Self-complementary AAV virus (scAAV) safe and long-term gene transfer in the trabecular meshwork of living rats and monkeys

Abstract

Purpose: AAV vectors produce stable transgene expression and elicit low immune response in many tissues. AAVs have been the vectors of choice for gene therapy for the eye, in particular the retina. scAAVs are modified AAVs that bypass the required second-strand DNA synthesis to achieve transcription of the transgene. The goal was to investigate the ability of AAV vectors to induce long-term, safe delivery of transgenes to the trabecular meshwork of living animals.

Methods: Single doses of AAV2.GFP and AAV2.RGD.GFP/Ad5.LacZ were injected intracamerally (IC) into rats (n = 28 eyes). A single dose of scAAV.GFP was IC-injected into rats (n = 72 eyes) and cynomolgus monkeys (n = 3). GFP expression was evaluated by fluorescence, immunohistochemistry, and noninvasive gonioscopy. Intraocular pressure (IOP) was measured with calibrated tonometer (rats) and Goldmann tonometer (monkeys). Differential expression of scAAV-infected human trabecular meshwork cells (HTM) was determined by microarrays. Humoral and cell-mediated immune responses were evaluated by ELISA and peripheral blood proliferation assays.

Results: No GFP transduction was observed on the anterior segment tissues of AAV-injected rats up to 27 days after injection. In contrast, scAAV2 transduced the trabecular meshwork very efficiently, with a fast onset (4 days). Eyes remained clear and no adverse effects were observed. Transgene expression lasted >3.5 months in rats and >2.35 years in monkeys.

Conclusions: The scAAV viral vector provides prolonged and safe transduction in the trabecular meshwork of rats and monkeys. The stable expression and safe properties of this vector could facilitate the development of trabecular meshwork drugs for gene therapy for glaucoma.

Figure 1.

Transduction of scAAV2.GFP in the anterior segments of IC-injected rats. Representative frozen meridional sections, 10-μm thick, from rats' anterior segments at various time points. (A) scAAV2 transduction of the trabecular meshwork (TM) and iris (I) at 2 weeks after injection. (B, C) scAAV2 transduction of a variety of tissues including the TM, iris, ciliary body (CB), and corneal endothelium at 2.5 and 3 months after injection. (D) Contralateral eyes were injected with vehicle which served as a control. scAAV2.GFP transduced the TM of living rats efficiently. GFP expression lasted for at least 3 months (last point tried). SC, Schlemm's canal. Original magnification, ×200.

Figure 2.

scAAV2.GFP vector expression in the anterior segment of monkey 2. Stable, long-term expression, monitored serially and noninvasively with goniophotography, was evident in the tissues of the outflow pathways of the scAAV.GFP-injected eye. Images were taken with a fundus camera on the indicated postinjection days. Bottom right: anterior chamber angle, OS, shows persistent GFP expression (A, C white arrows) at 2.35 years. (A) Image obtained by custom microscope. (B) Image obtained with a Storz endoscope used externally with 2.5% hydroxypropyl methylcellulose as an interface. (C) Fundus camera image.

Figure 3.

Histologic assessment of scAAV2.GFP transduction of monkey trabecular meshwork. (A) GFP expression examined by direct fluorescence (top) and immunohistochemistry (bottom) on OCT-embedded, frozen sections from monkey 3 at 19 days after injection. (B) GFP expression examined by direct fluorescence on frozen sections from monkey anterior segment organ cultures perfused with 2 to 4 × 1010 VP of scAAV2.GFP for 7 days. scAAV2.GFP transduced all cell layers of the monkey trabecular meshwork as well as the anterior ciliary muscle. TM, trabecular meshwork; CM, ciliary muscle. Original magnification: (left) ×200; (right), ×400.

Figure 4.

scAAV2.GFP transduction of other anterior segment tissues in an IC-injected living monkey. Direct fluorescence of wholemounts and cryosections of the iris (A), and goniophotography and cryosection of the cornea (B) tissues of monkey 3 at 19 days after injection. Intense GFP transduction was observed in the posterior face of the iris as opposed to the anterior face which had little to no GFP. Strong GFP expression was also observed in the corneal endothelium, especially when assessed by goniophotography in the living animal. Original magnification, ×200.

Figure 5.

Effect of a single IC injection of scAAV2.GFP on the IOP of living rats. The rats were injected with equivalent volumes of scAAV2.GFP (OS; 3–6 × 109 VP) and vehicle (OD). IOPs were measured, before injection (t ≤ 0 days) and at weekly postinjection intervals, in sedated rats using an induction impact tonometer with a calibrated probe. Results were from two separate, representative experiments (n = 8 each) over the course of 92 and 75 days. (A) Time course of IOPs with tonometer values converted to mm Hg (mean ± SE). The IOP of the viral-injected eyes at the end of the experiment was not significantly different from baseline and/or from eyes injected with vehicle. (B) IOP parameters calculated in scAAV2.GFP- and vehicle-treated eyes. Integral IOPs (cumulative IOP exposure, in mm Hg-days) and peak IOP (maximum IOP elevation during the experiment) were not significantly different between virus- and vehicle-treated eyes. (C) Graphic representation of the integral IOP of the average IOPs in experiment 2 in eyes injected with scAAV2.GFP and vehicle. Dark gray area between the viral and vehicle control constitutes the IOP–integral difference.

Figure 6.

Heat maps of sets of genes expressed in HTM cells infected with scAAV2.GFP and untreated control cells. Primary HTM cells were infected with scAAV2.GFP (∼1000 VP/cell; scAAV2) and harvested at 7 days after injection. Untreated (UNT) cells grown in parallel were used as a control. RNA was hybridized to microarray chips and analyzed with gene expression software. Expression signals in each chip were compared to a median value. Change from the median is visually represented by a color assignment (scale at right). Each row represents the expression of a single gene, and each column contains the genes selected in each category. (A) Expression levels of the total number of genes altered more than twofold in scAAV2.GFP-infected cells. (B) Expression levels of oxidative stress genes generated from the ontology gene list. (C) Expression levels of immune response genes generated from the ontology gene list. (D) Expression levels of extracellular matrix (ECM) genes generated from the ontology gene list. Most of the altered genes in scAAV were downregulated from the UNT. Expression of genes in the selected gene lists was barely affected in the scAAV2.GFP-infected cells.

Figure 7.

Venn diagrams of genes altered more than twofold and immune response genes. Each circle represents one gene list category. Intersections indicate the number of genes that are shared between the two categories. (A) Genes shared by the lists of altered twofold and immune response genes. (B) Heat map of the five immune response genes which were altered more than twofold. Only one immunorelated gene (FAS) of the 456 was upregulated, whereas the other four were downregulated in scAAV2.GFP-infected cells. This adeno-associated vector had a negligible effect on immune response genes.

Figure 8.

PBMC proliferative response against the GFP in monkeys. PBMCs derived from the blood of monkeys 1 and 2 were plated at 2 × 105 cells/well and cultured without antigen or with GFP at 10 μg/well. A stimulation with PMA/ionomycin was used as a positive control. Cells were cultured in triplicate wells for 5 days, and proliferation was assessed by measurement of [3H]-thymidine incorporation. The results are presented as an average counts per minute ± SE or as a stimulation index (SI: cpm in stimulated wells/cpm in unstimulated wells). Statistical analysis was performed by Student's t-test. *P < 0.05.