Evidence of F.IX RNA, AAV vector DNA insubject Fmuscle. (A) Evidence of active transcription of F.IX RNA. Two independent primer pair PCR amplifications (216-bp and 408-bp amplicons) specific for human F.IX cDNA on patient muscle cDNA derived from isolated RNA. Three-week post-AAV2-CMV-F.IX–injected mouse (C57BL/6) muscle RNA-derived cDNA serves as a positive control. (B) Evidence of AAV vector genome in patient muscle. Primers amplifying a 242-bp genomic DNA segment from the CMV promoter of the vector to the exon 1/intron 1 junction of the transgene cassette reveal persistence of vector in injected patient muscle DNA. Three-week post-AAV2-CMV-F.IX–injected quadriceps mouse muscle genomic DNA serves as a positive control, while the contralateral uninjected quadriceps muscle serves as a negative mouse control.