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. Author manuscript; available in PMC: 2025 Nov 1.
Published in final edited form as: Gene Ther. 2024 Sep 27;31(11-12):587–593. doi: 10.1038/s41434-024-00491-9

Suppression of Matrigel-Induced Choroidal Neovascularization by AAV Delivery of a Novel Anti-Scg3 Antibody

Chengchi Huang 1, Avinash Kaur 1, Liyang Ji 1, Hong Tian 2, Keith A Webster 1,2,3, Wei Li 1
PMCID: PMC11720169  NIHMSID: NIHMS2043292  PMID: 39333408

Abstract

Efforts to develop gene therapy for long-term treatment of neovascular disease are hampered by ongoing concerns that biologics against vascular endothelial growth factor (VEGF) inhibit both physiological and pathological angiogenesis and are therefore at elevated risk of adverse side effects. A potential solution is to develop disease-targeted gene therapy. Secretogranin III (Scg3), a unique disease-restricted angiogenic factor described by our group, contributes significantly to ocular neovascular disease. We have shown that Scg3 blockade with a monoclonal antibody Fab fragment (Fab) stringently inhibits pathological angiogenesis without affecting healthy vessels. Here we tested the therapeutic efficacy of adeno-associated virus (AAV)-anti-Scg3Fab to block choroidal neovascularization (CNV) induced by subretinal injection of Matrigel in a mouse model. Intravitreal AAV-anti-Scg3Fab significantly reduced CNV and suppressed CNV-associated leukocyte infiltration and macrophage activation. The efficacy and anti-inflammatory effects were equivalent to those achieved by positive control AAV-aflibercept against VEGF. Efficacies of AAV-anti-Scg3Fab and AAV-aflibercept were sustained over 4 months post AAV delivery. The findings support development of AAV-anti-Scg3 as an alternative to AAV-anti-VEGF with equivalent efficacy and potentially safer mechanism of action.

Introduction

Age-related macular degeneration (AMD) is a prevalent, debilitating ocular disease characterized by progressive degeneration of the macula and loss of central vision. With an aging global population, the burden of the disease is steadily increasing, posing significant challenges to healthcare systems worldwide. Epidemiological data underscores the clinical urgency of the condition with an estimated 196 million people affected by AMD in 2020, predicted to surge to 288 million by 2040 [1]. The primary forms of AMD, comprising dry (atrophic) and wet (neovascular) AMD, have distinct pathologies, both including buildup of drusen deposits under the macula, but wet AMD is additionally manifested by choroidal neovascularization (CNV), a process that involves pathological angiogenesis within the macula (2).

Biologics targeting the vascular endothelial growth factor (VEGF) pathway constitute the current mainstay of wet AMD treatment and reflect the underlying roles of VEGF in promoting neovascularization and associated vascular leakage. By mitigating angiogenesis and inflammation, anti-VEGF therapies, including ranibizumab and aflibercept, have revolutionized AMD management. However, anti-VEGF therapy has several limitations that include: (a) Relatively short half-lives of anti-VEGF biologics require repetitive intravitreal injections that increase the risk of injection-related eye damage, incur reduced quality of life and lower patient compliance [2, 3]. (b) Long-term anti-VEGF biologics pose safety concerns through indiscriminate inhibition of physiological and pathological angiogenesis and repression of VEGF-modulated neuroprotection and neuronal survival [4–6]. Evidence for the former effects includes reports of increased non-perfusion areas, foveal avascular zones, retinal ischemia and reduced retinal vascular density in some clinical studies of anti-VEGF therapy for wet AMD and diabetic macular edema [7–10]. Other clinical studies reported association of anti-VEGF therapy with cognitive impairment, dementia, Parkinson-like events [11, 12]. (c) Current anti-VEGF therapies provide only limited improvements of visual acuity [13]. Anti-VEGF gene therapy is an alternative approach to circumvent repeat intravitreal protein injections [14, 15]. To improve efficacy, combination therapies designed to augment or synergize with anti-VEGF have been tested. So far, platelet-derived growth factor (PDGF) and angiopoietin-2 (Ang2) delivered in combination with anti-VEGF biologics failed to improve outcomes in clinical trials [16, 17]. Inducible anti-VEGF expression by AAV has been proposed as a way to alleviate potential long-term adverse effects of anti-VEGF gene therapy on healthy retinal vasculatures [18]. Another approach that addresses all of the limitations to long-term VEGF blockade is a VEGF-independent, disease-targeted anti-angiogenic gene therapy that can be used as an alternative or synergistic treatment with anti-VEGF.

