Figure 4.

MnTBAP Counteracts the Adverse Effects of rAAV-Mediated i.m. Gene Delivery

(A) Protocol outline. C57BL/6 mice received i.p. multi-injections of MnTBAP or an equivalent volume of PBS (80 mg/kg daily during 5 days). Two hours after the first injection of MnTBAP, mice were administered PBS or rAAV1_CMV_SGCA_HY vector (2.5 × 10⁹ vg per mouse) in the left TA. Four days post-vector injection, mice were intravenously injected with 1.5 mg L-012, a chemical agent that reacts with ROS to produce light. Fifteen days post-rAAV-vector injection, muscle integrity, transgene and CD8a mRNA expression, and transgene-specific T cell responses were analyzed. (B) Histological analysis of injected TA muscle from PBS- or MnTBAP-treated mice, 15 days following injection of rAAV vector or control PBS. Microscopy analysis of cryosections of rAAV-injected muscles after H&E staining (left panel) or anti-CD8 staining followed by enzymatic detection (right panel) was performed. Images are representative of one experiment out of 2, with 3–4 mice per group. Scale bars, 500 μm. (C) High magnifications of selected areas shown in (D). Scale bars, 150 μm. (D and E) Analysis of CD8a and SGCA transgene expression by RT-PCR in injected TA muscle 15 days following injection of rAAV vector or control PBS. Levels of CD8a (D) and SGCA (E) mRNA are expressed in arbitrary units (AU) relative to the endogenous murine acidic ribosomal phosphoprotein (PO) mRNA (n = 2; 3–4 mice per group). (F and G) Analysis of transgene-specific T cell responses: 8 days (upper panels) and 15 days (lower panels) post-injection of vector, splenic cells were harvested to measure the frequency of transgene specific CD4⁺ (F) and CD8⁺ (G) T cells by IFN-γ ELISPOT. Dots represent individual mice. Horizontal bars indicate the average values. All statistical analyses were performed using the Mann-Whitney test. *p < 0.05; **p < 0.005.