AAV9-Mediated Gene Delivery in CNS

The transgene expression cassette included AAV2 terminal repeats, the hybrid cytomegalovirus/chicken β-actin promoter and the bovine growth hormone polyadenylation sequence.^{5 (link)} This study used separate constructs with DNA encoding either GFP, human wild-type TDP-43^{5 (link)} or human UPF1 with an N-terminal myc epitope tag (mycUPF1).^{31 (link)} The addition of the myc epitope tag does not diminish UPF1’s function in a cellular assay of NMD.^{32 (link)} Additionally, a vector-only construct, that is, empty vector, was used to control for viral particles. DNAs were packaged into recombinant AAV9 as described previously.^{5 (link)} The AAV9 serotype was first described by Gao et al.^{33 (link)} and has enhanced gene transfer to the CNS.^{6 (link),9 (link),20 (link),34 (link)–37 (link)} Helper and AAV9 capsid plasmids used to generate AAV9 were from the University of Pennsylvania (Philadelphia, PA, USA).^{33 (link)} Viral stocks were sterilized using Millipore (Billerica, MA, USA) Millex-GV syringe filter, aliquoted and frozen. Viral genome copies were titered using dot-blot assay, and equal titer doses were obtained by diluting stocks in lactated Ringer’s solution (Baxter Healthcare, Deerfield, IL, USA). Through the course of this study, two batches of AAV9 TDP-43 and three batches of AAV9 mycUPF1 were used, and consistent results were obtained across the batches.