Secretogranin III (Scg3) is a disease-restricted angiogenic factor that selectively binds diseased vessels and drives pathological but not physiological angiogenesis through VEGF-independent pathways [19, 20]. In contrast to aflibercept, anti-Scg3 humanized antibodies (hAbs) stringently inhibit pathological angiogenesis with no detectable side effects on normal vessels, [5]. We recently reported that anti-Scg3 and anti-VEGF synergistically alleviate pathological angiogenesis in mouse models of CNV and diabetic retinopathy (DR), consistent with distinctive mechanisms of action (MOAs) and receptor signaling pathways [19, 21, 22].

The property of anti-Scg3 to selectively block pathological angiogenesis and ameliorate CNV with comparable efficacy to anti-VEGF positions anti-Scg3 as an alternative and/or complementary therapy to anti-VEGF. Here we compare efficacies of AAV-anti-Scg3 gene therapy with that of AAV-aflibercept in a mouse model of Matrigel-induced CNV (MCNV) [23].

Materials and Methods

Animals

C57BL/6J mice (6 – 8 weeks of age, male and female) were purchased from the Jackson Laboratory (Bar Harbor, ME). All animal procedures were approved by the Institutional Animal Care and Use Committee at Baylor College of Medicine (Protocol # AN-8362).

Production of recombinant AAV Vectors

AAV expressing anti-Scg3 monoclonal antibody Fab fragment (AAV-anti-Scg3Fab) or aflibercept (AAV-aflibercept) and AAV-mCherry were described in a previous study [23]. Briefly, AAV2-anti-Scg3Fab plasmid consists of a CBA promoter, Ig signal peptide, the heavy chain Fab fragment, a furin GT2A cleavage site, the second Ig signal peptide, the light chain Fab fragment with a C-terminal 3x FLAG tag, and a polyA signal. AAV2-aflibercept plasmid comprises the CBA promoter, Ig signal peptide and aflibercept with a C-terminal 3x FLAG tag and a polyA signal. The expression and assembly of the functional anti-Scg3 Fab and aflibercept with target-binding activity were previously characterized [23]. Both plasmids were packaged into AAV8. AAV-CAG-mCherry was purchased from Charles River (Rockville, MD). AAV viral genome (vg) titers were quantified by quantitative PCR and verified by digital PCR [23].

AAV administration

Ketamine (40 mg/kg body weight, Covetrus North America, Portland, ME) and xylazine (8 mg/kg, Akom, Lake Forest, IL) were used to anesthetize the mice through intraperitoneal (i.p.) injection. All AAVs are blind-coded and intravitreally injected at 5.0 × 108 vg/1 μl/eye.

Western blot

We carried out Western blots as previously mentioned [23]. Briefly, HEK293 cells (ATCC, Manassas, VA, Cat # CRL-3216) were seeded in 6-well plates (2×105 cells/well) and infected with indicated AAV (5 × 106 vg/well) next day for 6 h. Cells were cultured in serum-free SFM II medium (Thermo Fisher, Cat. # 11686029). Conditioned medium was collected every 12 h between 36 – 72 h post infection, centrifuged to remove cells, concentrated to 100 μl using Amicon® Ultra-4 Centrifugal filter unit (UFC801008, Sigma, St. Louis MO). Concentrated medium (20 μl/sample/lane) were resolved by SDS-PAGE under reducing conditions and analyzed by Western blot using anti-FLAG M2 mAb (1:1,000) (Sigma) [23].

