scAAV2-GFP

Neurodegenerative diseases, including Parkinson's disease, amyotrophic lateral sclerosis (ALS), and Alzheimer's disease, pose significant challenges to treatment development due to drug delivery limitations and toxicity issues. To minimize the possibility of transgene contamination in the blood, researchers designed a CNS-specific AAV system. This system utilizes a self-complementary AAV (scAAV) encoding a quadruple repeat target sequence of hematopoietic cell-specific miR142-3p at the 3′ untranslated region (UTR). As a representative therapeutic survival gene for Parkinson's disease treatment, researchers integrated DX2, an antagonistic splice variant of AIMP2, an apoptotic gene known to be associated with Parkinson's disease, into the vector. This configuration ensures that transgene expression is strictly localized to the CNS, even if the vector enters blood cells. A single injection of scAAV-DX2 significantly improved behavior and motor activity in animal models of Parkinson's disease induced by rotenone or 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Importantly, comprehensive preclinical data that adhered to Good Laboratory Practice (GLP) standards showed no adverse effects in treated animals.

For the SK-N-SH cell line, the infection rate after 24 hours of treatment with 1000 MOI of ssAAV-GFP was comparable to that after 12 hours of treatment with 1000 MOI of scAAV-GFP (Figure 1A). This phenomenon also showed a similar trend when treated at 10000 MOI (Figure 1B). Therefore, in all cell lines tested, scAAV2 has a better infection effect than ssAAV2. In addition, scAAV2 also has a higher efficiency of infecting SH-SY5Y than ssAAV2 (Figure 1C, D). To evaluate the infectivity of the scAAV2 viral system to the striatum region, a major target of Parkinson's disease, the researchers injected scAAV2-GFP intracranially through minimally invasive surgery, targeting the right part of the striatum region. Transgene expression was initially verified by detecting the GFP signal in the striatum region (Figure 1E). Strong GFP signals around the injection site in the spinal cord indicated that the GFP signal was observable to diffuse from the administration focus (Figure 1F). Next, they delivered scAAV2-DX2 virus to the substantia nigra, an area densely populated with dopaminergic neurons, by intracranial injection. Notably, DX2 expression reached a saturation point four days after in vivo injection of the viral vector in the intracranial region (Figure 1G). AIMP2 appeared to be widely and stably expressed in tissues, while DX2 expression showed fluctuations between tissues (Figure 1H). In particular, organs rich in immune or hematopoietic cells (e.g., thymus, spleen) showed relatively high DX2 expression compared with other tissues (Figure 1H). Endogenous expression of DX2 was rarely observed in the brain.

Figure 1. Efficacy test of scAAV2-GFP and scAAV2-DX2 in vitro and in vivo. (Lee M H, et al., 2024)