growth and retinal changes such as thinning of the eye is monitored to assess whether the compound may slow or prevent the progression of lesion growth. The compound may be a small molecule (e.g., an organic compound), a protein (e.g., an antibody or antigen-binding fragment thereof), a peptide, a nucleic acid (e.g., an siRNA, an antisense RNA, or an mRNA). The compound may be administered to the laser-treated eye through intravitreal injection, subretinal injection, suprachoroidal administration, intracameral administration, topical administration, or systemic administration (e.g., intravenous administration). In some embodiments, the compound, such as a protein or an mRNA, may be delivered to the eye through a vector such as a viral vector. In some embodiments, the viral vector is an adeno-associated viral (AAV) vector, such as an AAV vector with tropism for the eye (e.g., AAV2).

[0053] The therapeutic compound or a delivery vector (e.g., a recombinant AAV) may be administered prior to, contemporaneous with, or after laser application (e.g., within about six weeks). The compound or vector may be administered in a single dose or in multiple doses. Efficacy of the compound or vector to treat GA (slow or prevent progression of GA) may be assessed in terms of reduction of lesion growth and / or retinal thinning. Histopathology, FA,and OCT imaging can be used to assess efficacy. Routine visual acuity tests may also be used.III. Exemplary Embodiments

[0054] Nonlimiting, exemplary embodiments of the present disclosure are described below.1. A method for generating a non-human primate model for geographic atrophy (GA), the method comprising delivering laser with a power of 30-50 milliwatts (mW) for 30-60 seconds to one or more spots in a macula of a non-human primate, wherein each spot is outside the fovea of the macula and has a diameter of about 1 mm, thereby inducing one or more lesions in the macula.2. The method of embodiment 1, wherein the laser is delivered to two or more spots in the macula outside the fovea and the spots are spaced about 0.5 mm or more apart.3. The method of embodiment 2, wherein the two or more spots are four to six spots.4. The method of any one of embodiments 1-3, wherein the laser has a wavelength of about 480 nm to about 550 nm, optionally about 488 nm to about 540 nm.5. The method of embodiment 4, wherein the laser has a wavelength of 532 nm.6. The method of any one of embodiments 1-5, wherein the laser is delivered with a power of 40 mW for about 30 seconds.7. The method of embodiment 6, wherein the laser has a wavelength of 532 nm and is delivered to four to six spots in the macula outside the fovea with a power of 40 mM for 30 seconds.8. The method of any one of embodiments 1-7, wherein the laser is delivered through a scleral port.9. The method of any one of embodiments 1-8, further comprising monitoring lesion growth and / or retinal thinning at the one or more lesions over a period of about six weeks to about ten weeks.10. A non-human primate with geographic atrophy obtained by the method of any one of embodiments 1-9.11. A method of determining whether a compound is capable of treating geographic atrophy in humans, comprising:administering the compound to a laser-treated eye of the non-human primate of embodiment 10, andmonitoring lesion growth and / or retinal thinning in the eye, wherein reduced lesion growth and / or retinal thinning as compared to a control eye not treated with the compound is indicative of the compound’s capability of treating geographic atrophy in humans.12. The method of embodiment 11, wherein the administering step is performed before, concurrent with, and / or after the delivery of laser, optionally repeatedly.13. The method of embodiment 12, wherein the administering step is performed within about six weeks after the laser delivery.14. The method of any one of embodiments 11-13, where the geographic atrophy in humans is associated with macular degeneration.15. The method of embodiment 14, wherein the geographic atrophy in humans is secondary to age-related macular degeneration (AMD).16. The method of embodiment 15, wherein the AMD is dry AMD.17. The method of any one of embodiments 1-9 and 11-16, further comprising detecting choroidal neovascularization (CNV) formation in the laser-treated eye, optionally by fluorescein angiography imaging.18. The method of any one of embodiments 1-9 and 11-17, further comprising measuring complement activation levels in the laser-treated eye.19. The method of any one of embodiments 1-9 and 11-18, further comprising measuring vascular endothelial growth factor (VEGF) levels in the laser-treated eye.20. The method of any one of embodiments 1-9 and 11-19, wherein the non-human primate is a cynomolgus macaque.21. Laser for use in generating a non-human primate model for geographic atrophy (GA) in the method of any one of embodiments 1-9.22. The non-human primate with geographic atrophy of embodiment 10 for use in the method of any one of embodiments 11-20.