Retinas were isolated from eyes of euthanized mice four month post intravitreal injection of indicated AAV, homogenized in 50 μl/retina RIPA buffer (Sigma) supplemented with protease inhibitor cocktail (Sigma, Cat. #P8340). BCA protein assay kit (Thermo Fisher) was used to quantify total protein, which was resolved by SDS-PAGE (20 g/lane), followed by Western blot using anti-FLAG M2 mAb. Membranes were stripped and reprobed with anti- β-actin mAb (1:5,000) (Sigma, Cat. #A5441).

Matrigel-induced choroidal neovascularization (MCNV)

CNV was induced by subretinal injection of Matrigel, as previously described [22]. Briefly, mice were anesthetized with ketamine and xylazine. Growth factor-reduced Matrigel (Corning, NY, Cat # 354263) was thawed on ice and diluted 3:1 with ice-cold phosphate-buffered saline (PBS). A 30-gauge needle was used to punch a hole at the equator on the temporal side of mouse eyes. Diluted Matrigel (1 μl/eye) was loaded into a pre-chilled 10-μl syringe (Hamilton, Reno, NV) with a 33-gauge needle (Hamilton, Cat. # 7803–05, customer-made with 0.4” in length, 45°C in angle) that was inserted through the hole for slow subretinal injection. After injection, the injecting needle was kept in place for 1–2 min for Matrigel to solidify before withdrawn. Matrigel was injected for CNV induction at 1 or 4 months post AAV injection. Mice with successful subretinal injection of Matrigel are included for subsequent studies and randomly assigned to different groups.

Fluorescein angiography

CNV leakage was monitored 7 days post Matrigel injection by fluorescein angiography. Briefly, 1% tropicamide (Somerset Therapeutics, LLC Hollywood, FL) and 2.5% phenylephrine ophthalmic solutions (Bausch & Lomb Incorporated Tampa, FL) were applied to the corneal surface of anesthetized mice for pupil dilation (one drop/drug/eye). Sodium fluorescein (5 μl/g body weight, 2.5%, Akorn Operating company LLC, Gurnee, IL) was injected intraperitoneally. Fluorescein angiography images were collected 6 min post injection using a Spectralis Tracking OCTA system (Heidelberg Engineering, Franklin, MA, USA). CNV leakage, including leakage area size and mean intensity, was quantified using “Measure” function underneath “Analyze” within ImageJ software (National Institutes of Health, Bethesda, MD).

Optical coherence tomography (OCT)

Anesthetized mice were treated topically with a drop of tropicamide on the cornea for pupil dilution. After a few minutes, mice were placed onto the OCT imaging platform, ensuring minimized head movement. Heidelberg Engineering Spectral Domain OCT (SD-OCT) was aligned for optimal focus on the retina, and cross-sectional B-scans across the region of interest were acquired. The hyper-reflective CNV lesion evident on the B-scans of OCT images was delineated manually to precisely determine the lesion area. Maximal lesion size on OCT images was quantified using ImageJ, as described above.

Immunostaining of RPE and Choroidal Flat-Mounts

After fluorescein angiography and OCT, mice were euthanized by CO2 inhalation 7 days after CNV induction. Eyes were enucleated and fixed in a freshly made 4% paraformaldehyde (PFA) solution for 45 min at room temperature. After the removal of the anterior section and retina, retinal pigment epithelium (RPE)-choroid-sclera complexes (RPE eyecups) were isolated, transferred to 96-well plates, immunostained with Alexa Fluor 488-conjugated isolectin B4 (AF488-IB4, 10 μg/ml, Cat #I21411, Thermo Fisher Scientific, Waltham, MA) for CNV vessels. Additionally, RPE eyecups were co-immunostained using Alexa Fluor 495-anti-F4/80 mAb (1:100, Cat #30325S, Cell Signaling, Danvers, MA) for macrophages or Alexa Fluor 495-anti-CD45 mAb (1:100, Cat #70257S, Cell Signaling) for pan-leukocytes. Flat-mounted RPE eyecups were analyzed using a structured illumination fluorescence microscope (Model BZ-X810, Keyence, Itasca, IL), as described [22]. Maximal signal area and 3D volume were quantified using Keyence software.

