Lentiviral Vector Gene Transfer of Endostatin/Angiostatin for Macular Degeneration (GEM) Study

Abstract

Neovascular age-related macular degeneration (NVAMD) is a prevalent cause of vision loss. Intraocular injections of VEGF-neutralizing proteins provide benefit, but many patients require frequent injections for a prolonged period. Benefits are often lost over time due to lapses in treatment. New treatments that sustain anti-angiogenic activity are needed. This study tested the safety and expression profile of a lentiviral Equine Infectious Anemia Virus (EIAV) vector expressing endostatin and angiostatin (RetinoStat^®). Patients with advanced NVAMD were enrolled at three centers in the United States, and the study eye received a subretinal injection of 2.4 × 10⁴ (n = 3), 2.4 × 10⁵ (n = 3), or 8.0 × 10⁵ transduction units (TU; n = 15). Each of the doses was well-tolerated with no dose-limiting toxicities. There was little or no ocular inflammation. There was one procedure-related serious adverse event (AE), a macular hole, which was managed without difficulty and resolved. There was a vector dose-related increase in aqueous humor levels of endostatin and angiostatin with high reproducibility among subjects within cohorts. Mean levels of endostatin and angiostatin peaked between 12 and 24 weeks after injection of 2.4 × 10⁵ TU or 8.0 × 10⁵ TU at 57-81 ng/mL for endostatin and 15-27 ng/mL for angiostatin, and remained stable through the last measurement at week 48. Long-term follow-up demonstrated expression was maintained at last measurement (2.5 years in eight subjects and >4 years in two subjects). Despite an apparent reduction in fluorescein angiographic leakage that broadly correlated with the expression levels in the majority of patients, only one subject showed convincing evidence of anti-permeability activity in these late-stage patients. There was no significant change in mean lesion size in subjects injected with 8.0 × 10⁵ TU. These data demonstrate that EIAV vectors provide a safe platform with robust and sustained transgene expression for ocular gene therapy.

Keywords: age-related macular degeneration (AMD); equine infectious anemia viral (EIAV) vector; lentiviral vector; neovascularization; ocular gene therapy; subretinal injection.

Figure 1.

Aqueous levels of angiostatin (A) and endostatin (B) after subretinal injection of RetinoStat^®. Aqueous samples were obtained at baseline and at 1, 4, 8,12, 24, and 48 weeks after injection of 2.4 × 10⁴ transduction units (TU; cohort 1; n = 3), 2.4 × 10⁵ TU (cohort 2; n = 3), or 8.0 × 10⁵ TU (cohorts 3 and 4; n = 15). Endostatin and angiostatin were measured by semi-quantitative Western blot analysis and relative densitometry. Mean (±standard error of the mean) expression was low in cohort 1, but subjects in cohorts 2–4 had measurable levels of endostatin at week 1, which increased substantially at week 4, peaked at 57–81 ng/mL between weeks 12 and 24, and were maintained through week 48. Levels of angiostatin for cohorts 2–4 peaked at 15–27 ng/mL between weeks 24 and 48 and remained stable through week 48.

Figure 2.

Aqueous levels of endostatin and angiostatin after subretinal injection of RetinoStat in the long-term follow-up study. Aqueous samples were obtained every 6 months (scheduled up to 5 years, then annually thereafter). Endostatin (A) and angiostatin (B) were measured by semi-quantitative Western blot analysis and relative densitometry for patients with significant levels of expression during the main study. Expression remained stable or increased over time.

Figure 3.

Change from baseline central subfield thickness (CST) after subretinal injection of RetinoStat. Subjects had measurement of CST by optical coherence tomography at baseline and at 1, 2, 4, 8, 12, 16, 20, 24, and 48 weeks after subretinal injection of 2.4 × 10⁴ TU (A, cohort 1), 2.4 × 10⁵ TU (B, cohort 2), or 8.0 × 10⁵ TU (C, cohort 3; D and E, cohort 4) of RetinoStat. Subjects in cohort 4 are divided into those that did not receive any anti-VEGF injections (D) and those that received at least one anti-VEGF injection (E). There was little mean change (±standard deviation [SD]) in CST between baseline and week 48 in the 15 patients in cohorts 3 and 4 who had a subretinal injection of 8.0 × 10⁵ TU of RetinoStat (F).

Figure 4.

Change from baseline best corrected visual acuity (BCVA) after subretinal injection of RetinoStat. Subjects had measurement of BCVA at baseline and at 1, 2, 4, 8, 12, 16, 20, 24, and 48 weeks after subretinal injection of 2.4 × 10⁴ TU (A, cohort 1), 2.4 × 10⁵ TU (B, cohort 2), or 8.0 × 10⁵ TU (C, cohort 3; D and E, cohort 4) of RetinoStat. Subjects in cohort 4 are divided into those that did not receive any anti-VEGF injections (D) and those that received at least one anti-VEGF injection (E). There was little mean change (±SD) in BCVA between baseline and week 48 in the 15 patients in cohorts 3 and 4 who had a subretinal injection of 8.0 × 10⁵ TU of RetinoStat (F).