[0055] Unless otherwise defined herein, scientific and technical terms used in connection with the present disclosure shall have the meanings that are commonly understood by those of ordinary skill in the art. Exemplary methods and materials are described below, although methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present disclosure. In case of conflict, the present specification, including definitions, will control. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. Throughout this specification and embodiments, the words “have” and “comprise,” or variations such as“has,” “having,” “comprises,” or “comprising,” will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers. All publications and other references mentioned herein are incorporated by reference in their entirety, as if each individual reference were specifically and individually indicated to be incorporated by reference in its entirety. Although a number of documents are cited herein, this citation does not constitute an admission that any of these documents forms part of the common general knowledge in the art. As used herein, the term “approximately” or “about” as applied to one or more values of interest refers to a value that is similar to a stated reference value. In certain embodiments, the term refers to a range of values that fall within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater than or less than) of the stated reference value unless otherwise stated or otherwise evident from the context.

[0056] According to the present disclosure, back-references in the dependent claims are meant as short-hand writing for a direct and unambiguous disclosure of each and every combination of claims that is indicated by the back-reference. Further, headers herein are created for ease of organization and are not intended to limit the scope of the claimed invention in any manner.

[0057] In order that this invention may be better understood, the following examples are set forth. These examples are for purposes of illustration only and are not to be construed as limiting the scope of the invention in any manner.EXAMPLESExample 1: Generation of an NHP Model for Dry AMD

[0058] This Example describes a study that generated a non-human primate (NHP) model for dry age-related macular degeneration (AMD) by using laser photocoagulation to create lesions in the retina. In this study, animals were anesthetized and placed in dorsal recumbency. Topical proparacaine was applied to the eyes. The conjunctival fornices were flushed with a 1:50 dilution of betadine solution / saline and the eyelid margins were swabbed with undiluted 5% betadine solution. The eyes were draped and kept open by a wire eyelid speculum. A lateral canthotomy was performed using Stevens tenotomy scissors. A caliper was used to mark spots 3.0 mm posterior to the limbus on the superotemporal and / or inferotemporal sclera. Bipolar cautery was used to cauterize the sclera under the marked spots, followed by topical application of undiluted 5% betadine solution. Scleral fixation forceps were used to fix the globe position, while a microvitreoretinal blade with either a 25or a 23 gauge (g) valved cannula was inserted at each marked spot through the conjunctiva and sclera, and advanced into the vitreous humor. The trocar was positioned to face the posterior axis of the globe and then retracted to leave the scleral port in place. A direct contact surgical lens was placed on the cornea with sterile coupling gel. An endoilluminator probe was inserted through the scleral port to facilitate direct visualization of the posterior segment through the microscope. The endolaser probe was inserted through the 25 / 23g scleral port and advanced into the mid-vitreous. The laser spot was then focused onto the retinal surface and laser energy was delivered temporal to the macula, with great care to spare the foveal region. Laser spots were 1 mm in size and produced with a laser power of 40 mW and a duration of 30 seconds. Following the generation of the lesions, the endolaser, along with the endoilluminator probe, if utilized, were removed from the scleral port.Subsequently, the contact lens was removed from the cornea. The scleral port was then removed, and if a lateral canthotomy had been performed, it was sutured using 7-0 Vicryl™. The same procedure was then carried out on the contralateral eye in an identical manner.