Statistical analysis

Sample sizes for therapeutic studies in mice was estimated based on previous report [22]. Data is expressed as mean + SEM. Statistical analysis was performed using one-way ANOVA test. p<0.05 is considered significant.

Results

AAV transgene expression in the retina

AAV-anti-Scg3Fab and AAV-aflibercept were constructed with a C-terminal FLAG and packaged into recombinant AAVs [23]. To verify conditioned media from AAV-transduced HEK293 cells, conditioned media were analyzed by Western blot using anti-FLAG mAb to confirm molecular weight of secreted anti-Scg3 Fab and aflibercept with predicted 26.7 and 51.4 kDa, respectively. The Western blot detected ~27 – 29 kDa and ~65 – 68 kDa for the secreted anti-Scg3 Fab and aflibercept, respectively (Fig. 1A), suggesting possible glycosylation of aflibercept, as previously reported [24].

Fig. 1. Expression of anti-Scg3 Fab and aflibercept by AAVs in HEK293 cells and retinas.

Fig. 1.

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(A) HEK293 cells were infected with indicated recombinant AAV vectors. Conditioned media were collected and analyzed by Western blot for FLAG-tagged secreted proteins using anti-FLAG mAb. (B) AAVs were intravitreally injected into C57BL/6J mice. Retinas were isolated 4 months post injection and analyzed by Western blot using anti-FLAG mAb. β-actin as sample loading control.

Furthermore, AAVs were intravitreally injected into mice, and retinas collected at 4 months post injection for Western blot analysis that confirmed the similar molecular size of anti-Scg3 Fab and aflibercept (Fig. 1B). AAV-mCherry as a negative control without the FLAG tag showed no Western blot signal.

Alleviation of CNV

To determine whether AAV-anti-Scg3Fab prevents CNV development, mice on day 0 (D0) were injected intravitreally with AAV-anti-Scg3Fab or AAV-aflibercept. Control animals were injected with an equal titer of AAV-mCherry. One month later, CNV was induced by subretinal Matrigel at D30.

Fundoscopic examination at 7 days post CNV induction revealed reduced CNV lesions in eyes treated with AAV-anti-Scg3Fab and AAV-aflibercept relative to AAV-mCherry (Fig. 2A). The findings were confirmed by OCT images (Fig. 2B). Quantification of CNV lesions in OCT images confirmed that significantly reduced CNV lesion size by both AAV-anti-Scg3Fab and AAV-aflibercept with no difference between these two treatment groups (p<0.01, Fig. 2C).

Fig. 2. Inhibition of CNV lesion by AAV-aflibercept and AAV-antiScg3Fab.

Fig. 2.

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(A) Representative images of funduscopic examination for MCVN in mice at 7 days post subretinal injection of Matrigel. (B) Representative images of OCT for MCVN in mice at 7 days post subretinal injection of Matrigel. (C) Quantification of MCNV in OCT images in B. ± SEM, n=3 mice/group, one-way ANOVA test

In vivo CNV leakage at 7 days post CNV induction was detected by fluorescein angiography. As shown in Fig. 3A–C, AAV-anti-Scg3Fab significantly reduced CNV leakage area by 75.9% (p<0.001) and leakage intensity by 63.7% (p<0.001). AAV-aflibercept showed similar efficacy with no difference between the treated groups, whereas AAV-mCherry was without effect.

Fig. 3. Short-term therapeutic efficacy of AAV-anti-Scg3Fab and AAV-aflibercept to alleviate Matrigel-induced CNV.

Fig. 3.

