Figure 5.
MnTBAP Inhibits Primary and Memory Humoral Response against Capsid Allowing Efficient Readministration of rAAV Vector in Muscle
(A) Protocol outline. C57BL/6 mice received i.p. multi-injections of MnTBAP (80 mg/kg daily during 5 days) or an equivalent volume of PBS. Two hours after the first injection of MnTBAP, mice were administered i.v. injections with PBS or rAAV1_CMV_SGCA_HY vector (2.5 × 109 vg per mouse). Each mouse was subjected to a second injection of rAAV serotype 1 encoding the mSEAP reporter gene (2 × 109 vg of rAAV1_CMV_mSEAP vector per mouse) into the left TA, 28 days after the first injection of vector. (B and C) Analysis of capsid-specific primary humoral response: anti- rAAV1 IgG titers were measured by ELISA in serum samples harvested at day 21 (B) and day 28 (C) post-vector injection. Curves represent the mean ± SD of optical density (OD) obtained at the indicated dilutions of sera from 6 mice per group and compare anti-rAAV IgG response in MnTBAP-treated mice (open circles) with that in PBS-treated mice (closed circles). (D) Analysis of capsid-specific memory humoral response after rAAV1 readministration (rAAV1_CMV_mSEAP vector) in TA. Measurement of anti- rAAV1 IgG titers by ELISA was performed in serum samples from 6 mice per group, harvested 10 days after rAAV vector readministration. For (B)–(D), statistical analyses with the Mann-Whitney test were performed on AUC values, obtained from ODs at four dilution points for each mouse serum. (E) Histochemical detection of mSEAP in injected TA harvested 93 days after vector readministration (day 121). In the first panel, non-injected right contralateral TA is represented as negative control. The second panel shows injected left TA with the rAAV1_CMV_mSEAP vector alone as positive control of mSEAP expression in muscle. The third and fourth panels show administered left TA with the rAAV1_CMV_mSEAP vector in PBS- or MnTBAP-treated mice, respectively, and who received an initial systemic injection of rAAV1_CMV_SGCA_HY vector. Images are representative of one mouse out of 6 per group. Scale bars, 500 μm. (F) Serum level of mSEAP measured by a chemiluminescent assay from mice harvested 93 days after vector readministration (day 121). Dots represent individual mice (closed and open circles show PBS- and MnTBAP-treated mice, respectively). Statistical analyses were performed using the Mann-Whitney test. (G) Copy number analysis of readministered rAAV1 in injected TA by quantitative CMV PCR assay with endogeneous titin gene for normalization. Data are expressed in AUs relative to titin DNA. Each symbol represents a mouse (closed and open circles indicate PBS- and MnTBAP-treated mice, respectively). Statistical analyses were performed using the Mann-Whitney test. **p < 0.005.