[0059] Fluorescein angiography (FA) was conducted using the Heidelberg Spectralis® HRA / OCT system as follows. An intravenous catheter was placed in a suitable vein. 10% fluorescein sodium solution was aspirated into a 1 mL syringe at 0.05 mL / kg, and 0.9% sodium chloride was aspirated into the same syringe to a total of 1 mL. The diluted fluorescein solution was then injected as a slow bolus through the catheter into the vein. From the right eye, a one-minute funduscopic video was recorded immediately following initiation of injection under excitation with a blue laser confocal scanning laser ophthalmoscope. Following video recording from the right eye, a still image was taken from both eyes during the second minute post-injection. Then images were captured from both eyes each minute through the 6th minute post-injection.

[0060] Optical coherence tomography (OCT) imaging was conducted using the Heidelberg Spectralis® HRA / OCT system, where single, horizontal high-resolution line scan from optic nerve head through fovea, as well as a volume scan of the lesioned area, were performed. Confocal scanning laser ophthalmoscopy (cSLO) imaging, including blue-laser autofluorescence (BAF) images of the lesioned area, was captured using the Heidelberg Spectralis® HRA / OCT system.

[0061] Olink® proteomics profiling of aqueous and vitreous humor samples was conducted using the Olink® Explore 3072 platform (Cardiometabolic, Cardiometabolic II, Inflammation, Inflammation II, Neurology, Neurology II, Oncology, and Oncology II panels, with 2926 unique assays obtained from Olink® Proteomics AB, Uppsala, Sweden, and usedaccording to the manufacturer's instructions). The platform employs Proximity Extension Assay, which uses a matched pair of antibodies for a protein target, wherein each of the matched pair of antibodies is labelled with a unique oligonucleotide such that the oligonucleotides hybridize only when the antibodies bind to their intended target. The annealed product is then amplified by PCR and detected, in a multiplexed fashion, using NGS readout. The final assay readout is presented in Normalized Protein expression (NPX) values, which is an arbitrary unit on a log2-scale where a higher value corresponds to a higher protein expression.

[0062] Aqueous humor was collected as follows. Conjunctival forceps were used to fix the globe position while the tip of a 31g needle was inserted bevel -up into the sclera immediately posterior to the limbus at a 90-degree angle. The angle of the needle was then shallowed prior to being advanced into the anterior chamber between the iris and cornea. The syringe plunger was slowly withdrawn to aspirate aqueous humor.

[0063] Several laser settings were assessed in the eyes of three cohorts (Cohort 1, Cohort 2, and Cohort 3) of cynomolgus macaques with the 25-gauge articulating Alcon Vektor™ laser to identify an optimal setting at which outer retinal atrophic lesions would develop in the macula without CNV induced vascular leakage (Table 1). Parameters that were evaluated included: laser power (100 to 30 mW), spot size (0.5 mm or 1 mm), and duration of laser exposure time (30 to 60 seconds).Table 1. Laser Treatment Parameters Tested Across Three Cohorts

[0064] Cohorts 1 and 2 were used to optimize the laser setting during a 4 week in-life period, while Cohort 3 further tested the identified optimal settings with an extended in-life period of 8 weeks. The laser-induced lesions created groups of three or four spots in the macula that were superior and inferior to the fovea, while sparing the fovea (FIG. 1, Table 1). OCT imaging was performed at baseline, immediately following laser treatment and then weekly or biweekly, to assess retinal thickness and atrophy. Fluorescein angiography (FA) was performed at baseline and then weekly or biweekly to assess CNV development.

[0065] In Cohort 1, at laser settings of 100 mW and 80 mW, severe damage to the retina was observed with loss of all retinal layers and retinal or vitreal detachment at the site of laser application that persisted to week 4 (FIG. 2A and FIG. 2B). This was accompanied by hazy hyperfluorescence on FA imaging, indicating the occurrence of vascular leakage. Loweringthe laser power to 50 mW led to variable atrophy with either selective outer retinal atrophy with no vascular leakage, or more extensive atrophy of the retinal layers accompanied by vascular leakage that persisted to week 4 (FIG. 2F).