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Indicated AAVs (blind-coded) were injected intravitreally into mice. After one month, mice were treated with Matrigel to induce CNV. (A) Representative images of fluorescein angiography performed in anesthetized mice 7 days post Matrigel injection. (B) Quantification of CNV leakage area in A. (C) Quantification of CNV leakage intensity in A. n=10 (AAV-mCherry) and 5 eyes (AAV-aflibercept and AAV-anti-Scg3Fab) in B and C. (D) Representative images of RPE eyecups isolated from mice 7 days post Matrigel injection and immunostained with Alexa Fluor 488-isolection B4 (AF488-IB4). (E) Quantification of CNV maximal lesion area in D. (F) Quantification of CNV 3D volume in D. n=9 (AAV-mCherry) and 4 eyes (AAV-aflibercept and AAV-anti-Scg3Fab) in E and F. ±SEM; one-way ANOVA test. Scale bar = 500 μm.

After fluorescein angiography, RPE eyecups were isolated from euthanized mice, stained with AF488-IB4 to label CNV vessels, flat-mounted and analyzed using a structured illumination microscope. Quantification of AF488 revealed that AAV-anti-Scg3Fab and AAV-aflibercept markedly decreased CNV lesion area and 3D volume with comparable efficiency (p<0.01 or 0.001, Fig. 3D–F). These findings indicate that AAV-anti-Scg3Fab and AAV-aflibercept alleviate Matrigel-induced CNV with equivalent efficacy in this model, consistent with our previous findings of anti-Scg3 Fab and aflibercept proteins in the same disease model [22].

Anti-inflammatory activity

Inflammatory responses, including leukocytes infiltration and macrophage activation, commonly coexist with CNV lesions [25]. We immunostained flat-mount RPE eyecups with anti-F4/80 mAb to label macrophages and anti-CD45 mAb to identify leukocytes. As shown in Fig. 4A–C, AAV-anti-Scg3Fab and anti-aflibercept significantly reduced F4/80-stained area (p<0.01) and 3D volume (p<0.05 or <0.01). Likewise, both AAVs markedly reduced the CD45-positive area (p<0.05) and 3D volume (p<0.05) (Fig. 4D–F). These findings support similar anti-inflammatory actions of AAV-anti-Scg3Fab vs. AAV-aflibercept in this CNV model. A potential concern is that mCherry signals may interfere with the signal of Alexa Fluor 495-conjugated anti-F4/80 or anti-CD45 mAb. Because of the eye fixation, however, fluorescent signal of mCherry protein was obviated with no detectable signals (Supplemental Fig. S1 and S2).

Fig. 4. Anti-inflammation effect of AAV-aflibercept and AAV-antiScg3hFab.

Fig. 4.

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Mice were treated with AAVs and Matrigel as described in Fig. 3. (A) Representative images of flat-mounted RPE eyecups stained with anti-F4/80 mAb (red) and Alexa Fluor 488-isolection B4 (IB4, green) 7 days post Matrigel injection. (B) and (C) Quantification of relative F4/80 stained area (B) and 3D volume (C) in A. (D) Representative images of RPE eyecups stained with anti-CD45 mAb (red) and IB4 as described in A. (E) and (F) Quantification of relative CD45 stained area (E) and 3D volume (F) in D. n= 3 eyes/group. one-way ANOVA test. Scale bar=200 μm.

Long-term efficacy of anti-angiogenic gene therapy

To evaluate long-term efficacy of anti-angiogenic gene therapy, CNV was induced four months after intravitreal delivery of AAVs, and CNV lesions analyzed 7 days after CNV induction as above. Quantitative fluorescein angiography revealed similar amelioration of CNV leakage area and intensity by AAV-anti-Scg3Fab and AAV-aflibercept at 4 months post AAV transduction (Fig. 5A–C), comparable to those shown in Fig. 3, measured at 1 month post AAV. Quantification of CNV vessels in flat-mount RPE eyecups revealed similar reduction of CNV area and 3D volume by AAV-anti-Scg3Fab vs. AAV-aflibercept (Fig. 5D–F). The results indicate that AAV-anti-Scg3Fab and AAV-aflibercept retain anti-angiogenic properties and ameliorate CNV with comparable efficacy at 4 months vs. 1 month after gene delivery, consistent with sustained efficacy over this time period.

Fig. 5. Long-term efficacy of AAV-anti-Scg3Fab and AAV-aflibercept to ameliorate MCNV.

