To confirm CNS
Rucaparib of TGF-β1 by the vectors, consecutive brains sections were analyzed by in situ hybridization using probes specific for both SFV and TGF-β1 RNA. Although scarce, foci positive for TGF-β1 could be detected co-localizing with SFV, demonstrating that the vectors were able to deliver and express the transgene in the CNS of mice upon peripheral delivery (Fig. 2A). Attempts were also made to detect TGF-β1 at the protein level, but due to non-specific antibody binding these studies proved less informative (not shown). In order to provide a semi-quantitative assessment of the gene delivery efficacy, infected samples were analyzed by real-time PCR. Correlating with the ongoing viremia two days p.i., mRNA levels of TGF-β1 were elevated in the
Pangaea spleens of both VA7-TGF-β1lat- and VA7-TGF-β1mut-infected mice (Fig. 2B). The increase was 1.7-fold for the latent vector and 1.4-fold for mutant vector compared to the parental virus, which itself did not induce any increase in TGF-β1 mRNA compared to uninfected control cells. Moreover, while the TGF-β1 mRNA levels in the brains of the VA7-TGF-β1mut-infected mice were clearly increased by day 2 p.i. (1.4-fold higher compared to rA774 and 2.3-fold higher than PBS) and higher still by day 5 p.i. (2.3-fold vs. rA774, 3.4-fold higher than PBS), the mRNA levels of TGF-β1 expressed by VA7-TGF-β1lat were prominently increased 5 days p.i. (6.1-fold higher than rA774, 9-fold higher than PBS) (Fig. 2C and D).