[0066] In Cohort 2, laser settings of 50 mW, 40 mW, and 30 mW with durations of 30 or 60 seconds of laser exposure, as well as 0.5 mm to 1 mm spot sizes, were evaluated (FIGs.3A-D) To minimize the number of animals used, we tested multiple laser settings in single eyes were tested, noting lesion positions and corresponding laser settings. To identify the optimal laser settings needed to create a representative geographic atrophy (GA) lesion, selective damage to the outer retina and retinal pigment epithelium (RPE) was monitored, using in-life OCT imaging. At 50 mW, variable results were obtained. However, with a spot size of 1 mm and a laser setting of either 30 mW for 60 seconds or 40 mW for 30 seconds, selective atrophy of the retinal ONL and RPE was achieved (FIG. 3B and FIG. 3D). These settings were further evaluated in Cohort 3 for reproducibility.

[0067] In Cohort 3, lesions were generated using either 30 mW for 60 seconds or 40 mW for 30 seconds and the study duration was extended to 8 weeks to assess lesion growth (FIGs. 4A-D and FIGs. 5A-J). The laser settings tested resulted in precise atrophy of the outer retina-ONL and RPE (RPE hyperfluorescence). Moreover, animals in Cohort 3 showed minimal inflammation and no vascular leakage after week 1. Lesion surface area measurements of the 12 lesions created with 40 mW for 30 seconds and a spot size of 1 mm revealed a significant increase over time (FIGs. 6A-C), with an average lesion growth of 0.72 mm2at week 8. Retinal thickness within the lesion and outside the lesion were measured in each eye, with a significant reduction in retinal thickness observed by week 8 (FIG. 7). In contrast with a laser setting of 30 mW at 60 seconds, no increase in lesion growth was observed, while there was a significant decrease in retinal thickness, albeit lower than that seen using a laser setting of 40 mW for 30 seconds (FIGs. 6A-C and FIGs. 7A-B).

[0068] Olink® analysis was performed on aqueous humor from three of the animals in Cohort 2 (3001, 4001, and 4501; both OD and OS) that were exposed to lower laser powers of 30 mW to 50 mW, and had selective atrophy of the outer retina. The expression of several complement proteins significantly increased (average of six eyes) in the aqueous humor at 2-or 4-weeks post-laser, compared to pre-laser levels (FIGs. 8A-E). VEGF levels were analyzed in the Olink® datasets, and there was no significant increase relative to pre-laser, further validating this model as a dry AMD model (FIGs. 9A-C)

[0069] Overall, analysis of the three cohorts demonstrated a dose-dependent effect of laser power on retinal ablation and vascular leakage. Laser powers >50 mW caused severeretinal thinning, vascular leakage, and choroidal ablation. In contrast, laser settings of 30 mW and 40 mW achieved selective outer nuclear layer thinning without vascular leakage. A laser setting of 40 mW for 30 seconds and a 1 mm spot size was observed to be optimal for the generation of geographic atrophy-like lesions that demonstrated growth overtime, creating a representative NHP model of late stage dry AMD or GA.

Claims

CLAIMS1. A method for generating a non-human primate model for geographic atrophy (GA), the method comprising delivering laser with a power of 30-50 milliwatts (mW) for 30-60 seconds to one or more spots in a macula of a non-human primate, wherein each spot is outside the fovea of the macula and has a diameter of about 1 mm, thereby inducing one or more lesions in the macula.

2. The method of claim 1, wherein the laser is delivered to two or more spots in the macula outside the fovea and the spots are spaced about 0.5 mm or more apart.

3. The method of claim 2, wherein the two or more spots are four to six spots.

4. The method of any one of claims 1-3, wherein the laser has a wavelength of about 480 nm to about 550 nm, optionally about 488 nm to about 540 nm.

5. The method of claim 4, wherein the laser has a wavelength of 532 nm.

6. The method of any one of claims 1-5, wherein the laser is delivered with a power of 40 mW for about 30 seconds.

7. The method of claim 6, wherein the laser has a wavelength of 532 nm and is delivered to four to six spots in the macula outside the fovea with a power of 40 mM for 30 seconds.