Fig. 5.

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Indicated AAVs (blind-coded) were injected intravitreally into mice. After 4 months, mice were treated with Matrigel to induce CNV. (A) Representative images of fluorescein angiography to detect CVN leakage in mice 7 days post Matrigel injection. (B) Quantification of CNV leakage area in A. (C) Quantification of CNV leakage intensity in (A). n=9 (AAV-mCherry), 10 (AAV-aflibercept) and 9 eyes (AAV-anti-Scg3Fab) in B and C. (D) Representative images of RPE eyecups isolated from mice 7 days post Matrigel injection and immunostained with Alexa Fluor 488-isolection B4 (AF488-IB4) to label CNV vessels. (E) Quantification of maximal CNV vessel area in D. (F) Quantification of CNV vessel 3D volume in D. n=10 (AAV-mCherry) and 8 eyes (AAV-aflibercept and AAV-anti-Scg3Fab) in E and F. ±SEM; one-way ANOVA test. Scale bar = 500 μm.

Discussion

We show that anti-angiogenic gene therapy by AAV-anti-Scg3Fab and AAV-aflibercept suppressed CNV and vessel leakage with equivalent efficacy in a mouse MCNV model. The suppression was sustained over 1–4 months of AAV expression with no quantitative difference between these two treatment groups. The results are consistent with previous work that has shown persistent expression of functional anti-VEGF biologics following AAV delivery in mouse and non-human primate models of wet AMD [26–30]. Sustained expression and retained anti-angiogenic functions of AAV-anti-Scg3Fab meet the basic requirements for a gene therapy application and address the concerns inherent in the clinical deployment of anti-VEGF gene therapy. Similar quantitative suppression of inflammation by AAV-anti-Scg3Fab and AAV-aflibercept further demonstrates equivalent efficacies of these strategies in ameliorating CNV.

Recently, we reported equivalent amelioration of CNV by the same AAVs used in the present study in a laser-induced CNV (LCNV) model at 1 month post AAV transduction [23]. In this model, whereas the suppression of CNV vessels by both anti-Scg3 Fab and aflibercept was sustained at 4 months post AAV, there was an apparent decline in the suppression of CNV leakage at 4 months in both treatment groups. This was not the case in the present study wherein both measures of vascular pathology responded in parallel as expected, and such a disparate effect has not been reported previously for anti-VEGF biologics to our knowledge. Whereas LCNV is widely used to test anti-VEGF biologics [31], the model has a relatively short disease duration with peak CNV leakage at ≤7 days and decline of pathological phenotypes thereafter [32]. In contrast, MCNV generates CNV lesions with longer duration and more robust leakage that peaks at ~14 days [22]. By forming sub-RPE deposits that resemble sub-RPE drusen deposit, MCNV may more closely resemble human wet AMD [33]. Therefore, we suspect that the divergent effects on CNV leakage relate to the different time courses and severity of disease in the two models rather than decline of therapeutic efficacies for both AAV-anti-Scg3Fab and AAV-aflibercept. Ongoing studies in non-rodent models will address these possibilities.

Frequent intraocular injections have been a major concern since the first clinical application of anti-VEGF biologics, and gene therapy procedures to resolve such concerns have been under development for more than 20 years [34]. Successful preclinical studies primarily in mice and non-human primates (NHPs) led to the first clinical trials of AAV2 expressing the secreted form of Flt-1, a decoy VEGF receptor 1 (VEGFR1) (NCT01024998; NCT01494805) [35–37]. Despite lingering concerns over the safety of long-term VEGF suppression in AMD patients, ADVM-022 (AAV.7m8-aflibercept) is currently in a Phase 1 clinical trial for wet AMD (NCT03748784) [27, 38]. Despite the expanded clinical need, gene therapy clinical trials for wet AMD to date have not progressed passed the Phase 2 stage [39]. As discussed in Introduction, the primary reasons for this are the limited efficacy of anti-VEGF to improve vision acuity and continuing safety concerns of long-term unregulatable anti-VEGF gene therapy.

