scAAV9-GFP

Central nervous system (CNS) gene therapy holds promise as a powerful approach to treat a wide range of neurological disorders. Adeno-associated viral vectors based on serotype 9 (AAV9) have demonstrated that they are potent candidates for delivering gene therapy throughout the brain and spinal cord via intravenous, intrathecal, cisternal, and intracerebroventricular (i.c.v.) administration. Icv delivery of self-complementary AAV9 has been previously studied in neonatal mice, with only a single dose delivered. However, more information on the dose-response relationship of transduction efficiency in adult animals is needed before clinical trials can be considered. In the current study, three doses of self-complementary AAV9 were administered to adult rats. High levels of transduction were observed in the hippocampus, cerebellum, and cerebral cortex, and transduction increased with increasing doses. Both neurons and astrocytes were transduced. No evidence of astrogliosis was found at the doses tested. The results of this study will inform dosing studies in large animal models prior to clinical trials.

Here, adult Sprague-Dawley rats were injected unilaterally with scAAV9-GFP intracerebroventricularly and sacrificed 3 weeks later. Three doses were evaluated: 3.1 μl (low dose, 3.7X10¹⁰ vg), 15.5 μl (mid dose, 1.9X10¹¹ vg), and 77.5 μl (high dose, 9.3X10¹¹ vg). In addition, a fourth group was included that used the lowest dose of virus (3.7X10¹⁰ vg) delivered in the highest volume (77.5 μl) to account for differences in delivery volume (low dose/high volume). Figure 1 shows representative images of green fluorescent protein (GFP) staining of the brains of rats treated with the high dose. Two distribution types are visible at low magnification. In the cerebral cortex and cerebellum, cells were evenly distributed in these areas. Qualitative analysis showed that GFP expression was similar in the injected and uninjected hemispheres, with the exception of the area near the injection track, which showed higher GFP expression levels. In contrast, limbic regions such as the hippocampus and striatum showed different transduction patterns on both sides of the brain following unilateral intraventricular injections. Cervical spinal cord sections stained positive for GFP, especially in the high-dose group (Figure 1c). However, there was little evidence of transduction of cell bodies in the ventral gray matter. Instead, most staining appeared to be from axonal projections of neurons outside the spinal cord.

Figure 1. Widespread transduction of the CNS occurs following i.c.v. scAAV9 delivery. Sprague-Dawley rats were killed 3 weeks after receiving i.c.v. injection of scAAV9-GFP or saline. (Donsante A, et al., 2016)