8. The method of any one of claims 1-7, wherein the laser is delivered through a scleral port.

9. The method of any one of claims 1-8, further comprising monitoring lesion growth and / or retinal thinning at the one or more lesions over a period of about six weeks to about ten weeks.

10. Use of a laser to model geographic atrophy lesions in a non-human primate, wherein the laser is to be delivered with a power of 30-50 milliwatts (mW) for 30-60 seconds to one or more spots in a macula of the non-human primate, wherein each spot is outside the fovea of the macula and has a diameter of about 1 mm.

11. The use of claim 10, wherein the laser is to be delivered to two or more spots in the macula outside the fovea and the spots are spaced about 0.5 mm or more apart.

12. The use of claim 11, wherein the two or more spots are four to six spots.

13. The use of any one of claims 10-12, wherein the laser has a wavelength of about 480 nm to about 550 nm, optionally about 488 nm to about 540 nm.

14. The use of claim 13, wherein the laser has a wavelength of 532 nm.

15. The use of any one of claims 10-14, wherein the laser is to be delivered with a power of 40 mW for about 30 seconds.

16. The use of claim 15, wherein the laser has a wavelength of 532 nm and is to be delivered to four to six spots in the macula outside the fovea with a power of 40 mM for 30 seconds.

17. The use of any one of claims 10-16, wherein the laser is to be delivered through a scleral port.

18. The use of any one of claims 10-17, wherein lesion growth and / or retinal thinning in the laser-treated eye is to be monitored over a period of about six weeks to about ten weeks.

19. A non-human primate with geographic atrophy obtained by the method of any one of claims 1-9.

20. A method of determining whether a compound is capable of treating geographic atrophy in humans, comprising:administering the compound to a laser-treated eye of the non-human primate of claim 19, andmonitoring lesion growth and / or retinal thinning in the eye, wherein reduced lesion growth and / or retinal thinning as compared to a control eye not treated with the compound is indicative of the compound’s capability of treating geographic atrophy in humans.

21. The method of claim 20, wherein the administering step is performed before, concurrent with, and / or after the delivery of laser, optionally repeatedly.

22. The method of claim 21, wherein the administering step is performed within about six weeks after the laser delivery.

23. A method of evaluating the efficacy of a compound in treating geographic atrophy in humans, comprising:obtaining a macula image of a laser-treated eye of the non-human primate of claim 10 after the laser-treated eye has been treated with the compound,analyzing the image for lesion growth and / or retinal thinning, wherein reduced lesion growth and / or retinal thinning as compared to a control eye not treated with the compound is indicative of the compound’s efficacy in treating geographic atrophy in humans.

24. Use of the non-human primate with geographic atrophy of claim 10 for determining whether a compound is capable of treating geographic atrophy, wherein reduced lesion growth and / or retinal thinning in the compound-treated eye of the animal as compared to a control eye not treated with the compound is indicative of the compound’s efficacy in treating geographic atrophy in humans.

25. The method of any one of claims 20-23, or the use of claim 24, where the geographic atrophy in humans is associated with macular degeneration.

26. The method or use of claim 25, wherein the geographic atrophy in humans is secondary to age-related macular degeneration (AMD).

27. The method or use of claim 26, wherein the AMD is dry AMD.

28. The method or use of any one of claims 1-18 and 20-27, further comprising detecting choroidal neovascularization (CNV) formation in the laser-treated eye, optionally by fluorescein angiography imaging.

29. The method or use of any one of claims 1-18 and 20-28, further comprising measuring complement activation levels in the laser-treated eye.

30. The method or use of any one of claims 1-18 and 20-29, further comprising measuring vascular endothelial growth factor (VEGF) levels in the laser-treated eye.

31. The method or use of any one of claims 1-18 and 20-30, wherein the non-human primate is a cynomolgus macaque.

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