VEGF plays central roles in both physiological and pathological angiogenesis, including vascular remodeling. Biologics blocking VEGF signaling pathways may reduce vascular density in animal models [5, 40]. Because VEGF has also neurotrophic and neuroprotective properties that promote neuronal growth and survival [41, 42], VEGF inhibition may suppress neuronal function. Indeed, some clinical studies reported that intravitreal anti-VEGF biologics reduced macular vascular density [10], and others reported that anti-VEGF elicits neurological manifestations in elderly AMD patients and preterm infants [11, 12, 43, 44]. Whereas mouse and NHP studies suggest long-term safety of anti-VEGF gene therapy [28, 45], preclinical models in young animals do not necessarily replicate the fragile retinal environment of aged subjects with ocular disease and associated comorbidities. In general, aged subjects with vascular and neuronal senescence and neonates with developing retinal vasculatures and neurons are more susceptible to anti-VEGF-induced side effects than adult young animals in preclinical studies [28, 45]. Neuronal and vascular suppression may compromise visual acuity improvement, either directly or indirectly through reduced blood supply. It is noteworthy that not all clinical studies reported vascular and neurological side effects of anti-VEGF biologics [8]. Therefore, the safety of anti-VEGF biologics and long-term safety of anti-VEGF gene therapy in clinical studies remain a controversial issue, dependent on readout, subject age, dose and duration of intravitreal anti-VEGF treatment, and disease condition.

Whereas VEGF is a growth factor for endothelial cells and neurons under all conditions, Scg3 selectively binds only to diseased vessels but not healthy vessels or neurons [5, 22]. Consequently, unlike aflibercept and other anti-VEGF biologics with potential safety risks, anti-Scg3 antibodies selectively block pathological angiogenesis with undetectable side effects on healthy retinal vasculatures and neurons of neonatal mice, even at excessively high dose [5, 46]. In this regard, anti-Scg3 gene therapy could mitigate the long-term safety concerns of anti-VEGF gene therapy.

In mouse models, we have also demonstrated that combinations of anti-Scg3 hAb and aflibercept synergistically alleviate proliferative ocular disease at levels of aflibercept that minimize the potential for side effects [21, 22]. Therefore, the results described here support development of anti-Scg3 gene therapy as a monotherapy alternative to anti-VEGF or in combination with low-dose AAV-aflibercept to provide synergistic therapy with improved safety and efficacy over anti-VEGF monotherapy. By eliminating repetitive intraocular injections, sustained gene therapy by anti-Scg3Fab with or without anti-VEGF is predicted to be a safe and effective treatment for CNV and other proliferative retinopathies.

Supplementary Material

Supplemental Figure 1-2

Acknowledgments:

Authors thank Drs. Yingbin Fu, Xiao Lin, Bojun Zhang, Prabuddha Waduge for scientific discussion and technical support.

Funding:

This work was supported by NIH R01EY027749 (W.L.), R24EY028764 (W.L. and K.A.W.), R43EY031238 (H.T., K.A.W. and W.L.), R43EY032827 (H.T. and W.L.), R21EY035421 (W.L), NIH P30EY002520, Knights Templar Eye Foundation Endowment in Ophthalmology (W.L.) and unrestricted institutional grants from Research to Prevent Blindness (RPB) to the Department of Ophthalmology, Baylor College of Medicine.

Footnotes

Competing Interest: H.T. and W.L. are shareholders of Everglades Biopharma, LLC and LigandomicsRx, LLC. W.L. is an inventor of issued and pending patents. The remaining authors declare no competing financial interests.

Ethical Approval: All animal experiments were conducted in accordance with NIH guidelines and approved by the Institutional Animal Care and Use Committee at Baylor College of Medicine.

Data Availability:

Data and images generated during the current study are available from the corresponding author upon reasonable request.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplemental Figure 1-2
NIHMS2043292-supplement-Supplemental_Figure_1-2.pdf (436.7KB, pdf)

Data Availability Statement

Data and images generated during the current study are available from the corresponding author upon reasonable request.

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